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STANDARDIZING EMBRYO RESCUE TECHNIQUE IN TRITICUM SYSTEM TO GENERATE NEW VARIABILITY
ABSTRACT
THESIS SUBMITTED FOR THE DEGREE OF
Boctor of $i|tlo£io9i)P M
BOTANY
GHAZALA PRAVEEN
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (INDIA)
2002
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India has been a major importer of wheat grain till 1960s. It was
with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64
and Sharbati Sonora, Kalyansona, Sonalika, Arjun, WL 711, HD 2329,
PBW 343 etc, the green revolution resulted increasing wheat production
to the extent that India became 2"* largest wheat producing country in the
world in 1999. One of the major drawbacks of the green revolution is that
genetic erosion of the indigenous genotypes/native germplasm pool
occurs due to dependence on few selected types only and thus making the
crop more vulnerable to various diseases and pests. The new varieties
released every year do not last long as new pathotypes appear and also
old resistant genes get defeated due to development of virulence against
the existing ones. Since success of any crop improvement program very
much depends on the extent of genetic variability present in the base
population, it is required to broaden the genetic base by enriching the
crop with more and more desirable genes.
Wild relatives of wheat are a valuable reservoir of genes which are either
absent in cultivated wheat or their diversity is narrow particularly with respect to
pest resistance, tolerance to biotic and abiotic stresses and various morphological
traits including the end use properties for making bread and pasta as well as other
products. Therefore, the wild species can be used as a potential source to add
novel variability and affecting the genetic enrichment program through wide
crossing. However, in most of the wide crosses the embryo aborts at various
levels of development, Hence, the technique of embryo rescue involving excision
and culture of embryos aseptically had been quite successful in transferring the
useful traits from alien sources to the background of cultivated species.
During the present course of study, before going straight for rescuing
embryo, an attempt has been made to develop a suitable regeneration protocol
Ahslracl 2
and to select out the better responding genotype in vitro. Although, several
other factors can affect the callus induction and plant regeneration from
rescued embryo. Besides, an established protocol can also help in going for
transformational studies through various methods, inducing somaclonal
variation, screening of genotypes against various stresses and in vitro
mutational studies.
In lieu of this, an effort was made to standardize a regeneration protocol,
using the cultivars of both 71 durum and T. aestivum species of wheat. Later on,
the embryos of the wide crosses were rescued to test the protocol on selected
varieties and media.
The salient results/observations recorded from the present study have
been summarized here under:
> Two varieties each for bread wheat (WH 542 and UP 2338) and durum
wheat (Bijaga Yellow and Gulab) were screened on four callus induction
media CI (MS+2.4-D, 2mg/l), C2 (MS + 2.4-D, 4mg/l), C3 (MS+ 2,4-D,
2mg/l + lAA. 0.5 mg/1) and C4 (B5 + 2,4-D, 2mg/l). The embryos were
excised at 10, 12, 14, 16 and 18 days post pollination/days post anthesis.
12 and 14 days calli of bread wheat and 12, 14 and 16 days old calli of
durum wheat were found optimum. Maximum frequency of callus
induction was recorded on C1 (embryogenic) than on C2 (loose and non-
embryogenic)
> Five media viz., Rl (left on the same medium and exposing to light), R2
(MS + Kinetin, 2 mg/1), R3 (MS + Kinetin, 2mg/l + lAA, 0.5 mg/1), R4
(MS + BAP, 2 mg/1) and R5 (MS +BAP, 2ml/l + lAA, 2mg/l) were tested
to achieve regeneration from calli produced on CI, C2, C3 and C4 media.
> Three media viz., Rl, R2 and R5 were found to give good regeneration in
all the varieties, while R3 and R4 proved quite poor in this regard.
Abstract 3
> The regenerated plantlets were grown on three media viz., SI (left on the
same medium where they were induced), S2 (MS + GA3 0.5 mg/1 +30 g/1
sucrose) and S3 (GR free MS + 20 g/1 sucrose) up to 3 to 4 leaf stage.
Among these, the S3 medium was found better than other two media.
> The regenerated plantlets were transferred on a range of rooting media
and the medium containing MS salts without vitamins + NAAimg/1 +
sucrose 20g/l) was found better responding than rest.
> The dark condition during callusing and light during regeneration phase
proved quite effective. Interestingly, the alternate light and dark
treatment using 20 watts electric bulbs (16/8 hrs light/dark) could induce
regeneration in calli produced on MS + 2, 4-D (2mg/l) medium.
> A temperature of 25±2°C was found optimum, while the further rise in
temperature resulted in the browning of callus.
> The plantlets were hardened in small pots filled with agropeat and soilrite
in equal proportions and put into growth chamber at 15±1°C and 90 per
cent relative humidity. Established plants were transferred in bigger pots
containing soil, sand, FYM and agropeat in equal proportions.
> Following the above mentioned protocol, six additional varieties each of
bread wheat (CPAN 3004. KRL 1-4. PBW 343, PBW 373. Raj 3765 and
HD 2329) and durum wheat (HD 4530. HI 8381, Raj 1555. PDW 215.
PDW 233 and MACS 2846) were screened to check both the validity of
protocol and selection of better responding genotypes.
> The age of immature embryo, genotype, media composition and culture
conditions all affected the regeneration potential of the explant.
Abstract 4
> The above mentioned protocol was further extended to grow hybrid plants
from the crosses involving wide relatives.
> Five varieities each of bread wheat (WH 542, UP 2338, HD 2329, PBW
343 and Raj 3765) and durum wheat (Bijaga Yellow, Raj 1555. Gulab,
PDW 215 and PDW 233) were taken as female and 20 species of wild
triticum and Aegilops species [T. urartu (A"), T. hoeticum (A**), Ae.
speltoides(S), Ae. longissima (S), Ae. sharonensLs{S^^^), Ae. hicornis (S^),
Ae. squarrosa (D), Ae. caudata (C), Ae. markgrafii (C), Ae. umbellulata
(U), Ae. cylindrica (CD), Ae. ventricosa (D"), Ae. kolschyi (US), Ae.
peregrine (US), Ae. triuncialis (UC), Ae. ovata (UM"), Ae. lorentii
i\JM\ Ae. triaristata (UM'), Ae. geniculata{\JM°), T. araraticufn(AG)]
were used as pollen parent.
> The hand emasculated spikes were pollinated twice with pollens collected
from the alien species. The pollinated spikes were sprayed with 2. 4-D +
GA3 solution for four consecutive days starting from the day of
emasculation.
> The hybrid embryos were scooped out at 10, 12, 14 and 16 days with
slight variation in some crosses and cultured aseptically following the
above mentioned protocol.
> The embryos rescued after 12 to 14 DPP were found to give optimum
regeneration in most of the crosses.
> The regenerated hybrid plants were established in the field with various
levels of seed settings.
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STANDARDIZING EMBRYO RESCUE TECHNIQUE IN TRITICUM SYSTEM TO GENERATE NEW VARIABILITY
THESIS SUBMITTED FOR THE DEGREE OF
Boctor of $))tIo£iop{)p M
BOTANY VV
W
- > \
< ^ . '
GHAZALA PRAVEEN
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (INDIA)
2002
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T5841
PLANT TISSUE CULTURE & CYTOGENETICS LABORATORIES
Department of Botany, Aligarh Muslim University, Aligarh.202 002 (INDIA)
CERTIFICATE
Certified that the thesis entitled "STANDARDIZING EMBRYO
RESCUE TECHNIQUE IN TRITICUM SYSTEM TO GENERATE
NEW VARIABILITY " embodies the original research work carried out by
Ms. GHAZALA PRAVEEN under our guidance and supervision. The
work has not been submitted elsewhere for the award of any other degree or
diploma and can be submitted in fulfillment of the requirement for the
award of the degree of Doctor of Philosophy in Botany of AUgarh Muslim
University, Aligarh.
Dr. Mohammad Anis Supervisor Plant Cytogenetics and Tissue Culture Lab Department of Botany, Aligarh Muslim University AUgarh 202 002 (India)
y^^r^y^
Dr. Jag Shoran Co-Supervisor
Principal Investigator Crop Improvement Division
Directorate of Wheat Research (Indian Council of Agricultural Research)
Kamal-132 001 (India)
VedlccubeJi
to
Ii4i^e6sezC by tke^ dUcoutrL&i of gireaor Mt*uLei, BlffeMy
LH/ 1906 i^^OAiateA'.
"tf t Huw Loolc into tke^juiwre^ t aiH/jhiresee-
the' tmte' COIMIHO wke*v « ^ skaH be^ oJbLe^ to
UKLte^ uv onZ' tmriety the best atiaiUy, the best
cropping poci^tr, the best strcuit MUL SO o^t
After alt4wst cu ctntuyy oftUajit brttdlna n/e^ are^ still uv the
Huddle but the iMiMMlnatioH/ of com^eteMt ploMt breeders
like B(£fzH/ stiMudatts lu to specMiatefurther:
AC1CN'0M/L<E(Dg^M<E!Nr
To Begin xintfi I Bow in reverence to the JiCmigfity, as it is indeed!Kis BCessings,
wfiicfi (edrm to complete this piece ofworf{^
It is my proud privilege to ej(press my deep sense of gratitude and sincere thanf{s to
my supervisor (Dr. MohammadJinis, ^aderin the (Department of (Botany, JiCigarhMusCim
University, Jidgarh and my co-supervisor (Dr. Jag Shoran, (PrincipaC, Scientist, (Directorate
of 'Wheat (Research, 'Karrw.C for Benevolent guidance, constant watchfuC interest and
heCping attitude throughout the present investigation.
I am ex}reme[y thankful to (Dr. S. !Kagarajan, 'E^Q^oject (Director, (Directorate of
'Wheat (Research for alfoiving me to carry out my ej^periment at the (Directorate of'Wheat
(Research, T(amaC My thanlifuCruiss is also ej(tended to (Prof Saeed^. Siddiqui, Tji-
chairman, (Department of (Botany and (Prof SamiuC[ah, Chairman, (Department of (Botany
Jlligarh MusCim 1)niversity, JiCigarhfor mailing things convenient for me to Cay down my
eo(;periment at T^amaC
It is my great pleasure to record my deep sense of gratitude and irufeBtedness to (Dr.
(B.S. Tyagi, Senior Scientist, for his ej(ceClent guidance, meticuCous supervison and healthy
criticism throughout the present investigation and preparation of manuscript, vAthout
which it would have not Been possiBle to Bring the thesis in the present form.
I ackriowCedge (Dr. (D.S. Chauhan, (P.I.-Agronomy, (Dr. (5.%, Misra,
(P.I.-QuaCity and (Basic Sciences, ©r. S. Sharma, (P.I.-(P[ant (Protection and
(Dr. 9^. Singh, (P. I.-Computers and Statistics for extending their heCp whenever required.
I also express my sincere thanl{s and gratitude to (Dr. (Raj "Kumar for providing
faciCities aruf infrastructure for the experimental wor^(Dr. (B.S- MaCi^ requires a special
mention for procuring necessary manpower
The moral support from (Dr. (Rjitan Tiwari, (Dr. < 7C ^upta, SeniorScientists and
my friends ^agan (Bajwa, Moushami (Dutt aruffatima M. Sultana proved to Be afource
of Inspiration and zest for me.
/ am very mucfi tfianfiJuCto (Dr. Sfiafiid ^hmacC and Mr. (Rajendra % goCafor
their support, immense care, irritating criticism (?) and unconditionaC fieCp. Jissistance
extended to me 6yMr. T^eeraj Tyagi, Mr. Harsh Qhoudhary, Mr. <Pradeep %umar, (Dr.
AnwarShahzad, Mr. Mohd<Faisa(, Mr. M. KashifJfusain, Mr. 9/aseemJLhmadisdu(y
acf^nowtedge. Co-operation of Mr. "Kjizi 'Fariduddin and (Dr. Jlrif "Wad is also
acfinowCedged.
Services extended 6y Mr. ([(fljendra Sharma, (Photographer and Mr. yiagar,
Librarian needs a speciaC mention. The personal attention given By Mr. JlniCforthe entire
computerjoS related to my thesis is commendaBle.
I also stand oSCiged to aU those people -who woriied Behind the curtain with great
sincerity and utmost seCfkssness to maf{e this project a success and to whom I cannot
ej(press individuaCgratitude.
(Research associateship provided By the (Department of(BiotechnoCogy, !New (DeChi
through (Directorate of'Wheat (Research, 'KiamaCis duly acfijiowledged.
(Finally, no words can match the emotionaC succor and amorous Cove which I got
from my (Parents, sistersShaBana, Sameena,Arzoo, SheeBa, BrotherAamirandMu^rram
JIfi during the entire course of my study. It was that intangiBle support mechanism which
helped me in proving my self as a denoted scholar and worthy memBer of concerned
fraternity.
f ( X ^ ^ ^
(ghazaCa (Praveen)
Contents Chapter Title Page No
1. Introduction
1.1 Wheat crop improvement, genetic variability and alien gene transfer
1.2 Wide crossing: history and prospects
1.3 Wild Triticum and goat grass : a useful reservoir
1.4 Constraints to wide crossing
1.6 Study undertaken
2. Review of Literature
2.1. Embryo culture
2.2. Wide hybridization
2.3. Constraints to wide hybridization
2.4. Stratagies to exploit alien sources
2.4.1. Choice of promising and compatible parents
2.4.2. Crossing procedure and pre/post pollination treatments
2.5. Embryo rescue
2.5.1. Immature embryo culture
2.5.1.1. Stage of scooping out
2.5.1.2. Culture pathways
2.5.1.3. Media manipulation
2.5.6. Alternative methods
2.6. Alien genes transferred in wheat
3. Materials and Methods
3.1. Procurement of wheat germplasm
3.2. Alien germplasm
3.3. Harvesting of immature embryos and wide crossing
3.4. /« v/Yro culture
3.4.1. Basal culture medium
3.4.1.1 Immature embryo culture
3.4.1.2. Rescued embryo culture
3.4.2. Growth regulators
3.4.3. Preparation of culture medium
1-9
4
6
7
9
10-22
11
16
16
17
17
18
19
19
19
20
20
21
22
23-30
23
24
25
26
26
26
26
26
26
Chapter
3.5.
3.6.
3.7.
3.8.
4. Results
4.1.
4.2.
4.3.
Asepti
Title
ic technique
Sterilization and plating of plant material
Incubation of cultures
Hardening of the plantlets
Standardization of regeneration protocol
4.1.1.
4.1.2.
4.1.3.
4.1.4.
4.1.5.
4.1.6.
Callus induction
4.1.1.1. Effect of callus induction media
4.1.1.2. Influence of age of embryos (DPP)
Regeneration
4.1.2.1. Bread wheat
4.1.2.2. Durum wheat
Interaction of callus initiation and regeneration medium
4.1.3.1. Bread wheat
4.1.3.2. Durum wheat
4.1.3.3. Statistical inference
Shoot proliferation and plantlet elongation
4.1.4.1. Bread wheat
4.1.4.2. Durum wheat
4.1.4.3. Statistical inference
Rhizogenesis in regenerants
4.1.5.1. Inference
Hardening and acclimatization
Selection of the better responding genotypes
4.2.1.
4.2.2.
4.2.3.
Callusing
Precocious germination
Regeneration
4.2.3.1. Regeneration on R1 medium
4.2.3.2. Regeneration on R2 medium
4.2.3.3. Regeneration on R5 medium
4.2.3.4. Selection of the varieties
Embryo rescue and wide crossing
Page No
28
28
29
29
31-61
31
31
31 T "1
34
34
37
37
39
39
39
39
41
41
41
43
43
45
45
45
47
47
47
47
50
53
Chapter Title Page No
5. Discussion 62-71
5.1. Basal culture media 62
5.2. Growth regulator 63
5.3. Precocious germination 64
5.4. Age of embryos 65
5.5. Growth condition of cultures 66
5.6. Genotype 67
5.7. Wide crossing and embryo rescue 68
6. Summary and Conclusion 72-74
Bibliography i-xxi
Annexure I-III
List of Tables
No. Title Page No.
Table 1: Alien wheat sources for specific traits 6
Table 2: Factors influencing in vitro responses of immature 12 embryos of wheat
Table 3: Disease resistance genes incorporated in wheat gene 22 pool from alien sources.
Table 4: Varieties used in standardization of regeneration 23 protocol from immature embryos and selection of genotypes to be used in wide crossing.
Table 5: Varieties selected for wide crossing program 24
Table 6: Wild species used in crossing program 24
Table 7: Recipe of different MS medium's used (final volume 1 27 liter).
Table 8: Effect of age of embryos (days post pollination, DPP) 32 and media composition on callus induction and precocious germination (data recorded 25 days after culture).
Table 9: Percent regeneration on different media in bread wheat 3 5 varieties (data recorded 25 days Qftet tuLtuYe).
Table 10: Percent regeneration on different medium from durum 36 wheat varieties (data recorded 25 days after culture)
Table 11: Influence of media composition and age of embryos 3 8 (days post pollination, DPP) on effective regeneration in bread wheat.
Table 12: Influence of media composition and age of embryos 40 (days post pollination, DPP) on effective regeneration i.e. regeneration per 10 explant cultured in durum wheat.
Table 13: Number of shoots (having at least 3 nodes) per callus 42 and per 10 ex-plant (data recorded 20 days after sub culture)-
Table 14: Rooting efficiency of in vitro regenerated shoots of 44 bread and durum wheat cultivars •
No. Title Page No.
Table 15: Per cent callus induction and precocious germination 46 from the immature embryo ex-plant of bread and durum wheats on MS+2,4D (2mg/l) over different days post anthesis (dpa)
Table 16: Per cent regeneration on Rl medium from calli 48 induced on CI (data recorded 25 days after transferring on to regeneration medium)
Table 17: Per cent regeneration on R2 medium from calli 49 induced on CI (data recorded 20 days after culture)
Table 18: Per cent regeneration on R5 medium from calli 51 induced on CI (data recorded 25 days after transferring on regeneration medium)
Table 19: Comparative chart for callusing and regeneration 52 potential of some bread and durum wheat varieties
Table 20: Wide crosses performed to standardize embryo rescue 54 technique
Table 21: Differential responses to callus induction and 57 regeneration of rescued embryos of different age group
Table 22: Cross combinations where dissectible embryos could 61 not be obtained
Table 23: Rescued embryos showing no response to callus 61 induction
List of Figures
Fig. 1: Genetic gain in yield after insertion of alien genes
Fig. 2: Possible strategy for exploitation of genetic resources and advanced genetic
technique in a long term breeding program.
Fig.3: Different types of induced calli
Fig.4: Morphogensis in different types of calli
Fig. 5: Different development stages
Fig.6: Hardening and acclimatization of plantlets
Fig.7: Normal and abnormal embryos rescued from wide crosses
Fig. 8: Regeneration from embryos along with peculiar hybrid spikes.
Fig. 9: T. urartu crosses at different stages
Fig. 10: Different wild hybrid plants established in field through embryo rescue
ZNTKOPUCTtON
1. Introduction
Wheat is an important and most ancient cereal crop in the world. The
variability present in the cultivated and wild relatives of wheat, traces its
origin to the evolution of angiosperms. Over the period, the wheat has
undergone various divergent and convergent evolutions leading to the modern
polyploid form. It belongs to the genus Tritlcum that comprises around 500
species. The commonly cultivated species are bread and durum wheats, the
dicoccum being cultivated only in certain pockets including the peninsular
part of India. Wheat is cultivated in one or the other part of the globe
throughout the year. In India aestivum, durum and dicoccum wheat cultivation
is in practice. T. aestivum acquires prime position with more than 90% share,
while T. durum, the second major species, contributes around 4% of total
wheat production.
Wheat is a unique gift of nature to mankind as it can be molded into
innumerable products like chapaties, bread, cakes, crackers, biscuits, pastries,
pizzas, patties, pasta, noodles, macaroni, sphagetties etc. Wheat is consumed
by nearly 35% of world population and contributes 20% food calories. In
India the availability of wheat has increased from about 79g to more than
185g/head/day despite doubling the -. ^ population since 1961. The
increase in protein availability due to reduced availability of pulses from
69g/head/day in 1961 to dihoxxi 38g/head/day has been compensated by the
increased availability of wheat. It is also reported to contribute more calories
and protein to the world's diet than any other cereal crop (Hanson et al.,
1982), sharing about 40% of total cereal protein production. The major
protein fractions are albumin, globulin, protease, gliadin and glutelin.
Moreover, it is also good source of vitamins of the B complex group and E.
The common bread wheat (6x) and durum wheat (4x) both are true
breeding natural hybrids. Bread wheat is an allohexaploid (2n=6x=42),
Inlrocluclion 2
composed of three genomes A, B and D. derived from three diploid wild
species native to the Middle East. The donor of A genome is Triticum urartu
Turn. The source of B genome is Aegilops speltoides Tousch. The D genome
came from Aegilops squarossa L. [= Ae. tauschii Coss, T. tauschii (Coss.)
Schmalti]. The cytoplasm of wheat is known from B genome donor. Durum
wheat is the fore runner of bread wheat, an allotetraploid (2n=4x=28) with
two genomes A and B. It is a result of natural hybridization between T. urartu
and Ae. speltoides. The hexaploid wheat evolved latter via natural
hybridization of durum wheat with a third wild grass Ae. squarrosa which
contributed its D genome (Jauhar, 199 ) . This extraordinary series of
hybridization produced both the durum and the bread wheat that sustain
mankind.
India used to be a major importer of wheat grain till 1960s. It was with
the advent of semi dwarf wheat varieties like Lerma Rojo, Sonara 64,Sharbati
Sonora, Kalyansona, Sonalika, Arjun, WL 711, HD 2329, PBW 343 etc. a
green revolution set in increasing wheat production to the extent that India
become 2"'' largest wheat producing country in the world in 1999. Now, it is
the time to think about sustaining this increase in yield per se and improving
the grain quality for export purpose. One of the major drawbacks of the green
revolution is that genetic erosion of the indigenous genotypes/native
germplasm pool occurs due to dependence on few selected types only and thus
making the crop more vulnerable to various diseases and pests. The new
varieties released every year do not last long as new pathotypes appear and
also old resistant genes get defeated due to development of virulence against
the existing ones. Therefore, there is always an urge to incorporate new genes
for different biotic and abiotic stresses.
Since success of any crop improvement program very much depends on
the extent of the genetic variability present in the base population, it is
required to broaden the genetic base by enriching the crop with more and
more desirable genes. Even long term breeding objectives can not be realized
unless more genetic variability is generated. Further, several international laws
Introduction J
like Intellectual Property Rights (IRP). General Agreement on Trade and
Tariffs (GATT), Plant Variety Rights (PVR), etc. are either been framed/or
likely to be framed which may restrict the inflow of germplasm being
provided by other countries. Hence, national program should be self-sufficient
and least dependent on the external sources.
1.1 WHEAT CROP IMPROVEMENT, GENETIC VARIABILITY AND ALIEN GENE TRANSFER
With the development and adoption of high yielding wheat varieties
with narrow genetic base over large growing areas, wheat crop has become
more vulnerable to biotic and abiotic stresses. Also, tremendous variability
existing in the indigenous landraces and wild germplam has been eroded and
facing extinction. As a result, the plant breeders find less and less appropriate
germplasm with desired traits among cultivated crops. The eroding genetic
base of the cultivated wheat has lead the research workers to investigate the
possibility of utilizing the genetic variation present in the wild relatives
(Feldman and Sears. 1981).
At present, there is lack of genes for some of the important characters
to be used in wheat breeding. Particularly, it is true for resistance to wheat
diseases that are either absent in the common wheat genebank or their
diversity is insufficient to support the wheat breeding program.
There are a number of ways to create and add new variability in
the wheat germplasm. The wild crosses are one such opportunity available
with the wheat geneticists. The secondary gene pool of wiJd wheat and alien
species of Aegilops, Agropyron, Elymus and other related genera that have
been reported to constitute a rich repository of resistance to various diseases,
stresses, quality characters etc. and can effectively contribute to genetic
enrichment program (Dhaliwal and Gill 1982; Gill et al., 1983; Tomerlin et
al., 1984; Mosemen et al., 1985; Warham et al., 1986; Jauhar 1993, 1995,
199/; Jiang et al., 1994; ThieJe et al, 2002).
It has been noted that the productivity has taken jump in India by the
development of many varieties since green revolution and these varieties
Introduction 4
made a mosaic which reduced the pest risks (Fig.l). The recently released
varieties like PBW 343 etc. have IB/IR translocations thus have diverse gene
pool. The incorporation of rye segment into wheat could lead to a wonder
combination of traits and resulting high yields and wide adaptation. In fact,
alien gene transfers through wide hybridization, coupled with cytogenetic
technique, may be the alternative option for creating superior wheat
germplasm.
Fig 1: Genetic gain in yield after insertion of alien genes
1965 1970 1975 1980 1985 1990 1994 1995 1998 S227 K.Sona WL711 Arjun HD2329 CPAN3004 UP2338 PBW3A3 HD2687
Winter x Spring (1B/1R types types)
1.2 WIDE CROSSING: HISTORY AND PROSPECTS
Wide crossing altogether is not a very new concept. In 1876, when
Stephen Wilson humbly presented some completely sterile ears of wheat x rye
hybrid, a beginning was made. Despite this early successful effort, the
potential of wide hybridization in agronomic species was not appreciated until
after the rediscovery of Mendal's laws and chromosome engineering
techniques (Sears, 1972). First successful transfer of a single trait of
economic relevance from a related wild species into cultivated wheat was
/nlrodiiclion 5
carried out by Sears (1956). He deliberately transferred a small insertion by
translocating a part of Ae. umbellulata to chromosome 6B of bread wheat
carrying resistance to leaf rust (Lr9). Later on by exploiting the homeologous
relationship between the genomes of related species and those of aesiivum and
durum wheat it became possible to incorporate genes or chromosome
segments from several of the related genera/species (Table 1).
Apart from the practical motivation to introgress genes from the alien
species into wheat, intergeneric hybrids offer promise to provide basic
cytological. evolutionary or phylogenetic information about the parental
species (Jauhar and Joppa, 1996; Bommineni et al., 1997). These wide crosses
are also a means of producing haploid plants by preferential chromosome
elimination of the pollen parent in early stage of development (Laurie and
Bennett, 1986; Inagaki and Mujeeb-Kazi. 1995).
During the evolution of the Triticeae the basic primeval genome of
seven pairs of homologous chromosomes has been modified following
speciation and allopolyploidization. However, a degree of similarity or
"homoeology' (Huskin, 1931) has been retained, so the genetic material may
be transferred to T. aestivum directly from closely related species with one or
more genomes in common with bread wheat. The triplication of much of the
genetic material in hexaploid i.e. AA, BB and DD (Sears 1954, 1966)
provides two advantages to alien gene transfer. Firstly, the wheat can tolerate
the loss or addition of whole chromosome usually without drastically
affecting the viability of the plant (Merker, 1992; Jauhar, 1993), and
secondly, duplicate genes present in the other two genomes can often mask
the effects of a deleterious genes present on an alien chromosome substituted
for a given wheat chromosome from the third genome. These factors coupled
with cytogenetic technique lead to the transfer of a number of desirable genes
into cultivated wheat, and set wheat ahead from other cereal crops in terms of
the possibilities for the introduction of genetic material from other species
(Ceoloni, 1987; Jiang el al., 1994).
Introduction 6
Table 1: Alien wheat sources for specific traits
Traits Species
Leaf and stripe rust
Karnal bunt
Foliar blights
Moisture stress
Drought tolerance
Heat tolerance
Salt tolerance
Herbicide resistance
Apomixis
Male sterility
Biomass
Harvest index
Grain number
Grain weight
Grain quality
Lysine content
Storage protein
T. monococcum, T. boeticum, T. urartu, Aegilops
bicornis, Ae. speltoides, Ae. squarrosa, Ae. ovala,
Ae. triaristata. Ae. lorentii
T. boeticum, T. urartu, Ae. tauschii, T. araraticuin,
Ae. caudate, Ae. sharonensis
T. spelta, T. dicoccum
T. timopheevii
Ae. kotschyi, Ae. umbalullata, Ae. squarrosa
Ae. speltoides, Ae. triuncialis
Agropyron, Elynius, Secale cereale
Ae. bicornis, Ae. cylindrical
Elymus spp.
Ae. squarrosa
T. monococcum, T. dicoccum, Ae. speltoides.
Ae. Tauschii
T. monococcum, T. boeticum, T. urartu, Ae. bicornis,
Ae. tauschii
T. turgidum
T. turgidum, T. carthlicum, T. tlicoccoides
T. monococcum, T. boeticum, T. urartu, Secale cereale
T. boeticum, T urartu
T. dicoccoides
1.3 WILD TRITICUM AND GOAT GRASS RESERVOIR
A USEFUL
Extensive use has been made of a number of wild species in Aegilops
and Triticum (Gale and Miller 1987), high degree of homo/homoeology
between Triticum aestivum and its diploid and triploid progenitors and
Aegilops species make it an excellent material to cross with. They are also
sources of A, D and B genomes and constitute a rich repository of desirable
genes (Table 1). Therefore, the probability of introducing alien variation from
these genomes is much higher than from others (Kimber, 1993). Wild
Introduclion 7
Trilicum and Aegilops, both are annual, divided into different sections ranged
from diploid to octaploid in some cases. In literature, it has been shown that
a number of useful genes have been extracted from them till date (Table-4).
Siill ihey have a potential to be taken as the genetic stock for the specific trait
improvement program or pyramiding the desired genes.
1.4 CONSTRAINTS TO WIDE CROSSING
I he challenge is to incorporate this "new" germplasm routinely into
existing wheal crop. The ways and means of exploiting them are depicted in
fig. 1. Hybrid production occupies the initial critical step as generally various
barriers encountered during production of wide crosses frustrated such
jtlempts. Some times the strong crossing incompatibility makes direct
h\ bridizatii)ii between wheat and other Trilicum and Aegilops species
difficult. Also in most of the cases of interspecific and intergeneric hybrids,
seed setting is very low as either embryo aborts at certain stage of
de\elopment or their subsequent development is arrested. The reason to
degeneration of embryos prior to full maturity may be (1) the inability of the
endosperm to carry out its normal role in supplying nutrients to the
developing embryo, or (2) the maternal tissue being hostile or antagonistic to
the development of the embryo. In other words embryo culture is useful when
the embryo has the ability to mature but is prevented from doing so (Ceoloni
ci a/.. 1993). In others, embryo does not abort but seed setting is very low. In
such cases, in vitro clonal propagation of hybrid embryos can generate more
plants to be used for further studies (Vasil, 1990).
The successful rescue of embryos paved the way for a series of
application of this technique to recover interspecific hybrids in a large
number of genera including Hordeum, Triticale, Agropyron and Trilicum.
The important examples of intergeneric hybrids obtained via embryo rescue
are barely x rye (Fedak 1986). wheat x barley (Koba et al., 1991), wheat x
maize (Lam-ie el al., 1990), Hordeum x Elymu.s (Lu and VanBothmer 1990;
Balyan and Fedak 1991), Elymus x Trilicum (Lu and Vanbothmer. 1990), T.
aeslivum x Aegilops (Ter-kuile el al. 1988), T. durum x Aegilop.s (Djenadi.
Introduction 8
2000) etc. Disease resistance has been successfully introduced from Triticum
timopheevi to bread wheat via embryo rescue technique (Bhowal et al., 1993).
Embryo rescue was also used to obtain seeds from T. durum x T. timopheevi
(Claesson e /a / . , 1990).
Land Races - Wild Species Obsolete cv. Present cv.
Traditional Multiple Crosses
Wide Crossing Embryo Rescue
Protoplast fusion
New Genetic Variation
Wide Base Population Breeding Lines
Cycles of Recurrent Selection at Different Locations
In vitro selection , somaclonal variation, gametocedal variation,
transgenosis
Varieties
Fig.2: Possible strategy for exploitation of genetic resources and advanced genetic technique in a long term breeding programme (after Porceddu et al., 1988)
Introduction 9
Although a number of other examples can be cited, it suffices to
emphasize that embryo culture, an easy and old tool, has not been fully
exploited. More efforts should be made to employ this method for obtaining
intergeneric and interspecific hybrids, and for increasing the genetic diversity
in wheat.
1.6 STUDY UNDERTAKEN
Agronomically useful hybrids, even between closely related species,
are often unattainable because of sexual incompatibility. In many instances
fertilization takes place, but the developing embryo aborts at various stages of
development. The technique of embryo rescue which involves the excision
and culture of the young hybrid embryos, has proven to be very useful in
growing such embryos to maturity and in obtaining hybrid plants.
Furthermore, genetic and field evaluation of the hybrids can be accelerated by
generating multiple plantlets of the embryos in culture.
Keeping the above in view the following objectives were taken to:
1. standardize the regeneration protocol of immature embryos and
hardening procedure of the regenerants.
2. selecting the better responsive genotypes of both aestivum and durum
wheat to be used as female/recipient plant in the crossing program.
3. attempt the crosses with alien donor plant
4. find out the time to scoop embryos from hybrids.
5. culture rescued embryo in vitro.
6. develop full plant from hybrid embryos which otherwise aborts
normally.
RBt/I£^ OV^ UTBkATUtiM
2. Review of Literature
Among the cultivated forms of wheat ^Triticum aestivum L.em. Thell.
(2n=6x=42,BBAADD) and T. durum desf (2n=4x=28, BBAA)] are natural
allopolyloids. Their cytoplasm is known to come from B genome (Ogihara and
Tsunewaki. 1988; Dvork and Zhang, 1990) and donor of B genome is similar to
Aegilops speltoides Tausch. (Sarkar and Stabbins, 1956). At times species of the
Aegilops, section sitopsis such as Ae. searsii, Ae. longissima or Ae. sharonesis was
also considered as a possible source of B genome (Kimber and Feldman, 1987). The
source of A genome is now thought to be T. urartu Tum. rather than T. monococcum
L. ssp. boeticum Boiss. (Waines et al., 1993). The source of D genome is Ae. lauschii
Coss, more commonly called Ae. squarrosa L. in the literature (Kihara, 1924, 1944;
McFadden and Sears, 1944). The wild progenitors of cultivated wheat comprises a
rich repository of the desirable traits which could potentially be transferred into
cultivated wheat through sexual hybridization (Harlan, 1976; Plucknett et ai, 1987;
Lange and Balkema-Boomstra, 1988; Ceoloni et al., 1993). However, due to cross
incompatability in many of the wide crosses either embryo gets aborted or seed
setting is very low. By aid of embryo rescue a sufficient number of progenies can be
obtained to use for further studies (Vasil, 1990).
The literature reveals that the in vitro culture response of Triticum is very
much genotype specific (Maddock et al. 1983, Machii et al, 1998). Although several
other factors can affect the callus induction and plant regeneration from the rescued
embryo, if we have a better responding genotype as one of the parents, the chances of
getting regenerants becomes high (Ou et al, 1989). Therefore, screening of the wheat
genotypes for culture responses and having a reliable protocol is a prerequisite before
we go for embryo rescue of wide crosses. Keeping these facts in view, the current
research problem is being reviewed imder three major aspects of embryo culture, wide
hybridization and gene transfer.
Review of Literature 11
2.1. EMBRYO CULTURE
Since the early reports on plant regeneration from wheat callus by Adachi and
Katayana (1969) and Shimada et al. (1969), the techniques have been continuously
improved. Although wheat was previously thought to be recalcitrant, recent work has
established that immature embryos of wheat form embryogenic callus in a
reproducible manner and multiple plants can also be obtained from these calli.
Regeneration of plants from callus cultures occur by one of the two pathways,
embryogenesis and organogenosis. In wheat, regeneration by both pathways is
possible in same callus (Mathis and Boyd, 1986; Mathias, 1990; Kothari et al., 1998).
Organogenic type of regeneration was reported in early experiments (Chin and Scott,
1977; O'Hara and Street, 1978; Sears and Deckard, 1982). Histological investigations
have also shown the existence of organogensis in both Triticum aestivum (De Buyser
et al. 1988; He, 1989) and in T. durum (Bennici et al, 1988). Shoots were also
reported to originate from "Green spots" (Ivanov et al, 1998, Machii et al, 1998).
Embryogenic callus of wheat can be characterized by glistening pale yellow surface
and compact appearance, sometimes may be white and opaque which later on may
become nodular with pale yellow and green buds. On this type of white callus,
formation of typical embrj'oids of wheat were first observed by Ozias-Akins and
Vasil(1983a,b).
Somatic embryos or embryoids are morphologically and physiologically
similar to zygotic embryos (Scott et al, 1990). Some workers (Haccius, 1978; Vasil
and Vasil, 1982) consider that embryoids like zygotic embryos originate from single
cells and thus if mutation has occurred or exogenous gene have been introduced,
whole regenerants are genetically modified. However, others (Werinicke et al, 1982)
have proposed that embryoids are broadly based and hence of multiple cell origin.
Nonetheless, the multiple celled proembryogenic tissues may still have originated
from a single cell (Maddock, 1985; Williams and Maheshwari, 1986). In contrast,
organogenesis may produce chimeras and thus formation of plants through
organogenesis are not very much preferred for clonal propagation, mutation research,
genetic analysis, transformation and breeding (Vasil, 1987).
Review of Literature 12
Embryogenesis in wheat is known to be affected by genotype (Chu et ai,
1984; He et ai, 1986), phytohormone concentration and type (Hunsinger and Schauz,
1987) and interaction between hormone and sugar concentration (Ozias-Akins and
Vasil, 1983b). It can be greatly enhanced by culturing on two fold concentration of
MS medium (Ozias-Akins and Vasil, 1983b), on medium containing a high
concentration of salts (Galiba and Yamada, 1988; He et al, 1989) or on medium
without zinc (He, 1989). However, the relative frequency of embryogenesis,
organogenesis and the effect of explant, media, and culture conditions, on the balance
between the two pathways are unclear. The salient information available in literature
on various factors influencing in vitro response of immature embryos of wheat has
been reviewed in following table.
Table 2 : Factors influencing in vitro responses of immature embryos of wlieat
Factor Age (daj 10-14 10-15
16-22
19-20 >22 Up to 25
Used to {S post pollination)
Optimum age
fast growing and regenerable calli
85% callus and regeneration optimum age precocious germination callusing on medium having 2,4-D -i- ABA
Reported by
Wang et ai, 1993; Arun et ai. 1994
Shimada, 1978; Sears & Deckard, 1982; Magnussan & Bomman, 1985; Ozygen et al, 1998
Zhang, 1986
Karadimova e/a/., 1985
He era/., 1988 Quarashi e/a/, 1989
Size of embryo (mm) ~ 1.0 mm
1.0-1.5mm
>0.5-2mm
0.9-1.8 <0.9-1.8
optimum callus
better callus
no effect on callus morphogenesis, affect rate of morphogenesis epiblast callus scutellum callus
Ozias-Akins & Vasil, 1982; Bohorova er a/., 1995 Liang et al, 1988; Redway et a/., 1990 Maddock etal, 1983
He era/. ,1986 He era/., 1988
Orientation of embryo Scutellum upward check precocious germination Scutellum downward
Eapen & Rao, 1985; Ivanov et al, 1998
Hcetal, 1986
Review of Literature 13
Basal culture medium MS
B5 N6 LS P-medium
Hussain and Islam, 1993; Ganeva et al, 1995; Zair et al, 1995; Kothari et al., 1998; Ozygen et al, 1998; Karaca & Burun, 1999 Chawla & Wenzel, 1987; Tuberosa et al, 1988; Chawla, 1989
Jia & Zhao, 1993; Bohorova et al, 1995 Gaponekoe/a/., 1985a, b; Heyser e/a/., 1985 hazQxetal, 1983; Sharmae/a/., 1998
Phytohormones 2,4-D(0.1mg/l) 2,4-D (0.5 mg/1) 2.4-D(l mg/1) 2,4-D (1 mg 1.5/1) 2,4-D (2 mg/1)
2,4-D (2.5-5mg/l) 2.4-5-t + Kinatin
lAA (2.5 mg/1) NAA (2.5-5 mg/1) Dicamba
Picloram
NAA+BAP lAA
Kinetin
BAP
lAA+Kinetin lAA+BA
no callusing no callusing Callus Callus Callus
reduced callus with rooting Optrimum callus but reduced regeneration no callusing no callusing better than 2, 4-D for callus induction plantlets less vigorous and difficult to establish in pot
double amount of callus then on 2,4-D good callus and regeneration in regeneration medium induce rooting
no effect on callus induction inhibited callus induction
callus formation improve plant regeneration
induce plant regeneration
used in root formation regeneration and rooting as well
Heetal, 1986 Rashid & Quaraishi, 1989 Maddock etal, 1983 Abdrabou & Moustafa, 1993 Sears & Deckard, 1982,; Mathias et al, 1986; Ozyg en etal, 1998 Chauhan «& Singh, 1995 Lazeretal, 1983
Ozias-Akins & Vasil, 1982 Ozias-Akins & Vasil, 1982 Hunsinger & Schauz, 1987
Paperfuss & Carman, 1987
Lazeretal, 1983
Anshulika e/a/., 1999 Quaraishi, 1989
Ahloowalia 1982
Butonko & Dzhardemaliev, 1986 Lazeretal, 1983 Bajaj, 1985; Shrivastava et al, 2000
Mathias etal, 1988; Hussain & Islam, 1993 Ivanov et al, 1998 Bohorova e a/., 1995
Review of Literature 14
Zeatin
Zeatin + lAA Auxin/2,4-D free basal medium Gbberelic acid (GA3) ABA (0.25 mg/1)
ABA (0.1 mg/1)
Ethylene
AgN03
inhibit callus induction promote growth reduce embryogenesis
improve shoot regeneration frequency inhibit root formation beneficial for shoot formation Regeneration
enhance rooting in callus no effect on regeneration
vigorous precocious germination help in inducing callus increase embryogenesis little effect on callus induction strongly inhibit precocious germination inhibit shoot formation inhibit ethylene action
Ahloowalia, 1982
Carman, 1989
Gosch Wackerle et al, 1979
Tanzarella & Greco, 1985 Ozias Akins «& Vasil, 1982 Symillidese/o/., 1995; Taghvaie/a/., 1998 Ozias-Akins & Vasil, 1982
Machiie/a/., 1998
Brown era/., 1989 Morris et al, 1986; Quarashi etal, 1989
Papenfuss & Carman, 1987 Pumhauser e/a/., 1987
Organic additives Yeast extract Casein hydrolysate
Coconut milk
Potato extract
Aspergine Proline
Gultamine
Tyrosine
promote callusing
induce embryogenesis
suppress precocious germination promote initiation, growth and regeneration of callus promote callusing no significant improvement better callus induction
prolong totipotency better callus induction
root formation improve regeneration potential
regeneration medium
Zamora & Scott, 1983 Shimadaero/, 1969
Ozias-Akins & Vasil, 1983b Ahloowalia, 1982; Mathias & Simpson, 1986 Zhaire/a/., 1995
Karaca & Burun, 1999 Machii et al, 1998; Malhotra et al, 1999
Tuberosa e/a/., 1988
Sharma et al, 1998; Anshulika etal, 1999 Ivanov era/, 1998
Shrivastava et al, 2000; Shrivastava & Chawla, 2001 Hussain & Islam, 1993
Carbohydrate Sucrose
Glucose Maltose
(2%) Symillides et al, 1995; Ivanov et al, 1998; (3%) He et al, 1989; (9%) Sharma et al, 1998
(2%) Ozias-Akins & Vasil, 1983a (6%)Machiiera/, 1998
Review of Literature 15
Gelling agent Agar Agarose Gelrite Liquid medium
(0.8%) Jauhar, 1991; (0.7%) Ivanov etal., 1998
(0.6%) Inagaki & Tahir. 1990a; Symillides et ai, 1995 (2%)Machiie/a/., 1998 Dolezel er a/.. 1980
Effect of environmental conditions on culture establishment Growth condition of explant plant have a significant effect on culture response
Larkin et al, 1984; Carman et ai, 1988
No marked effect Mathias, 1990 Green house grown embryosshows poor response Purnhauser e/a/., 1987
Growth condition of cultures Dark during callus induction Light during callus induction
Dark during regeneration phase Light during regeneration phase
calli grow faster and morphogenic decrease regeneration potential for longer time
promote precocious germination
promote regeneration
Papenfuss & Carman, 1987; Higgins & Mathias, 1987 He et al, 1986; Lazer et al., 1983
Mathias etal., 1986
Ozias-Akins & Vasil, 1983c
Genotype Influence callus induction Influence callus growth rate Frequency of embryogenesis Plantlet regeneration frequency
Mathias & Simpson, 1986; Machii et al., 1998 Lazer etal., 1983 Arun et al., 1994; J'Aiti et al., 1999
Karadimova et al., 1985
Cytoplasm Influence in vitro response Mathias & Fukui, 1986
Genie control stimulate callus growth and embryogenesis
not a sole factor to better culture response 6 & 7 chromosomes of Secale cereale induce higher regeneration frequency 7B, 7D and ID are important to regulation Rhtl and Rht2 loci, modify callus growth and embryogenesis
Langridge et ai, 1991; Fennell et al., 1996 Henry Buyser, 1990 Lazer era/., 1987
Langridge er a/., 1991 Mathias & Atkinson, 1987; Yamamoto & Miuri, 1995; Franzone et al., 1998
Review of Literature 16
2.2. WIDE HYBRIDIZATION
The history of hybridization of alien species with Triticum goes back
to 1876, when Wilson made the first hybrid involving wheat x rye. However,
Rimpau (1891) obtained seeds in a wheat x rye hybrid to represent the first
triticale. The systematic work on wide hybridization was started only after the
first decade of last century when many hybrids involving Triticum and
Aegilops species were produced. The pioneering work of Anton Kruse in
attempting hybridization with Triticum aestivum x Avena sativa (Kruse,
1969), Hordeum vulgare x Secale cereale (Kruse, 1967) and H. vulgare x T.
aestivum (Kruse, 1973) played an important role in accelerating the research
in the area of intergeneric hybridization involving T. aestivum or T. durum
with species of Agropyron, Aegilops, Elymus, Haynaldia, Hordium etc.
2.3. CONSTRAINTS TO WIDE HYBRIDIZATION
Assortments of barriers are inherent in an attempt to utilize exotic
germplasm for crop improvement. Successful sexual hybridization involves a
sequence of events including pollen germination, pollen tube growth,
fertilization, embryo and enodsperm development and seed maturation.
Stebbins (1950) has divided these hybridization barriers into two main groups
namely, pre-fertilization and post-fertilization barriers. The pre-fertilization
category includes external barriers like geographic isolation and internal
barriers like apomixis, pollen pistil incompatibilities and prevention of
fertilization. Post-fertilization barriers are a greater hindrance to
hybridization and can be a result of ploidy differences, chromosome
alterations, incompatible cytoplasm, seed dormancy and hybrid breakdown
etc. These barriers can be overcome by making alterations in choice of
parents, emasculation procedure, pre and post pollination treatments and
embryo excision and culture (Mujeeb-Kazi and Kimber, 1985).
Laibach (1925) was the first to use embryo culture successfully in
cross of Linum austriacum x L. perenne. Since then several refinements have
been made in embryo culture/embryo rescue technique. It paved a way to the
Review of Literature 17
successful hybridization of a number of otherwise incompatible intergeneric
and interspecific crosses. Currently embryo rescue holds great promise not
only for effecting wide crosses but also for obtaining plants for inherently
weak embryos, obtaining haploid plants as well as shortening the breeding
cycle and overcoming hybrid necrosis.
2.4. STRATAGIES TO EXPLOIT ALIEN SOURCES
2.4.1. CHOICE OF PROMISING AND COMPATIBLE PARENTS
Introgression of alien variability is demarcated into two distinct areas
of long-term intergeneric and short-term interspecific hybridizations. These
are separated on the basis of wheat/alien genomic similarity and level of
genetic recombination. Interspecific approaches are favored for genetic
introgression (Mujeeb-Kazi and Asiedu. 1995). When more diverse or distant
hybridization were initiated, hybrid production constraints were increased
manifold and arrays of manipulative techniques were required. Moreover,
wider the crosses are, lesser the stability of the transferred gene (Dhaliwal et
al, 1993). Additionally, genotypes, ploidy levels, cross direction and
environment all become contributors to hybrid production success (Sharma
and Gill, 1983; Mujeeb-Kazi and Kimber, 1985). A hybrid production
procedure in which wheat is the maternal parent is being routinely adopted by
various authors with significant success (Mujeeb-Kazi et al, 1987).
Alien diploids have been difficult to hybridize with wheat. Such
crossability barriers can be overcome either by reciprocal crosses or by first
doubling the alien diploid and using the induced autotetraploid as the pollen
parent (Mujeeb-Kaziitfl/ji^dy 1995). Specific examples of intergeneric hybrids
produced by this procedure are: T. aestivum x A. cristanum (4x), T. aestivum
X Psathyrostachys juncea (4x), T. turgidum x P. juncea (4x), in which each
hybrid possesses 35, 35, and 28 chromosomes respectively.
Certain wheat cultivars are more crossable than others (Jauhar, 1993).
Crossibility of wheat and rye, for example, is controlled by two recessive
Review of Literature 18
alleles Krl and Kr2 on chromosome 5A and 5B respectively (Riley and
Chapman, 1967), prevents their crossability with Secale cereale (Lein, 1943)
and Hordeum bulbosum L. (Snape et ai, 1979; Tanner and Falk, 1981).
Moreover, wheat cultivars that give high seed set in crosses with rye are
reported to produce high seed set after pollination with several species of
Triticum, Secale, Agropyron, Elytrigia and Elymus.
2.4.2. CROSSING PROCEDURE AND PRE/POST POLLINATION TREATMENTS
In most of the wide crosses, the wheat is generally used as female
parent, unless cytoplasmic characters have to be transferred. In routine, spikes
of wheat are manually emasculated and receptive stigma is pollinated at least
twice with the pollen of the donor parent. Some times hot water is being used
to emasculate the spikes (Inagaki et al. 1997). The spikes are immersed in
hot water set at 43°C for 3 minutes one day before pollination and pollinated
after cutting the upper part of the florets but leaving the anthers.
The emasculated florets are recommended to treat with growth
hormones or immunosuppresant (Kruse, 1967). The 1:1 mixture (v/v) of 2,4-D
(25mg/l) + GA3 (75mg/I), one day before and one day after pollination was
recommended to be used (Jauhar, 1991, 1993). A hormone solution containing
GA3 (75mg/I) + 2,4-D (5mg/l) was used by Jauhar and Peterson (1996) and
Bommineni et al. (1997) only one day after pollination. Riera-Lizarazu and
Mujeeb-Kazi (1993) used aqueous solution of 50mg/l 2,4-D + 150mg/l GA3,
only one day after pollination. However, Mujeeb-Kazi and Asiedu (1990)
used only GA3 (75 ppm aqueous) up to 4 days post pollination. These authors
recommended treatment of growth hormone by spraying the solution on the
pollinated spike or flooding the cup by hormone solution. While some other
researchers (Inagaki and Tahir, 1990b; Inagaki and Mujeeb-Kazi, 1995,
Inagaki et al, 1997) needle injected lOOmg/1 2,4-D solution in the upper
nodes of the wheat clum the pollinated spike. Even some of the other
researchers used 2,4-D in combination of AgN03 (O'Donoughae and Bennet,
1994; Almouslem et al, 1998), and reported it to give better seed set then
Review of Literature 19
2,4-D + GA3 solutions. Almouslen e( al. (1998) also mist sprayed the spike
with tween-20 (1% v/v) as surfactant before spraying hormone solution.
2.5. EMBRYO RESCUE
To make interspecific and intergeneric hybrids with bread wheat, the
latter is generally used as the female parent and embryo rescue is practiced to
save the aborting embryos and to rear them to the seedling stage. It is a wider
term and includes immature embryo culture, ovule/ ovary culture and floret
culture. In some cases even spikelet and spike culture.
2.5.1. IMMATURE EMBRYO CULTURE
Hybrid embryos are rescued by aseptically removing embryos prior to
their abortion and culturing them directly onto an artificial media. An
important aspect of embryo culture is a priori knowledge of the in vitro
system for the species of interest and has a suitable culture system for the
species being hybridized (Collins et al, 1993).
2.5.1.1. Stage of scooping out
The successful rescue is influenced by the age of the embryo at the
time of its culture. Two basic stages of the embryo growth exist with regard
to nutritional independence i.e. heterotrophic and autotrophic stage
(Raghavan, 1976). The heterotrophic stage of growth is the period during
which embryo depend on the endosperm for its nutrition and extends from
fertilization up to approximately the heart stage and termed as pro-embryo.
During the heterotrophic stage, the embryo usually requires presence of
growth regulators to allow for its proper development (Raghavan and
Srivastava, 1982). The autotrophic phase of growth begins at the late heart
stage. This stage is significant for in vitro culture, as embryo becomes
sufficiently independent from endosperm for its further growth. The growth
requirements for artificial culture for these embryos are minimal and also they
can withstand the isolation shock (Collins et al, 1993). The culture of young
embryos is also more difficult due to their size, damage during dissection and
sensitivity to osmotic change (Maheshwari and Rangaswamy, 1965).
Review of Literature 20
The Stage at which embryos can be rescued depends on the time at
which endosperm aborts and the time at which subsequent embryo
degeneration results. For example. Gill et al. (1981) recorded that in cross of
Aegilops squarrosa x T. boeticum, endosperms aborted at about 10 days and
embryos aborted by 14 days following pollination. Hybrids were obtained by
rescuing embryos 10 days after pollination.
2.5.1.2. Culture pathways
There are two different pathways to be followed for culture of rescued
embryos. One is by going through direct germination of embryos and second
through dedifferentiation and subsequently regenerating the whole plant.
Alien gene transfer mediated by callus culture promotes multiple cytological
variations. The alien exchanges through callus culture have been
demonstrated in wheat x rye hybrids (Lapitan et al, 1984, 1986) and are
being applied to other intergeneric hybrids {T. aestivum x Ae. variabilis) with
poor F2 wheat/alien chromosomal recombination (Ter-Kuile et al., 1988).
Amphipliods of intergeneric hybrids represent an important means of
germplasm distribution and organized alien genetic component exploitation,
particularly for stable amphiploids. It has been rather difficult to induce
amphiploidy in several intergeneric hybrids by colchicine treatment. In an
observation by Ter-Kuile et al. (1988), it has been anticipated that the
regenerated Fi hybrids of T. aestivum x Ae. variabilis and T. turgidum x Ae.
variabilis demonstrated seed set in an otherwise self sterile population. Callus
induced doubling may prove advantageous for combinations otherwise hard to
double (Mujeeb-Kazi and Asiedu, 1990).
2.5.1.3. Media manipulation
The successful production of plants from the cultured embryos largely
depends upon the maturation stage and composition of the medium. Culture
medium requirements are almost same for rescued embryos as for normal
immature embryos. Some authors recommend amino acids (organic nitrogen)
Review of Literature 21
rich LS/MS medium to be used for culturing very small rescued embryos
(Valkoun et al, 1990). Most culture media include an inorganic source of
nitrogen, however, the addition of an organic form has been shown to have a
stimulating effect on embryo growth and development (Monnier, 1978).
The form in which organic nitrogen is supplemented is also important.
Although aspergine is found to be beneficial in some experiments (Lea et al.,
1978), glutamine is generally superior for a wide range of genera (Mok et al..
1978). However, glutamine alone does not always give the best response. A
mixture of amino acids may be more effective in some instances especially
for very small embryos. An undifferentiated nitrogen source such as casein
hydrolysate, coconut milk, potato or yeast extract (Bajaj, 1990) has been
shown to be beneficial.
MS is the most extensively used medium for rescuing embryo
(Gaponeko et al., 1985a,b). Orchid agar (Jauhar, 1991, 1995), Taira and
Lartor's (1978) medium (Mujeeb-Kazi and Asiedu, 1990), Lanssmier and
Scoog (1965) medium (Gaponenko et al., 1985a; Valkaun et al., 1990) have
also been used.
2.5.6. ALTERNATIVE METHODS
Embryos were also reported to culture on the endosperm of other
species or one of the parent's endosperm to rear them to plantlet. This method
helped to obtain a number of intergeneric & interspecific crosses like
Hordeum x Triticum (Kruse, 1973, 1974a, b; Williams and De Lautour, 1980,
1981; Williams & Williams, 1983). In some other, where technically it is not
possible to scoop out an embryo, ovule culture (Stewart, 1981; Comeau et al.,
1988, Agnihotri, 1990; Nikova and Zogorska, 1990), spikelet culture (Zhang
et al., 1996) or even spike/detached tiller culture (Inagaki et al., 1997;
Monika ,1999; Cherkaoui et al., 2000) were deployed.
Review of Literature 22
2.6. ALIEN GENES TRANSFERRED IN WHEAT
Since the report of Kruse (1967, 1969, 1973, 1974) there has been
heightened interest world wide in the production and exploitation of
interspecific/intergeneric hybrids. The enormous array of genetic stock
available in secondary gene pool of Triticum has been tapped for a number of
desirable characters. Some important ones are enlisted in following table.
Table 3: Disease resistance genes incorporated in wheat gene pool from alien sources.
Gene Donar plant Reported by Resistance to Puccinia graminis Disease : Black rust, stem rust Sr2 Sr9e Sr l3 Sr l4 Srl7 Sr24 Sr25 Sr26 Sr27 Sr31 Sr37 Sr38 Sr43
T. dicoccum T. dicoccum T. dicoccum T. dicoccum T. dicoccum A^ropyron elongatum A. elon^atum A. elon^atum S. cereale S. cereale T. timopheevii Ae. ventricosa A. elongatum
Hare & Mcintosh, 1979 Luig& Watson, 1967 Knott, 1962; Mcintosh e/a/., 197?-Knott, 1962 E. S. Macfedden (unpublished) Sears, 1973; Mcintosh et al., 1977 Sears, 1973; Mcintosh e a/., 1977 Mcintosh era/., 1977 Acosta, 1963; Marais & Marais, 1994 Friebee/o/., 1996 Mcintosh & Gyarfas, 1971; Gyarfas, 1978 Banana etai, 1969 Kibirige-Sebunya & Knott, 1983; Kim et al., 1993; Friebeera/., 1996
Resistance to Puccinia recondite Disease : Brown rust/ leaf rust Lr9 Lrl9 Lr21 Lr22a Lr24 Lr25 Lr26 Lr28
Ae. umbellulata A. elongatum Ae. squarrosa Ae. squarrosa A. elongatum S. cereale S. cereale Ae. speltoides
Sears, 1961, 1972 Browder, 1973a Rowland & Kerber, 1974 Dyck & Kerber, 1970; Kolmer, 1997 Browder, 1973b Heun and Friebe, 1989 Mettin, 1978 Mcintosh er a/., 1982
Resistance Puccinia striiformis Disease : Stripe rust/Yellow rust Yr5 Yr8 Yr9 YrlO Yrl5
T. spelta Ae. comosa S. cereale T. spelta T. dicoccoides
Macer, 1966 Riley era/., 1968 a, b Zeller, 1973, Mettin et al., 1978 Kema&Lange, 1992 Gerechter-Amitai et al., 1989
MATBtOAUANP MBTHOPS
3. Materials and Methods
3.1. PROCUREMENT OF WHEAT GERMPLASM
Literature reveals that in vitro response of wheat for
regeneration is genotype specific. Therefore, it is a prerequisite to
establish a reliable protocol for regeneration of genotypes to be used in
wide hybridization program. A range of varieties of both bread as well
as durum wheat (Table 4) were selected by keeping in view the best
variety available and acreage covered. Seeds were provided by
Germplasm Resource Unit, Directorate of Wheat Research, Karnal
India, and were sown in polyhouse under controlled environmental
conditions in three rows plot.
Table 4:Varieties used in standardization of regeneration protocol from immature embryos and selection of genotypes to be used in wide crossing.
Bread wheat Durum wheat
1.CPAN3004 1. Bijaga Yellow
2. HD 2329 2. Gulab
3. KRL 1-4 3. HD4530
4. PBW 343 4. HI 8381
5. PBW 373 5. MACS 2846
6. Raj 3765 6. PDW 215
7. UP 2338 7. PDW 233
8. WH 542 8. Raj 1555
Wide Hybridization Program
After screening the genotypes, three varieties each of bread and
durum wheat (Table 5) were selected to use as a female parent in the
wide crossing program. Varieties were sown in staggered manner at 10
days interval in five rows each to maintain the continuous availability
Materials and Methods 24
of female plant. Five sowings were done by starting in first weak of
November.
Table 5: Varieties selected for wide crossing program
Sr. No. Bread wheat Sr. No. Durum wheat
1.
2.
J .
4.
5.
WH 542
UP 2338
PBW 343
Raj 3765
HD 2329
1.
2.
J .
4.
5.
Raj 155 5
Bijaga yellow
Gulab
PDW 215
PDW 233
3.2. ALIEN GERMPLASM
Many accessions of diploid and tetraploid Triticum and Aegilops
species were used for alien gene resource taken as a male donor plant
in the wide crossing program (Table 6).
Table 6: Wild species used in crossing program
Sr. Species No.
Accessions Sr. available No.
Species Accessions available
2.
J .
4.
5.
6.
7.
8.
9.
10.
T. urartu (A")
T. boeticum (A )
Aegihps speltoides (S)
Ae. longissima (S')
Ae. sharonensis (S " )
Ae. bicornis (S )
Ae. squarrosa (D)
Ae. caudate (C)
Ae. markgrafi (C)
Ae umbellulata (U)
17
2
44
2
2
7
87
2
2
2
11
12
13
14
15
16
17
18
19
20
Ae. cylindrica (CD) 30
Ae. ventricosa (D") 1
Ae. kotschyi (US) 4
Ae. peregrina (US) 5
T. triuncialis (UC) 34
Ae. ovata (UM°) 9
Ae. lorentii ( U M ' ' ) 3
Ae. triaristata (UM') 11
Ae. geniculata (UM°) 2
T. araraticum (AG) 1_
Seeds were procured from Germplasm Resource Unit, Directorate
of Wheat Research, Karnal India. The wild species were grown in net
house, maintaining 2.0 ft distance between rows to avoid any
Materials and Methods 25
contamination and seed dispersal. Additional photoperiod of six to
eight hours was provided with bulbs of lOOOWt to facilitate flowering
of alien species. To break the dormancy, the seeds were stored at -4°C
for vernalization.
3.3. HARVESTING OF IMMATURE EMBRYOS AND WIDE CROSSING 10 to 18 days old embryos were cultured in the present study to
evaluate the optimum age of embryos. Only primary florets from the
central portion were left on the spike to be used for excision of
explant. Rests of the florets along with awns were removed before
taking the spike to the laminar flow bench.
The spikes quarter to half came out from flag leaf were used for
hand emasculation. The upper and the lower spikelets were plucked out
and from the left outs the secondary and the tertiary florets were also
removed. The spikelets were than cut slightly below the center with the
help of sharp scissors. Anthers were removed by forceps avoiding any
injury to the gynoecium. Utmost care was taken not to leave any anther
and secondary or tertiary floret to avoid selfing. The spikes were then
covered by butter paper envelope to secure humidity and resist
undesirable pollination. When the stigma became feathery, pollination
was done for two consecutive days. Pollinated spikes were sprayed by
0.05% 2,4-D + 0 .1% GA3 solution in the evening, for four days starting
by the day the spike was emasculated.
The crosses were made in the replication of 20-25 spikes per
cross in five days i.e. 3 to 4 spikes every day for each cross
combination. The spikes were continuously examined and three days
prior to the day the florets starts getting dried, were recorded as the
day of abortion of the embryo. The embryos were rescued from the rest
of the spikes accordingly. For making replications each spike was
divided into three and these three portions were taken at alternate days.
Three spikes per cross was left in the field as control.
Materials and Methods 26
3 . 4 . In vitro C U L T U R E
3.4.1. BASAL CULTURE MEDIUM
3.4.LI Immature embryo culture
Growth and morphogenesis of plant tissues in vitro are largely
governed by the composition of culture media. In the present study, the
revised MS medium (Murashige and Scoog, 1962) and the B5 medium
(Gamborg et al., 1968) were used as the basal medium in
standardization of the medium for embryo culture along with different
hormonal combinations. The details of the ingredients along with
recipe for the preparation of stock solutions of these mediums were
provided in annexure.
3.4.1.2. Rescued embryo culture
Two different paths were adopted to culture rescued embryos,
one through direct germination of embryos and another through
dedifferentiation into callus. To induce callus MS + 2,4-D (2 mg/1). For
regeneration, Rl method, used with normal immature embryo i.e., left
on the same medium and exposed to light provided by 20 Wt. electric
bulbs (Philips India). Calli were also transferred on the CM, EC and
then on R2 or R5 (Table 7) to get regeneration. Regenerated plantlets
were transferred on S3 and finally plants with two to three leaves were
transferred on Rt4 for root development (table 4).
3.4.2. GROWTH REGULATORS
2,4-D alone or in combination with lAA was used to induce
callus. Kinatin and BAP alone or with lAA were used to achieve
regeneration. Kinatin/Kinatin+GAs helped in elongation of regenerants.
NAA/IAA was used to get healthy roots. Preparation of stock solutions,
storage, sterilization procedure etc., was provided in annexure.
3.4.3. PREPARATION OF CULTURE MEDIUM
To prepare one-liter culture medium, 250ml double distilled
water (ddw) was taken in conical flask. All the ingredients were added
26
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Materials and Methods 28
to it according to table 4, except agar and final volume finished to
600ml. pH was adjusted to 5.8 by adding IN NaOH/1 N HCl. In another
one liter flask 8gm agar was dissolved in 400ml water. This melted
agar was than mixed to the 600 ml solution and homogenized by
stirring. In test tubes measuring 150 x 25mm, 15ml of the medium were
dispensed while in Erlenmeyer flasks (100ml capacity) 40-45 ml of the
medium was dispensed. The culture vessels were plugged by non-
absorbent cotton wool wrapped in single layered muslin cloth. Medium
and accessories were sterilized by autoclaving at 121°C temperature
and 15 psi pressure for 15 minute. The medium was autoclaved in
aliquots of 250 ml in one liter flask for 20 minutes when to be used in
petridishes. After autoclaving, the medium, in the flasks was allowed
to solidify as such and slants were made in test tubes. For pouring in
petridishes the medium were cooled to 50°C and poured in sterilized
petridishes on laminar flow bench. The autoclaved and solidified
medium was left on room temperature for four to five days to check
microbial contamination and afterward stored in refrigerator. This
media could easily be used till one month.
3.5. ASEPTIC TECHNIQUE
The aseptic techniques are necessary to create aseptic
environment to avoid contamination. The technique involves
sterilization of working bench, glassware, instruments, plant material
inoculation and incubation. Laminar airflow hood were sterilized by
UV-lights for one hour and wiping the surface with 70% ethanol before
any operation under the hood. The glassware, blotting sheets, cotton
wool etc. to be used in different processes were wrapped in butter
paper or aluminium foil and sterilized by autoclaving at 15 lbs/inch^
pressure (121°C) for 20 minutes.
3.6. STERILIZATION AND PLATING OF PLANT MATERIAL
The spikes were surface sterilized by 70% ethanol (v/v) and
taken to the laminar airflow cabinet. The seeds were threshed out on
28
the laminar and treated with freshly prepared 0 .1% solution of mercuric
chloride plus a drop of Tween 20 for 2 to 5 minutes, followed by a
quick wash in 70% ethanol and 6 to 7 rinses by autoclaved distilled
water. The embryos were scooped out aseptically under the Stereo-
zoom microscope taking utmost care that no piercing or injury occur to
the embryos and plated on the culture medium with scutellum side
upward.
3.7. INCUBATION OF CULTURES
Temperature was maintained on 25 ± 2°C during entire course of
study with a relative humidity of 50 to 60%. Calli vv ere incubated in
dark. The diffuse white lights were provided by 100 |j,mol m"'S"'Wt.
Philips, India cool tube rods and yellow lights by 20 Wt. Philips, India
bulbs.
3.8. HARDENING OF THE PLANTLETS
Plants with well-developed white roots were kept on hardening
to transfer into field. Different procedures were assumed.
> The plantlets were first transferred to the liquid medium. The test
tubes were covered by poly bags enriched with humidity by
spraying water. Established ones were transferred to soil mixture
containing equal proportions of soil, farm yard manure (FYM) and
sand, in growth chamber at 70 to 90 relative humidity and 25 ± 2°C
temperature.
> The plantlets grown on the root induction medium were transferred
to MS half strength medium + 1 0 mg sucrose + 6 g agar and then to
the '/4 MS medium + 2 gm sucrose + 4 g agar. Further plants were
transferred to liquid medium supported by blotting papers. The
established plants were transferred to soil mixture containing equal
amount of soilrite, perlite and soil.
> The plantlets with developed white roots were taken out of the agar
medium and washed carefully so as to remove every trace of media.
29
Materials and Methods 30
Plants were than transferred to 1:1 mixture of agropeat and soilrite
plantlets were irrigated by MS medium containing inorganic
constituents only. The plants were kept in growth chamber at 1 5 ±
1°C temperature and 80 to 90% relative humidity till two to three
fresh leaves emerged out. Thereafter these were transferred into the
field/bigger pots containing soil, sand, FYM and agro peat in equal
proportions.
30
RBSUITS
4. Results
EXPERIMENT 1
4.1. STANDARDIZATION OF REGENERATION PROTOCOL
Extensive studies were carried out on the effect of different basal culture
media, growth regulators and age of embryos (days post pollination, DPP) on the
initiation and growth of callus and regeneration. For standardizing the regeneration
protocol, the four wheat varieties, two each oiaesiivum and durum were utilized.
4.1.1. CALLUS INDUCTION
A wide range of variation was observed for callus induction when embryos of
different age groups (10-18 DPP) were cultured on following four callus induction
media Cl (MS+2,4-D, 2mg/i), C2 (MS+2,4-D, 4mg/l), C3 (MS+2,4-D, 2mg/l+lAA,
lmg/1), C4 (B5+2,4-D, 2mg/l). 25 to 35 embryos of each age group were cultured on
all the combinations of the media.
4.1.1.1. Effect of callus induction media
Mean values for callus induction frequencies in case of wheat varieties UP
2338, WH 542, Bijaga Yellow and Gulab were always significantly better on Cl
(83.00, 73.33, 78.18 and 78.00 percent) and C2 (83.33%, 87.35, 84.26 and 81.44
percent) in comparison to C3 (62.65, 57.60, 62.59 and 62.67 percent) and C4 (62.35,
55.60, 63.04 and 68.67 percent respectively). Differences between mean callus
induction on CI and C2 were non-significant (at 5% P) except for Bijaga Yellow
where it was significantly higher on C2 (Table 8). In general, 62.50-100% embryos on
Cl, 66.67-100% on C2, 37.93-76.67% on C3 and 46.15-83.33% on C4 transformed
into callus. All the embryos belonging to bread wheat varieties UP 2338 (12 DPP) and
WH 542 (12 and 14 DPP) transformed in to callus on C2 medium. 100 % callus were
also obtained from 12 days old embryo of WH 542 on Cl medium. Whereas, a
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Fig 3. Explanations
Different types of induced calli
A: Loose and white non embryogenic callus.
B: Same with improved texture callus mass.
C: Callus showing initiation of green sectors.
D: Typical organogenic callus with emergence of shoot primordia.
E: Typical embryogenic callus showing somatic embryos at different developmental
stages.
F: Embryo showing precocious germination and poor quality callus.
Results 33
maximum of 97% and 90% callus induction was recorded in durum wheat varieties
Bijaga Yellow (12 DPP) and Gulab (12 and 14 DPP, respectively) on C2 medium
itself.
With regard to quality, calli on CI were glistening white yellow to pale
yellow, compact, opaque, nodular and embryogenic (preferable) (Fig. 3 E) whereas
those of C2 were transparent white, loose, watery and non-embryogenic (non-
preferable) (Fig. 3A & B) . The calli induced on C3 and C4 were loose to compact,
yellow-white, coarse, friable and non-embryogenic with occasional green sectors
(Fig. 3, C, D).
4.1.1.2. Influence of age of embryos (DPP)
Optimum age of embryos for induction of callus for both aestivum and durum
were found to be 12-14 and 12-16 DPP respectively (Table 8). No significant
difference (at 5% P) were observed for mean percent callus from 12 and 14 days old
embryos of variety UP 2338 (84.43 and 86.47%) and WH 542 (83.33 and 82.44%).
Differences among mean callus induction frequency of 12, 14 and 16 DPP embryos of
Bijaga Yellow (80.42, 83.33 and 81.55%) and Gulab (80.00, 82.50 and 76.80%) were
also non significant.
4.1.2. REGENERATION
Calli were transferred on different MS based mediums i.e. Rl (left on the
same medium and exposed to 16/8hrs light/dark, R2 (MS + Kinetin, 2mg/l), R3 (MS
+ Kinetin, 2mg/l + lAA, 0.5mg/l), R4 (MS + BAP, 2mg/l + lAA, 0.5mg/l), R5 (MS +
BAP, 2mg/l) media to get regeneration. Experiments were designed to evaluate the
effect of regeneration medium, callus initiation medium and age of explants on
regeneration frequency and multiple shoot formation (Fig. 4). Callus induction
medium was recorded to have very influential effect on regeneration especially when
exposed to light (Rl). As for all the four varieties tested, calli induced on CI
exhibited quite appreciating regeneration, while there was no response from calli
induced on C2. C3 and C4. Therefore, while performing statistical analysis Rl was
omitted from regeneration data on C2, C3and C4 induced callus.
Rc'siihs 34
4.1.2.1. Bread wheat
Both callus induction as well as regeneration media had a significant influence
on regeneration frequency. Calli of UP 2338 induced on CI, C2, C3 and C4 reported
to give 30-90, 20-56, 21-68 and 29-63 percent respective regeneration, whereas
WH 542 exhibited 33-100, 20-56, 19-63 and 20-60 percent regeneration, respectively.
Maximum regeneration was observed from calli induced on CI medium.
Similarly on R2, R3, R4 and R5 media, 33-90, 23-63, 20-56 and 30-92 percent
calli of UP 2338 and 33-100, 18-57, 20-67 and 30-90 percent calli of WH 542
exhibited multiple shoot formation, respectively. In both the varieties mean frequency
of regeneration on R2 and R5 were significantly higher than R3 and R4, irrespective
of the callus initiation medium or age (Table 9). Plantlets on R3 and R4 were also thin
fibrous and delicate in comparison to that of R2 and R5. Albino plants were recorded
from calli of WH 542 induced on C2 and regenerated on R3 medium. Regeneration
on Rl for both UP 2338 and WH 542 were good from calli induced on CI. ( ^<Q-^)
Age of embryos also exerted a significant influence (at 5% level) on
regeneration frequency. In both the varieties, 10 DPP embryos gave maximum
regeneration among all the treatment groups. Frequency of regeneration was found to
decrease with increasing age of embryos.
4.1.2.2. Durum wheat
Callus induction and regeneration medium, as well as age of the embryos, all
exerted a statistically significant influence on regeneration. Calli induced on CI, C2,
C3 and C4 medium exhibited a respective regeneration frequency of 25-91, 20-63,
14-50 and 14-67 percent from Bijaga Yellow and 27-92, 11-55, 14-50 and 17-63
percent from Gulab. Maximum regeneration was always recorded on CI medium
(Table 10).
Regeneration on Rl was recorded to be 61-91 and 75-92 percent from Bijaga
Yellow and Gulab from calli induced on CI, whereas calli on the C2, C3 and C4 gave
negligible regeneration on exposing to light. A regeneration frequency of 33-88,
u o u V Li
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•9
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8 8
5 5
8 «
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9 9
*r\ ^
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^ ^
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9 9
8 8
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8 I
CD
lO —
r-i —
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= 8
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2 «
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8 8
9 g
9 9
O
r«1
* 8 *n
lj ti 9
* «
8 O
8 ?
2 Z
1 « 3 S
g 9
Fig 4. Explanations
Moqjhogenesis in different types of calli
A: Organogenic calhis with poor regeneration efficiency.
B: Callus showing regeneration pockets.
C&D: Regeneration from embryogenic calli (arrow showing germinating embryoids).
E: Organogenic callus (showing shoot as well as root formation).
F: Calli exhibiting both somatic embryoids (arrow) and shoot buds (green colored).
Results 37
14-63^ 14-54 and 20-86 percent from Bijaga Yellow and 25-86, 11-57. 13-43 and 20-
83 percent from Gulab were gained on R2, R3, R4 and R5 respectively.
10 DPP embryos gave significantly better regeneration from CI derived calli.
However, 12 DPP embryos were also better responding on other media compositions.
Embryos were found to be loosing their regeneration potential along with their
growing age.
4.1.3. INTERACTION OF CALLUS INITIATION AND REGENERATION MEDIUM
To study the interaction of callus initiation and regeneration medium, a
hypothetical regeneration per 10 explants cultured was calculated as,
% callus induction x % regeneration (•/,) 10
Both regeneration frequency and regeneration per 10 explants was plotted for
the same DPP embryos and analyzed. By computing this regeneration per 10 explants,
effective regeneration can also be comprehended.
4. L3.1. Bread wheat
Callus induction frequency from immature embryos of UP 2338 was quite
high on both CI and C2 media. Mean callus initiation on C2 was higher in
comparison to CI. However, these calli when transferred to regeneration medium, the
calli induced on CI tend to produce multiple shoots more frequently in comparison to
that of C2. This ultimately affected the effective regeneration which was always high
from calli of CI (Table 11). A constant maximum regeneration was attained on both
R2 and R5 as well.
Mean frequency of callus initiation for WH 542 was higher on C2 in
comparison to CI, C3 or C4 and mean regeneration was maximum from calli induced
on CI (Tables 9 and 10). Though the frequency of callus initiation was higher on C2,
the calli were watery, loose and non-embryogenic. When such type of calli were
transferred to regeneration medium, root induction preceded the shoot initiation and in
most of the calli no shoot was formed at all. Regeneration per 10 explants cultured
ii
e **-R U i)
CI
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<N
rs
o 1^ f S
ILJ
-H oo
* Tj-
~~. o <> rsi
o o — •
• ^ <N <N
* f ^ r v j ^ s o OO * ^ ^ .
^ ' '
— <N r*^ Tj-CJ O O U
r - — r j (N o o o o -H -H < CD CN — O oo v . fN
•+ CD m O
( N
-H D:
o r-i
oc r-j r n
r j rs) —
— »
o o o o -H -H
< m ^ TT
-H
a r*-l
+1 m CT-
(N r-j ro
•r. • O ^ 44
ft oo
O „ „ fS +1 o
— r* (N r j r^
13 2
CO
-H
— <N f* ^ CJ O U U
o -H <
r - l
o -H ffl
rv j
O -H ca
rs ]
O
+1 C3
f S — ^
•» r-l —
» r-l rf^ ( ^ >r, r n O _ _
-o oc a- -H
+1
\D — r-l O O O +1 -H -H
< E C r s 'fL \c
O r*^ O
— — O
Fig 5. Explanations
Different development stages
A: Embryogenic callus of varityGulab.
B,C & D; Multiple shoot proliferation from subcultured calli
E; Emergence of roots from green sectors of watery calli.
F,G & H: Developmental stages of regenerated plantlets.
Results 39
revealed that effective regeneration was always higher from CI induced calli than the
calli from C2, C3 and C4 (Table 11). Multiple shoot formation on both R2 and R5
was equally good and showed maximum regeneration.
4.1.3.2. Durum wheat
By analyzing the callus induction and regeneration responses of embryos of
Bijaga Yellow (Tables 8 and 10), it was apparent that though maximum callus
induction could be obtained on C2, maximum and effective regeneration
(regeneration per 10 explants cultured) was higher from the callus induced on CI
(Table 12). R2 and R5 were equally responding for both regeneration frequencies as
well as induction of multiple shoots.(Fig-5)
Frequency of callus induction from embryos of Gulab was higher on C2,
whereas calli induced on CI showed maximum regeneration. Effective regeneration
was also significantly higher from calli induced on C1. Both R2 and R5 gave a good
number of induced multiple shoots.
4.1.3.3. Statistical inference
For all the four varieties tested, effective regeneration was always maximum
from the calli induce on CI in every age group. Regeneration on Rl was quite
appreciating from calli on CI medium (Tables-11 and 12). R2 as well as R5
responded well irrespective of callus induction medium. Regeneration from R3 and
R4 were ought to be very poor.
4.1.4. SHOOT PROLIFERATION AND PLANTLET ELONGATION
For further proliferation of regenerated calli three different pathways were
deployed: SI, (left on the regeneration medium itself), S2, (MS + GAs 0.5mg/l +30g/l
sucrose) and S3 on (GR free MS). Number of plantlets per callus was recorded
irrespective of the age of embryos. Only calli induced on CI were subjected to study
for this parameter. Mean values of shoots per callus were calculated from five calli
and depicted in Table 13.
4.1.4.1. Bread wheat
c B O
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«/-! r«-i f N r<-i
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r- >o o r^ vo m <N f-n o o o o -H -H -H -H < m CQ D3 r-- (N r^i r--o — r-- -^ T t ^^ i — r v j
u-i • * * ^ ' ^ oo
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« r* — r* ' ^ I ^ oo fN rn -H ^ W rr; r^ =
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^ r* rN rn ;
» w-i r- o "- r- fN ta- rn -H
•it- r*-) '*-, OO J , I - sO r^ — ^ '"! r j — r4 sD
c:3 o o o
-H -H -H -H <C CD 'wi U o o " - . o r~ o O i-n r 1 r - i — —
— O O
^ ; f N " "
* <-> — ^ ^ o Tt r- o -H r- „ o - :f^
r-i — V-, O* -H ^ <N W - ^
HI — (N r^ -"t i i
(J U U U S — (N m -^ "
u u u u 2
. • I * • o • o m vo M
g S 00 f a H — m ^ CT>
UJ 00 -H
— r-J m TT ^
U U U U S
O • * r^ -H O — 00 BJ
•^ <-i r-; 2S
« ov r^ - r ^ r t r^ 00 r^ -H <^ — d d -P
• " 1 • • • „ o • oo r- pj _j. w-i ^ vO . 11
fN
— <N r-1 I f "
o u u u 2 — <S r-1 ^ 4i
o o u o S
Q U _J
— D
> a j
^ iy -1
.^ Ki
1 —
1)
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> i
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l ^
^ 5p 'i/i
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C13
u
-5 Q
J D r^
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( c .SP
ly)
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A X
Results 41
Regenerated calli of UP 2338, when transferred to S3, got established
maximum number of shoots per callus. However, when left on the same medium few
plantlets got dried, thereby reducing the number of plantlets. Regenerants when
transferred on S2, turned brown and only a few healthy plants were able to survive.
Maximum plantlets (20.4) were obtained from calli regenerated on R2 and later on
transferred to S3. Number of plantlets per 10 explants cultured led to obtain the
maximum plantlets on transferring to S3 from calli regenerated on R2 (140.56),
followed by R5 (125.94) and Rl (115.37).
Responses of WH 542 to shoot proliferation medium were in accordance to
UP 2338. Maximum plantlets (26.0) were obtained on S3 from calli regenerated on
R2. Maximum number of plantlets per 10 embryos were achieved on S3 itself from
the calli regenerated on R2 (188.76) followed by R5 (136.60) and Rl (132.00).
However, maximum regenerants were obtained on Rl medium (Table 13).
4.1.4.2. Durum wheat
Variety Bijaga Yellow was found to give maximum shoots per callus on S3
medium from calli induced on R5 (16.8) followed by R2 (15,4) and Rl (14.6).
However, mean values for plantlets per 10 explants were maximum on Rl (66.70)
followed by R5 (61.92) and R2 (56.78).
Although maximum regeneration from variety Gulab was obtained on Rl,
mean values for both shoots per callus and number of plantlets per 10 embryos were
found to be maximum from calli induced on R5 (Table 13).
4.1.4.3. Statistical inference
Statistically, S3 was found to give maximum number of shoots per callus and
differences among SI, S2 and S3 were found to be significant.
4.1.5. RHIZOGENESIS IN REGENERANTS
Healthy, white adventitious roots are prerequisites before going for hardening
and acclimatization of plantlets regenerated in vitro. During the present study it was
u S
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N O N O ( N — o o o o o N O r N i — NO ^ 00 r-~
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o • o <^ d -H
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2i ^ ^ r
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0 0 ° <N 00 [:; — ' i in CN) .
<-!• ':s' o - <;/ -H i -H -H ;ti
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NO
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in NO
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1^ *n
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NO
d
O NO
_ . O O J ^ - H - ^ R N O C S ^ - H ~_'ninH£~Z^NOin'j!;g
—" <N O^ O^
« 1^ » NO * O O N C C ^ , r ^ o * •<^ — - ^ N O o o O N O i n o — 4 | r N 4 N O > n N C i n ^ ' ^ i n r n r o i n N O ^ ^ i n r n r N i t n ' ^ NO 1 ^ NO f^
rN| r<-i T t a i CJi Qi
CNJ ( ^ Tj- i n D i D i D i Ci:
C/2
-H c
u
D-D
li 3
Q CO 3
Q U _ 1
'— <u > V
~~ s ^y~,
1 .
re c o LM
u-'•5
> N
• ^
c re o
( • p
on t/i
^ o
JD C
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C
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tc
60
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CQ
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re en u '7,
X) ^
re re
> > re re
> > 3 3
A A
Results 43
observed that among all the varieties tested, roots were also induced along with shoots
on regeneration medium itself, but quality was very poor. Such type of plantlets
failed to survive the hardening and acclimatization process. Therefore, to achieve a
good rooting plantlets with 2 to 3 leaves were transferred on a range of medium
having different growth hormones, sucrose content and with or without added
vitamins (Table 14). Most of the roots induced on Rt 1, 2, 3 and 5 were thin, fibrous
green and could attain a length of about 8-10 cm with lesser lateral branches. These
were therefore not suitable to go for plant hardening. However, roots emerged on Rt 4
medium were white, healthier and thick with lateral branches. These were considered
to be suitable as responded suitably during hardening and acclimatization of plantlets.
4.1.5.1. Inference
On the basis of the experiment conducted during investigation by examining
the role played by the medium having MS salts (without vitamins) + NAA (1 mg/1) +
Sucrose (20 g/1), without adding vitamins was found to be ideal for root induction and
subsequent hardening of the plantlets.
4.1.6. HARDENING AND ACCLIMATIZATION
Three different pathways were adopted for hardening and acclimatization of
plantlets (Chapter 3, Materials and methods). The success rate of established plants
was only 10 percent when plantlets were hardened using the first method about 20-30
percent plantlets survived in the second method. Decrease in success through these
methods may be attributed due to contamination during several sub-culturing. Only a
few plants withstood the shift from solid to liquid and then soil mixture route.
Nearly 50 to 90 per cent plants survived when hardened and acclimatized
through third method. This condition to success was having good root system and
stable low temperature and high humidity. Also plants did not have to withstand
several sub-culturing shocks passing through semi solid, liquid and soil phases.
u a >
'•C "a
JC
? B a u s
• a
73 C
"O
0) U
o
o o x: W5
0) 4 ^
B V DX)
? .i:
O >-> u B . _«
(U tt B o o &i
B _ 3
•5
B Ml
Vrf o o
'S "s U
•T/
• t
2^
f^i • « • «
2^
f S
r^
^ *
* * * * *
* * * *
*
*
00 m m CM
O-P
* * *
* * * *
* * * *
* *
*
i n
* * *
* * * * *
* * * *
* *
*
? _o
DO cO
m
•K-
*
* * * * *
* •»•
* *
*
3 O
^ 00
O ;-(
• 4 — 1
3
(U
15 5 0) ; - i
ca
(/) O
o
* * * * t/f
O o
3 O L^
(L)
1 ^
•K-* *
t/2
iG o o '-
o *^ CU 03
• Si I -fi
„ (D
o ? t, ' ^ i l o .a
lU *
> * * *
Xi
Fig 6. Explanations
Hardening and acclimatization of plantlets
A: Plantlets in liquid medium.
B: Semi-hardened plant in growth chamber.
C«&D: Hardened plants in field.
E&F: Established Ro plant of UP 2338.
Results 45
EXPERIMENT-2
4.2. SELECTION OF THE BETTER RESPONDING GENOTYPES
Adopting the abovementioned protocol, eight varieties of each, bread and
durum wheat varieties were screened for their ability to form callus and to explore the
regenerative potentiality of the calli.
4.2.1. CALLUSING
Among the 8 wheat varieties tested, cent per cent callus induction was
exhibited by 12 DPP embryos of WH 542. No marked difference was observed
among UP 2338, PBW 343, PBW 373, Raj 3765, HD 2329 and WH 542 for their
mean callus induction frequencies (Table 15). Comparatively poor callusing was
recorded from CPAN 3004 and KRL 1-4. Embryos aged 12, 14 or 16 DPP exhibited
higher callusing depending on the genotype.
In general, the durum wheat embryos were found to have lesser tendency to
transform into callus as compared to bread wheat. A maximum of 87 per cent
embryos of Bijaga Yellow and Gulab exhibited callusing to bread wheat. Mean
values for callus induction were higher in embryos taken from Bijaga Yellow, PDW
215 and Gulab (Table 15), followed by PDW 233 and Raj 1555 (both fall in same
significance level). Durum varieties HI 8381, MACS 2846 and HD 4530 responded
very poorly to callus initiation. Significantly better callusing was obtained from 12 to
18 DPP embryos depending on the genotype.
4.2.2. PRECOCIOUS GERMINATION
In the present study, older embryos (16 and 18 DPP) exhibited a tendency to
germinate precociously i.e. without attaining maturity. Maximum precocious
germination among bread wheat was recorded in varieties showing minimum callus
induction i.e. in KRL 1-4 (26%) and CPAN 3004 (20%) from 18 DPP embryos
followed by HD 2329, Raj 3765 and PBW 373. Minimum precocious germination
was observed in UP 2338 and WH 542 (Table 15).
Among durum wheat, HI 8381 and MACS 2846 showed maximum precocious
germination. Even 14 DPP embryo of HI 8381 germinated on callus initiation
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Results 47
medium. Embryos of Bijaga Yellow, Raj 1555 and Gulab showed lesser tendency to
germinate than PDW 215 and PDW 233.
4.2.3. REGENERATION
Ri R2 and R5 pathways were adopted to test the regeneration ability of the
cultivars.
4.2.3.1. Regeneration on Rl medium
Among bread wheat varieties cent per cent regeneration was obtained in case
of WH 542. PBW 343 and HD 2329. However, mean values for regeneration on all
the five days were significantly higher for WH 542 (92%) at 5% level of significance
(Table 16). Regeneration from UP 2338, PBW 343, PBW 373, Raj 3765 and HD
2329 was moderately higher and did not differ much from each other. CPAN 3004
and KRL 1-4 revealed poor regeneration.
Regeneration in durum wheat varieties Bijaga Yellow, Raj 1555, PDW 215,
PDW 233 and Gulab was quite high and their mean values did not show much
differences (Table 16). However, the response of HD 4530, HI 8381 and MACS
2846 was found to be poor. In general, age of embryo exerted a significant influence
on the regeneration frequency as the 10 DPP embryos always exhibited maximum
regeneration.
4.2.3.2. Regeneration on R2 medium
Among bread wheat varieties, on R2 medium, 100 per cent regeneration was
obtained in case of WH 542 and PBW 343 (Table 17). Response of UP 2338, PBW
343 and PBW 373 also got the same place as they fell in the same significant level
followed by PBW 373, Raj 3765 and HD 2329. Varieties CPAN 3004 and KRL 1-4
responded very poorly.
Durum wheat cultivars, Bijaga yellow, Gulab and Raj 1555 were found to be
ideal responding genotypes followed by PDW 215 and PDW 233 (Table 17). Rests
were poor in regeneration.
4.2.3.3. Regeneration on R5 medium
Bread wheat varieties UP 2338 and WH 542 were ideal on R5 medium
followed by PBW 343 and PBW 373 and subsequently by Raj 3765 and HD 2329
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Results 50
(Table 18). CPAN 3004 and KRL 1-4 were very poor in their response to this
medium.
Durum Wheat varieties PDW 233 and Gulab were found to be better followed
by Bijaga Yellow, Raj 1555 and PDW 233. Raj 1555 and PDW 215 were next in
their regeneration response. Rests did not show any encouraging response.
4,2.3.4. Selection of the varieties
On the basis of the present findings, among bread wheat varieties, WH 542
ranked the best for its regeneration potential followed by PBW 343 and UP 2338
(Table 19). However, these three varieties stood at the same level of significance
ladder (at 5%P). PBW 373, Raj 3765 and HD 2329 were found to be moderate in
their ability to regenerate. Performance of durum wheat varieties Gulab and Bijaga
Yellow was quite appreciating followed by Raj 1555 and PDW 233. Performance of
PBW 215 was also good.
Therefore, depending on their ability to regenerate following varieties of bread
and durum wheats were selected to be used in further crossing program.
Bread wheat Durum wheat
WH542
UP 2338
PBW 343
Raj 3765
HD 2329
Raj 1555
Bijaga yellow
Gulab
PDW 215
PDW 233
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Results 53
EXPERIMENT-3
4.3. EMBRYO RESCUE AND WIDE CROSSING
Five varieties each of bread and durum wheats were crossed with different
Triticum and Aegilops species (Table 20). Embryos were rescued at 10, 12, 14 and
16 days post pollination. Calli were induced on CI, MS + 2mg/l, 2,4-D and
regenerated on R2, MS + 2mg/l Kinetin or R5 2 mg 1'' BAP + 0.5 mg/1 lAA media .
All the three media were standardized in the laboratory.
In hybrids involving T. urartu as one of the parents, 14 DPP embryos of PBW
343 exhibited maximum callusing (48.48%) and 10 DPP of HD 2329 gave the least
(16.67%) among bread wheat varieties (Table 21). Moreover, these two combinations
again responded to maximum and minimum regeneration (66.67 and 46.88%,
respectively). In durums, the varieties Gulab (14 DPP) and Raj 1555 (10 DPP) with
urartu showed the highest (44%)) and the least (12.50%)) callusing, respectively.
However, there was not very apparent difference in crosses for regeneration. Much
older embryos (>16 DPP) of crosses with WH 542, UP 2338, Gulab, Raj 1555 and
PDW 233 were distorted in shap^F .' Embryos of Raj 1555 x T. urartu were so
vigorous in inducing callus that it required 30 to 40 vessels to maintain calli induced
from a single embryo. Plants established in the field were tall and long spiked but
with poor seed setting.(Fi^ 9)
Embryos of hybrids with T. boeticum were rescued at different DPPs viz.,
with WH 542 (10-16 DPP), PBW 343 (10-14 DPP) and Bijaga yellow and PDW 215
(9 to 15 DPP) (Table 21). The callus induction frequency in crosses involving bread
wheat varieties was recorded highest (47.06%)) and the minimum of 11.76 per cent
from 12 and 16 DPP in hybrids with WH 542 giving 42.86%) regeneration. The
variety PBW 343 carrying IB/IR translocation was found comparatively less
responding to callus induction and regeneration. Among crosses with durum, 14.29%)
(from 10 DPP) to 28.57% (12 DPP) calli of hybrid with Bijaga Yellow exhibited
callusing which either became brown or turned into roots. On the other hand,
embryos from the hybrids involving PDW 215 did not respond to callus induction
medium.
Embryos of hybrids with Ae. speltoides were scooped at 10 to 16 days from
hybrids with WH 542, 10 to 14 days PBW 343, 9 to 15 days UP 2338 and HD 2329
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Results 55
and 8 to 14 days post pollination from durums (Table 21). Success to callus induction
was found to 9.09 (lODPP, WH 542) to 30.77 per cent (13 DPP, PBW 343) and nill
regeneration (HD 2329) between 28.57% (UP 2338). Only the embryos of 10 and 12
DPP in durums responded to callus induction from 16.67 to 40.00%, with PDW 233
only regenerating genotype (25.00%). However, callus could b&«i)tdi?ied ^gpri 8 and
14 DPP embryos. Plants of the crosses with UP 2338 a^JPDW 233 got es tabh^d II ( /i..., -v-, ) \
in field with poor seed setting. ' > ^ _ ,' Sjj
Hybrid embryos of crosses with Ae. squarrosa werevjirj^ued either^ 10 to
14 DPP (with Gulab and Raj 1555), 10 to 16 DPP (with WH 542, UP''233'8; PDW 215
and PDW 233) or 10 to 17 DPP (HD 2329 and Raj 3765). Frequency of callus
induction ranged from no callusing (17 DPP. Raj 3765) to 60.00% (12 DPP, WH 542)
among aestivums and nil (10 and 14 DPP Gulab) to 54.55% (12 DPP, PDW 233)
among durums (Table 21). No marked difference was observed for regeneration.
However, no multiple shoots could be regenerated from calli of the crosses with Raj
3765 and Gulab. HD 2329 possessed distorted seed at 17 DPP. Hybrids involving
HD 2329, Raj 1555 and PDW 233 were raised to maturity with poor seed settingCFf^a).
Among the hybrids involving Ae. markgrafii, all embryos were rescued at
10, 16 DPP and in vitro responses of hybrids with only one durum variety Raj 1555
was found better (38.89 to 61.11%) callus induction and 63.64% regeneration) than
other with bread wheats showing 33.33 to 56.56%) callus induction and 56.76%)
regeneration from HD 2329 and 37.14 to 55.00 % callus induction and 50.00%)
regeneration from UP 2338 (Table 21). Plantlets of the cross with UP 2338 and Raj
1555 were grown to maturity but without any seed settingCFi'g lo).
Hybrid embryos with Ae. umbellulata were rescued at different days post
pollination i.e. at 10 to 14 DPP with Raj 1555 and PDW 233, 10 to 16 DPP with PBW
343 and 12 to 18 DPP with WH 542. No marked differences were observed among
hybrids of two varieties of bread and durum wheats, as both responded quite
vigorously to callus induction (35.00 to 62.50 and 25.00 to 53.85%) and regeneration
(53.33 to 62.07 and 54.07 to 55.00 % respectively) (Table 21). Hybrids involving Raj no
1555 and PDW 233 got established in field, however,feeed setting was observed.
Embryos of crosses involving Ae. cylindrica were rescued at different days
post pollinations viz., 9 to 15 DPP from crosses with Raj 3765; 10 to 15 with PBW
343; 10 to 16 with UP 2338 and HD 2329; 10 to 14 with Bijaga Yellow, Gulab and
Raj 1555; and 8 to 14 with PDW 215 and PDW 233. Frequency of callus induction
Re.sii/l.s 56
was quite good from embryos of hybrids with bread wheat (30 to 47.37%).
Regeneration was very poor with 20% from WH 542 and 24% from UP 2338. CalH
from other crosses could not be differentiated into shoots. 10 to 14 DPP embryos of
durum hybrids turned into callus (10.00 to 25.00%), while 8 DPP embryos didn't
responded (Table 21). All the calli turned into roots only. Some of the embryos of all
age groups of Gulab and 14 DPP of PDW 215 and PDW 233 got abnormal
protuberances. Hybrid HD 2329 x Ae. cylindrica attained maturity with poor seed
setting.
10 to 16 DPP embryos of the hybrids with Ae. ventricosa were cultured.
Only hybrids with WH 542 transformed into callus (10.00 to 25.00%) and regenerated
as well (33.33%)) (Table 21). No calli could be obtained from the hybrids with PBW
343 and Bijaga Yellow. Hybrid plants involving WH 542 could be raised but
exhibited poor seed setting.
In crosses with Ae. kotschyi 10 to 16 DPP embryos of the hybrids with single
bread wheat, PBW 343 and 9 to 15 DPP of durum were cultured. Although, 12.50 to
30.00% callus was obtained from PBW 343, however, none of the calli could turn into
shoots. The durum wheat hybrids exhibited better callusing (16.67 to 57.14% on 10
and 12 DPP, respectively) and even 52.63%) calli of Raj 1555 and 52.17% calli of
PDW 215 transformed into multiple shoots (Table 21). The calli of hybrid with
Gulab also turned brown producing roots only. The hybrids involving Raj 1555 and
PDW 215 were raised to maturity with very poor seed settingCKa \o\
Hybrid embryos with Ae. peregrina were rescued at 10 to 16 DPP . Among
bread wheat varieties, Raj 3765 was found better with maximum callus induction
(37.71%) froml2 DPP) and regeneration (44.44%)). Among durums. Raj 1555 was
found better with maximum callus induction (46.67%) from 14 DPP) and maximum
regeneration (47.37%) (Table 21). The hybrids with Raj 3765, Raj 1555 and PDW
233 got established with very poor or no seed setting.
10 to 18 DPP embryos of the hybrids involving y4c. triuncialis with bread
wheat and 9 to 16 DPP of durum were cultured with 22.22 to 64.29%) and 20.00 to
45.45%) success (Table 21). The young hybrid embryos of durums (9 and 10 DPP)
did not respond, however, all the calli became brown and no further development
could be achieved.
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Results 60
Hybrid embryos of Ae. geniculata as one of the parents were scooped at 10
to 16 days post pollination and cultured successfully. Among the crosses with bread
wheat, Raj 3765 was found better responsive for both induction of calli (a maximum
of 53.33% from 12 and 14 DPP embryo) and regeneration (60.87%). The frequency
of callus induction was recorded highest from the crosses with Gulab (54.55% from
12 and 14 DPP) and regeneration from Bijaga Yellow (52.63%) (Table 21). The
plantlets of the cross with Bijaga Yellow got established in field with no seed setting.
Hybrids involving/4e. ovata were also rescued at 10 to 16 DPP. The bread
wheat hybrid with WH 542 exhibited highest (63.75% at 14 DPP) calli formation and
better regeneration (62.88%) in comparison to hybrids with UP 2338. Among durums
the maximum (53.85%, 12 DPP) and minimum (37.50%, 10 DPP) callus induction
was attained in the hybrid embryos with Bijaga Yellow, whereas maximum
regeneration (60.87%) was recorded from PDW 233 (Table 21). Both callus induction
and regeneration were found good, inspite of having abnormal embryos (in shape)
from WH 542, Bijaga Yellow and PDW 233. The hybrid plants with WH 542, got
established in the field, with spikes having open florets at the time of pollination
(Fig. 8). However, very very poor seed setting was recorded.
Hybrids embryos with T. araraticum were rescued at 10 to 16 DPP. The
hybrid embryos with bread wheat variety UP 2338 did not respond to callus induction.
Whereas, 20.00 to 68.33% embryos of the hybrids with durum turned into callus
(Table 21). The organogenesis from all these hybrid embryos ended with the
formation of roots only.
None of the crosses with Ae. longissima and Ae. sharonensis (Table 22)
possessed dissectible embryos as the caryopsis either became white and epaque with
no content inside or green with watery fluid inside. Embryos of hybrids with Ae.
bicornis, Ae. caudatei Ae. lorentii and Ae. triaristata taken as male parent (Table 23)
were rescued at different days post pollination ranging from 8 to 18 days. However,
none of the embryos transformed into calli.
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Fig 7. Explanations
Normal and abnormal embryos rescued from wide crosses
A: 8 days old embryo of bread wheat.
B: 18 days old normal embryo.
C: 14 days old Gulab x T. urartu hybrid embryo (abnormally small size).
D: Hybrid embryo of UP 2338 x Ae. bicornis (trilobed).
E: Hybrid embryo of WH 542 x Ae. ovata (abnormal protuberance).
F: Hybrid embryo of Raj 1555 x Ae. peregrina (shapeless).
G: Hybrid embryo of PDW 233 x Ae. triuncialis (no distinct apical axis).
H: Hybrid embryo of PBW 2\5xAe. cylindrica ( twin embryos).
Fig 8. Explanations
Regeneration from embryos along with peculiar hybrid spikes.
A: UP233S\Ae. cylindrica.
B: PBW 343 X r. wrar/w.
C; In vitro raised branched spike from UP 2338 x T. urartii.
D. WH 542 X Ae. speltoides.
E: PDW 233 xAe. squarrosa
F: Hybrid spike at WH 542 x Ae. ovata showing open floret at the time of
pollination.
Fig 9. Explanations
AB&C: Regenerants induced via callus cultures of single embryo hybrid of
Raj 1555 X r. urartu.
D: Hybrid plants of PDW 233 x T. urartu.
E: Fi ears of Raj 1555 x T. urartu.
F: F2 ears of hybrid of Raj 1555 x T. urartu with female (left) and male
(right) parents.
Fig. 10 Explanations
Different wild hybrid plants established in field through embryo rescue
A: WH 542 xAe. ovata.
B: Raj 1555 x 7". iirartu.
C: UP 2338 xy t;. markgrafii
D: Bijaga Yellow x T. urartu
E; Raj \^S^xAe. kotschyi
VtSCUSSZON
5. Discussion
To keep pace with the global wheat demand, there is a need to break the
existing yield plateau, stabilize production at each level of production ladder, and
extend wheat cultivation to problem and marginal areas and breed wheat varieties
with specific industrial and nutritional requirements. This ultimately necessitates
broadening the genetic base through alien gene transfer and one alternative to
accomplish this in wide hybridisation. Literature reveals that agronomically useful
hvhrids, even between closely related species, are often unattainable due to sexual
incompatibility. In many instances fertilization however takes place, but the
de\ eloping embryo aborts at various stages of its development. The techniques of
embryo rescue which involves the excision and culture of the young hybrid
embryos to obtain hybrid plants can play vital role in creating variability and
pyramiding the useful genes (Collins and Grosser, 1984; Raghvan, 1986).
During the present course of study, before gomg straw for rescuing embryo,
it was tried to develop a suitable regeneration protocol and select out the better
responding genotype in vitro. Although, several other factors can affect the callus
induction and plant regeneration from rescued embryo. If we have a better
responding genotype as one of the parent, the chances of getting regenerants
become high (Ou et al, 1984). An established protocol can also help in going for
transformational studies through various methods (Becker et al, 1994; Karen et
al. 2000 and Huber et al, 2002) including somaclonal variation (Villareal et al,
1999; Benkirane et al, 2000), screening of genotypes against various stresses (Jia
and Zhao 1993; Wang et al, 1993) or in vitro mutational studies (Liu et al, 1993).
5.1. BASAL CULTURE MEDIA
Two types of basal culture media MS and B5 with same GR supplemented
i.e.. 2mg/l 2,4-D were used. Calli raised on MS were found to be embryogenic
and more regenerative, whereas, that on B5 were organogenic and have lesser
regeneration frequency. Both the media MS and B5 differe in nitrogen (NH4NO3
Discussion 63
& KNO3) and phosphorous source (KH2PO4 and NaH2P04), respectively. The
amount of CaCl2 , MgS04, KI, H3BO3, MnSo4, ZnS04 are also different. Any of
these compound(s) may have a. influence on role of culture responses and may lead
to poor or good regeneration. He et al. (1991) also noted that the ingredients of
basal culture media and their amount influence the in vitro responses. Nitrogen
source has been reported to affect the embryogenesis by Higashi et a/. (1996).
5.2. GROWTH REGULATOR
In the present course of study it was observed that growth hormones have a
vital role to play at each step to get regeneration. On the MS medium containing
2mg/l 2,4-D typical embryogenic calli were obtained characterized by glistening
pale yellow/yellow white surface, compact and opaque, which latter on became
nodular with buds. Embryogenesis from similar type of calli were also reported
by Ozias-Akins and Vasil, (1983a,b); Immonen, 1996; Farooq et al, 2001; Heyser
el ai, 1985; He et al, 1986 and Hunsinger and Schauz, 1987. Embryos transferred
on media containing 4mg/l 2,4-D were very vigrous, however, calli were loose
watery, fragile and transparent, more likely to transform into roots (Chauhan and
Singh, 1995). However, when immature embryos were planted on growth
hormone free MS media no callusing was observed. Addition of auxins were
found necessary to induce callus, synthetic auxins were supposed to disorient the
previously programmed patterns of cellular activity (Wernicke and Milkovits,
1984) and permit or stimulate cellular organization and embryoid formation in
some unknown way.
Poor callusing and regenerability at higher 2, 4-D concentration may be an
artifact of quantifying regeneration without allowing adequate time for
regeneration of the less mature embryos (Bregitzer et al, 1995). By using an
anti-auxin TIB A into medium Cistue et al (1999) concluded that by reducing
auxin, calli could be obtained from otherwise poor responding anthers, however
2,4-D was essential to elicit the response.
2, 4-D in combination with lAA leads to the formation of fragile calli with
green sectors, which latter on give rise to plant organogenically. Shoots were also
Discussion 64
reported to originate from such green sectors by Shimada 1978; Ivanov et al., 1998
and Machii et al., 1998. 2 mg/1 2,4-D was found to be optimum and supported by
Sear and Deckard et al., 1982; 1983; Mathias et al., 1986 and Ozyen et al., 1998.
No embryoids were recorded in calli induced on MS + 2,4D (4 mg/1), MS +
2,4D(4 mg/1) + lAA (1 mg/1) and B5 + 2,4-D (2 mg/1). This support the view that
embryogenesis in wheat callus culture was affected by phytohormone
concentration and type (He et al., 1986; Hunsinger and Schauz, 1987; Redway,
1990). In present study it seems that lAA inhibits the callus induction and also
deteriorates the quality. No callusing was observed on medium containing lAA.
This was also supported the view of Ozias-Akins and Vasil (1982). Embryogenesis
was preferred over organogenesis, as single cell origin of former leads to
genetically uniform plantlets, instead of chimeric ones (Haccius, 1978; Vasil and
Vasil, 1982; Vasil, 1987).
Addition of cytokinins like Kinetin and BAP stimulates shoot formation in
callus (Shrivastava el al., 2000). 2 mg/1 of kinetin was found to improve shoot
regeneration (Bosch Wackerle et al., 1979; Papenfuss and Carman, 1987),
however, in combination to 0.5 mg/1 lAA leads to rooting instead of shoots. On the
other hand 2 mg/1 BAP in combination to 0.5 mg/1 lAA gave better shoot
formation than BAP alone and lead to conclude the interactive effects of hormone.
BAP was also used by Hussain and Islam (1993) and Mathias et al. (1982) to get
regeneration. It was observed that callus induction media also have a profound
effect on regeneration ability of the calli in association to that of regeneration
media. It was in accordance to Ward and Jordan (2001), who postulated that
composition of callus induction media affects both, the percentage of callus
formation from embryos and subsequent frequencies of plant regeneration.
However, they found that regeneration medium had no effect on level of plant
regeneration, instead during our experimentation it was recorded that regeneration
frequency was also very much affected by composition of the regeneration
medium. R2 and R4 were found to give statistically better response than R3 and
R4.
Discussion 65
Shoot enlarged on GR free media with reduced sugar (20 mg/1) produced
maximum number of multiple shoots in comparison to that left on the same
medium on which they were induced. The reason can be, firstly the mechanical
injury faced by grown up plantlets with the 3-4 leaves during taking out from the
flasks; and secondly, additional nutrition provided by fresh subculture leads to the
development of each and every plantlet thereby increasing the number of
plantlets/callus. As plantlets established their own endogenously supplied growth
hormones, there was found no need to supply it artificially. Plantlets grown on
reduced sugar (S3 and Rt4) were having healthier shoots and roots. Reducing
sugar from the medium may be beneficial to plantlets by promoting
photoautotrophy in vitro and also by reducing the loss of plantlets due to
biological contamination of culture medium (Kozai, 1991, Seko and Nishimura,
1996).
During present course of study, addition of GA3 (0.5 mg/1) to the medium
caused browning at the base of the plantlets. This deteriorates the number of
shoots and even most of the plantlets get dried within 3 to 4 weeks. However, use
of GA3 was recommended to induce root formation (Ozias-Akins and Vasil, 1982)
and in regeneration medium (Bohorova et al, 1995). Callus induction rate,
regeneration frequency, number of plantlets regenerated were all found
independent of each other (Ozygen et al. 1998). Albino plant was recorded in
WH 542 from calli induced on C2 and regenerated on R3. The reason may be high
concentration of 2, 4-D (4 mg/l)in comparison to that of other initiation media (2
mg/1) and the cultivar may be more sensitive to changes.
5.3. PRECOCIOUS GERMINATION
Precocious germination is the ability of embryos to germinate without
attaining maturity. The phenomenon of precocious germination is not desirable as
it results in smaller and less vigorous seeds and poor quality of calli (Ozias Akins
and Vasil, 1982). In present course of study a very few zygotic embryos were
found to germinate precociously. Smaller embryos of 10, 12, 14 or even 16 DPP
do not germinate except durum wheat HD 4530 which germinate (3.33%) at 14,
DPP even. Culture of embryos by planting them with scutellum side upward, was
Discussion 56
used as a strategy to check the germination of embryos followed by Fenell et al. in
1996. Even than, some of the embryos of 16 and 18 DPP germinated. Minimum
germination was reported on medium supplemented with 4mg/l, 2,4-D than 2 mg/1
2,4-D. Media having lAA exhibited maximum germination. Several other
workers find decrease precocious germination with increased concentration of 2,
4-D (Ozias-Akins and Vasil. 1983; Carman et al., 1987). Papenfuss and Carman
(1987) used dicamba to suppress precocious germination with cent percent
success. However, calli and on dicamba containing medium were found necrotic
and even plantlets difficult to establish (Carman et al. 1987a). Distinct variations
were also observai among genotypes.
Carman et al. (1988) considered precocious germination of embryos as a
useful parameter for assessing the effect of callus medium on somatic
embryogenesis. Whereas, Immonen (1996) found it to be a cross specific/genotype
specific and therefore do not reflect the quality of callus induction medium. Bapat
el al. (1988) also treated the precocious germination of zygotic embryos as an
important characters, stating that genotypes that hui/e precocious germination of
zygotic embryos have greater embryogenic potential. However, findings of the
present study reflect a vice versa result.
5.4. AGE OF EMBRYOS
12 to 14 DPP embryos of aestivum wheat and 12 to 16 DPP of durum
wheat exhibited maximum callusing. Embryos less than 12 days were less likely
to transform into callus, however, older embryos (16 and 18 DPP) germinate
precociously. The similar trend was noted by Ward and Jorden (2001) in triticale.
They postulated that it is necessary for immature embryos to reach certain
physiological level to respond in vitro. The reason to this may be that 10 DPP
embryos were very small and delicate and get punctured/damaged during
dissection. 10 to 15 DPP embryos were reported to give fast and regenerable calli
by Shimada (1978), Magnussan and Bornman (1985) and Ozygen et al. (1998). 10
to 14 DPP was known to be optimum by Wang et al. (1993) and Arun et al.
(1994). Poor quality, lesser ability to regenerate and more tendency of having
precocious germination from older embryos can be attributed to the endogenous
Discussion 67
supply of the growth hormones from them. As they can hinder the growth of calli
into callus and influence latter development in one or the other way.
Age of embryos also exert an influence in multiple shoot production from
calli. Maximum regeneration was attained from 10 days old embryo in almost all
the genotypes. This was in contrary to findings of Ward and Jordan (2001), as
they reported that 10 DPP embryos have poor ability to regenerate when compared
with others. Older calli (18 DPP) exhibited minimum regeneration with weaker
plantlets and poor rooting as also reported by Redway et al. (1990). Whereas^He el
ul. (1988) reported increased frequency of precocious germination in older
embryos. These characters were also found to be genotype specific as even 14
DPP embryo of durum wheat cultivar HDj was reported to germinate precociously.
5.5. GROWTH CONDITION OF CULTURES
Following Mathias et al. (1986) and Carman et al. (1988) cultures were
maintained in dark to induce callus at 25 ± 2°C temperature. Higher temperature
(30°C) leads to the browning of callus and excessive root formation.
Light during the regeneration phase were recorded to have a marked effect
as the calli induced on 2 mg/1 2,4-D when exposed to light provided by bulb
(20wt) lead to multiple regeneration sometime in 100% calli, on the same medium
in which they were induced. The reason could be lowering of auxin concentration
near the callus mass leads to the germination of embryoids precociously (Bapat et
al, 1988), as no regeneration were observed from non-embryogenic calli initiated
on C2, C3 or C4. However, when callus were exposed to light provided by cool
florescent tubes no regeneration was obtained. Type of light might also have some
role to play in addition of type of calli. Arun et al. (1994), also reported
germination of embryos on callus induction medium itself.
Light induced precocious germination of somatic embryos were also
reported by Ozias-Akins and Vasil (1983c) and Papenfuss and Carman (1987).
Where as regeneration on 2,4-D free medium was reported by Zhang (1986),
Taghvaii et al. (1998) and Shrivastava et al. (2000), however, the type of light was
Discussion gg
not specified. Seko and Nishimura (1996) hypothesized that light is needed to
regulate morphogenic process of in vitro plantlets including shoot proliferation
and plantlet elongation,
5.6. GENOTYPE
In vitro responses were known to be genotype specific (Lang et al.. 1995;
Bankirane et al, 2000) in cereals along with wheat. Efficiency of callus induction
(Mathias and Simpson, 1986; Barkat and Abd-el-latif, 1995; Arzani and
Mirodjagh, 1999); Callus growth rate (Lazer et al., 1983), embryogenesis (Arun et
al.. 1994; J'Aiti et al., 1999) and plantlet regeneration (Karadimova et al.. 1985;
Machii et al.. 1998) have all reported to be in part genotype dependent. The fact
that on the same medium some genotypes produced embryogenic calli and
regenerated plants while others were completely incapable of regeneration
suggests that there are genetic components controlling this trait. Several
researchers have attempted to find location among the wheat genome responsible
for favourable tissue culture responses.
Chromosomes 7B, 7D and ID (Galiba et al., 1986), IRS (Langridge. et al.,
1991). IBS. 2BS, 6BL (Felsenburg et al., 1987). 4BL (Mathias and Fukui. 1986,
Mathias et al.. 1988), 2 DL, 2 AL, 2BS and 2 BL (Kaleikau et al., 1989a,b; Ben
Amer et al., 1992), mitochondrial DNA (Rode et al., 1986) or even cytoplasm
(Mathias and Fukui, 1986) have all been identified as possible regions responsible
for better callus induction and regeneration potential. Some others reported
different genes like reduced height/gibberellic acid insensitivity genes, Rht/Cai
(Mathias and Atkinson, 1987) semi-dwarfism allele, Rht 8 and day length sensitive
allele ppd 1 (Ben Amer et al, 1992) etc. to be responsible for in vitro responses.
It can be suggested that one or more of these genes are being expressed in the
highly regenerable genotypes on, one of the media (Fennel et al, 1996). Lazer
1981 also found genotype-medium interaction to be responsible for better in vitro
response.
Present course of study also supported the view of others that callus
induction and regeneration both was found to be genetically affected. In contrary
Discussion 69
Vasil (1987) and Redway et al. (1990) hypothesize that differences in cultivar
responses are physiological in nature and can be overcome by culture conditions
and media. However trend of responding of media composition for callus
induction, regeneration, number of shoots per calli or root formation were in
general found to be uniform, irrespective of the genotype.
5.7. WIDE CROSSING AND EMBRYO RESCUE
During the last four decades, there has been an extensive use of wild
relatives in wheat breeding programm, particularly outside the country. Recently,
the breeders are using these as the valuable source of variation for wider
adaptation, tolerance to biotic and abiotic stresses and finally for yield
contributing traits. However, this is not an easy job, since many factors are
involved in between to obtain the hybrids. There are even some genes like Krl,
Kr2, Kr3 and Kr4 located on chromosomes 5B, 5A, 5D and lA respectively,
which are known to reduce crossability with many species (Koba and Shimada,
1993).
In present study, greater emphasis has been paid to callus mediated embryo
rescue, as direct germination of the rescued embryos was reported in the literature
to lead precocious germination resulting into small, weak, lean and thin plantlets
(Ozias-Akins and Vasil, 1982). Therefore, the crosses which are difficult to make
and where the seed setting is very poor, even one successfully cultured embryo
may give rise to several hybrid plants and the chances of getting an amphiploid are
therefore much more (Immonen, 1996). In addition to alien genetic transfers
mediated by callus culture promote multiple cytological variations and could also
be exploited as an adjunct to Ph- mediated Fj introgeneric hybrid genetic
exchanges (Mujeeb-Kazi and Asiedu, 1990).
Ter-Kuile et al. (1988) supported the view by regenerating Fi hybrids of T.
aestivum x Ae. variabilis and T. turgidum x Ae. variabilis and demonstrated seed
set on an otherwise anticipated self sterile population. Moreover, the Callus
induced doubling may prove advantageous for combinations otherwise hard to
Discussion 70
double and also reported induced alien exchanges in wheat x rye hybrids (Lapitan
etal, 1984, 1986).
The hand pollinated spikes were sprayed with 2, 4-D + GA3 solution which
had a positive influence on developing seeds and embryo formation. In several
crosses with wheat x H. bulhosum and wheat x maize, Inagaki (1986, 1998)
could enhance embryo formation and then seed setting by spraying 2, 4-D +GA3
just after pollination. . This could be the reason of having better dissectible
embryos than in the experiments laid down earlier (unpublished).
In most of the crosses performed in the present investigations, the embryos
were rescued at 10, 12, 14 and 16 days post pollinations. 10 and 12 days old
embryos gave better callus induction than 10 or 16 days embryos. The possible
reasons for such behaviour may be that 10 days old embryos may require an
additional nutrition (Raghavan, 1976) or physical injury due to very small size
during dissection. On the other hand, most of the older embryos (> 16 DPP)
usually got distorted (may be due to initiation of degeneration) and callus
induction from such types of embryos was reported in very few cases. During the
present study, we did not need to enrich the medium instead 12 or 14 days
embryos could be cultured successfully to get regenerants.
Most of the calli regenerated on media containing kinetin (2 mg/1) or BAP
(2mg/l) + lAA (0.5mg/l) that led to produce multiple plantlets in many of the
crosses.
The calli in some of the hybrids like Bijaga Yellow x T. boeticum crosses
oi Ae. squarrosa (male) with Raj 3765 and Gulab, Ae. cylindrica (CD) with all the
five durums and T. araraticum (male) with Raj 1555, PDW 215 and PDW 233,
turned brown and no further development occurred in such cases. Also, in other
hybrids in which the wild species were taken as male parents namely, Ae.
speltoides (male) with HD 2329, Gulab and PDW 233, Ae. cylindrica (male) with
PBW 343, HD 2329 and Raj 3765 and all the hybrids involving Ae. triuncialis, the
calli became necrotic i.e. turned brown and did not respond at all to regeneration.
Seko and Nishimura (1996) and Fennell et al. (1996) reported this premature
Discussion 71
rooting or necrotic phenomenon of calli to be genotypic character. Since the
female parent of these crosses do not have such characters, it may be the trait of
the alien genotype or a cumulative effect of some other factors.
Embryos of hybrids involving Ae. bicornis, Ae. caudate and Ae. lorentii did
not respond at all. This may be due to complete incompetence of the genotypes
used. However, T. boeticum, Ae. speltoids, Ae. ventricosa and T. araraticum
showed partial incompetence with PBW 215, Raj 1555, PBW 343 and Bijaga
Yellow and UP 2338 respectively, as hybrid embryos of these species were
reported to induce callus with other cultivars. According to Endo (1988, 1979),
the presence of gametocidal chromosomes in Aegilops species, causes sterility and
abnormality in certain genetic backgrounds. The Occurrence of abnormal embryos
was found to be a common phenomenon particularly in older embryos (> 16 DPP).
The hybrid plantlets established in the field were seen to exhibit
intermediate morphology, casually giving plants a profuse tillering. Having
intermediate type of plant morphology is important as it proves the validity of the
cross (Tyaukgva, 2000).
During present experimentation, embryo formation with hybrids involving
bread wheat varieties PBW 343 and WH 542 exhibited maximum excisable
embryos with most of the alien genotypes. Verma et al. (1999) also found PBW
343 and WH 542 to respond better than others in wheat x maize crosses which
may be due to the presence of 'Veery' blood in these genotypes.
SUMMAHrAUV CONCLUSION
Summary and Conclusion 72
6. Summary and Conclusion
Wild relatives of wheat are a valuable reservoir of genes which are either
absent in cultivated wheat or their diversity is narrow particularly with respect to
pest resistance, tolerance to biotic and abiotic stresses and various morphological
traits including the end use properties for making bread and pasta as well as other
products. Therefore, the wild species can be used as a potential source to add
novel variability and affecting the genetic enrichment program through wide
crossing. However, in most of the wide crosses the embryo aborts at various
levels of development. Hence, the technique of embryo rescue involving excision
and culture of embryos aseptically had been quite successful in transferring the
useful traits from alien sources to the background of cultivated species.
In lieu of this, an effort was made to standardize a regeneration protocol,
using the cultivars of both T. durum and T. aestivum species of wheat. Later on,
the embryos of the wide crosses were rescued to test the protocol on selected
varieties and media.
The salient results/observations recorded from the present study have
been summarized here under :
> Two varieties each for bread wheat (WH 542 and UP 2338) and durum
wheat (Bijaga Yellow and Gulab) were screened on four callus induction
media CI (MS+2,4-D, 2mg/l), C2 (MS + 2,4-D, 4mg/I), C3 (MS+ 2,4-D,
2mg/l + lAA, 0.5 mg/1) and C4 (B5 + 2,4-D, 2mg/l). The embryos were
excised at 10, 12, 14, 16 and 18 days post pollination/days post anthesis.
12 and 14 days calli of bread wheat and 12, 14 and 16 days old calli of
durum wheat were found optimum. Maximum frequency of callus
induction was recorded on C1 (embryogenic) than on C2 (loose and non-
embryogenic)
Summary and Conclusion 73
> Five media viz., Rl (left on the same medium and exposing to light). R2
(MS + Kinetin. 2 mg/1), R3 (MS + Kinetin, 2mg/l + lAA, 0.5 mg/1), R4
(MS + BAP, 2 mg/1) and R5 (MS +BAP, 2ml/l + lAA, 2mg/l) were tested
to achieve regeneration from calli produced on CI, C2, C3 and C4 media.
> Three media viz., Rl, R2 and R5 were found to give good regeneration in
all the varieties, while R3 and R4 proved quite poor in this regard.
> The regenerated plantlets were grown on three media viz., SI (left on the
same medium where they were induced), S2 (MS + GA3 0.5 mg/1 +30 g/1
sucrose) and S3 (GR free MS + 20 g/1 sucrose) up to 3 to 4 leaf stage.
Among these, the S3 medium was found better than other two media.
> The regenerated plantlets were transferred on a range of rooting media
and the medium containing MS salts without vitamins + NAA^mg/l +
sucrose 20g/l) was found better responding than rest.
> The dark condition during callusing and light during regeneration phase
proved quite effective. Interestingly, the alternate light and dark
treatment using 20 watts electric bulbs (16/8 hrs light/dark) could induce
regeneration in calli produced on MS + 2. 4-D (2mg/l) medium.
> A temperature of 25±2°C was found optimum, while the further rise in
temperature resulted in the browning of callus.
> The plantlets were hardened in small pots filled with agropeat and soilrite
in equal proportions and put into growth chamber at 15±1°C and 90 per
cent relative humidity. Established plants were transferred in bigger pots
containing soil, sand, FYM and agropeat in equal proportions.
> Following the above mentioned protocol, six additional varieties each of
bread wheat (CPAN 3004, KRL 1-4, PBW 343, PBW 373, Raj 3765 and
HD 2329) and durum wheat (HD 4530, HI 8381, Raj 1555, PDW 215,
PDW 233 and MACS 2846) were screened to check both the validity of
protocol and selection of better responding genotypes.
Summary and Conclusion 74
> The age of immature embryo, genotype, media composition and culture
conditions all affected the regeneration potential of the explant.
> The above mentioned protocol was further extended to grow hybrid plants
from the crosses involving wide relatives.
> Five varieities each of bread wheat (WH 542, UP 2338, HD 2329, PBW
343 and Raj 3765) and durum wheat (Bijaga Yellow, Raj 1555, Gulab,
PDW 215 and PDW 233) were taken as female and 20 species of wild
triticum and Aegilops species [T. urartu (A"), T. boeticum (A**), Ae.
speltoides{S), Ae. longissima (S'). Ae. sharonensis{S^^), Ae. bicornis (S ).
Ae. squarrosa (D). Ae. caudata (C), Ae. markgrafii (C), Ae. umhelluluta
(U), Ae. cylindrica (CD), Ae. ventricosa (D"), Ae. kotschyi (US), Ae.
peregrina (US), Ae. triuncialis (UC), Ae. ovata (UM"), Ae. lorentii
(UM' ' ) , Ae. triaristata (UM'), Ae. geniculata{\]M°), T. araraticum{AG)]
were used as pollen parent.
> The hand emasculated spikes were pollinated twice with pollens collected
from the alien species. The pollinated spikes were sprayed with 2, 4-D +
GA3 solution for four consecutive days starting from the day of
emasculation.
> The hybrid embryos were scooped out at 10, 12, 14 and 16 days with
slight variation in some crosses and cultured aseptically following the
above mentioned protocol.
> The embryos rescued after 12 to 14 DPP were found to give optimum
regeneration in most of the crosses.
> The regenerated hybrid plants were established in the field with various
levels of seed settings.
BtBUOGKAPHY
Bibliography
Abdrawov, R.T., Moustafa, R.A.K. (1993). Effect of 2, 4-D concentration and two levels sucrose on callus induction and plantlet formation in two wheat cultivars. Annal. of Agril. Sci. (I), Special Issue, 41-46.
Acosta, A.C. (1963). The transfer of stem rust resistance from rye to wheat. Dissertation Abstracts, 23 : 34-35.
Adachi, T. and Katayama, Y. (1969). Callus formation and shoot differentiation in wheat tissue culture. Bull. Fac. Agric. Univ., Mujazaki, 40 : 77-82
Agnihotri, A.,Shivanna, K.R., Raina, S.N., Lakshmikumaran, M.S., Prakash, S., Jagannathan, V. (1990). Production of Brassiconapus x Raphanobrassica hybrids by embryo rescue : An attempt to introduce shattering resistance into B. Napus, Plant Breeding 105 : 292-299.
Ahloowalia. B.S. (1982). Plant regeneration from callus cultures in wheat. Crop Sci.. 22:405-410.
Almouslem, A.B., Jauhar, P.P., Peterson, T.S. Bommineni, V.R. and Rao. M.B. (1998). Haploid durum wheat production via hybridization with maize. Crop Sci., 38 (4): 1080-1087.
Anshulika, L., Singh, N. and Chawla, H.S. (1999). Regeneration response from different explants of high yielding genotypes of wheat (Triticum aestivum L.). Crop Imp., 26(1): 5-9.
Arun, B., Tiwari, K.N., Singh, B.D. and Dari, R.S.O. (1994). High frequency embr>'ogenesis in some elite Indian cultivars of wheat, Triticum aestivum L. em Thell. Ind. J. of Expe. Biol., 32 (11): 835-837.
Arzani A. and Mirodjagh, S.S. (1999). Response of durum wheat cultivars to immature embryo culture, callus induction and in vitro salt stress. PI. Cell, Tiss. Org. Cult., 58(1): 67-72.
Bajaj, Y.P.S. (1985). Somalonal variation and the cryopreservation of germplasm in wheat. Am. J. Bot., 72(6): 874.
Bajaj, Y.P.S. (1990). In vitro production of Triticale {Triticum x Secale) through embryo culture. In : Bajaj, Y.P.S. (ed.) Biotechnology in Agriculture and Forestry, Vol (3): Wheat. Springer Warlag Pub, Berlin, pp 218-228.
Balyan, H.S and Fedak, G. (1991). A study on genome relationships in Hordedum jubatum, Elymus trachycaulus and Elymus Canadensis. Cytologia, 3 : 4310436.
Bibliography [\
Bapat, S.A., Joshi, C.P. and Mascarenhas, A.F. (1988). Occurrence and frequency of precocious germination of somatic embryos is a genotype-dependent phenomenon in wheat. Plant Cell Rep., 7 : 538-541.
Bariana, H.N., DriscoU, C.J., Long, P.M. and Vickery, R.S. (1969). Gene similarity of the trichinae and the study of segmental interchanges. Nature, 222 : 897-898.
Bariana, H.S. and Mcintosh, R.A. (1993). Cytogenetic studies in wheat XV. Chromosome location of rust resistance genes in VPMl. Genome 36 : 476-482.
Barkat. M.N. and Abd-el-latif, T.H. (1995). Somatic embryogenesis and plant regeneration in callus from mature and immature embryo cultures of wheat. Alexandria J. Agri. Res., 40 : 113-129.
Becker, D., Brettschneider, R. and Lorz, H.(1994). Fertile transgenic wheat from microprojectile bombardment of scuteller tissue. Plant J., 5 : 299-307.
Ben Amer, I.M.. Worland, A.J., Schlegel, R. (1992). In vitro culture variation of wheat and rye caused by genes affecting plant growth habit in vivo. Euphytica, 61 : 233-240.
Benkirane, H.. Sabounji, K., Chlyah, A. and Chlyah, H. (2000). Somatic embryogenesis and plant regeneration from fragments of immature inflourescences and coleoptile of durum wheat. PI. Cell Tiss. Org. Cult., 61(2): 107-113.
Bennici, A., Caffari, L., Dameri, R.M., Gastaldo, P. and Profumo, P. (1988). Callus formation and plant regeneration from immature Triticum durum Desf embryos. Euphytica, 39 : 255-263.
Bhowal, J.G.,Guha, G. and Vari, A.K. (1993). A cytogenetic study to transfer disease resistance genes from Triticum timopheevi to bread wheat. J. genet. Breed. 47 : 71.76.
Bohorova, N.E., Ginkel, M. Van., Rajaram, S., Hoisington, D.A. and Van Ginkel, M. (1995). Tissue culture response of CIMMYT elite bread wheat cultivars and evaluation of regenerated plants. Cereal Res. Commun., 23(3) :243-249.
Bommineni, V.R., Jauhar, P.P., Peterson, T.S., Chibbar, R.N. and Almouslem, A.B. (1997). Analysis of hybrids of durum wheat with Thinopyrum junceiforme using RAPD markers. Theor. Appl. Genet., 95 : 757-763.
Bregitzer, P., Robert, D., Campbell and Ying, Wu. (1995). Plant regeneration from barley callus : Effects of 2, 4-dichlorophenoxyacetic acid and phenylacetic acid. PI. Cell, Tiss. Org. Cuh., 43 : 229-235.
Bibliography [[[
Browder, L.E. (1973)a. Probable genotype of some Triticum aestivum agent derivatives for reaction to Puccinia recondita f sp. tritici. Crop Science, 13 : 203-206.
Browder, L.E. (1973)b. Specificity of the Puccinia recondita f. sp. tritici : Triticum aestivum "Bulgaria 89" relationship. Phytopathology, 63 : 524-528.
Brown. C . Brooks, F.J., Pearson, D. and Mathias. R.J. (1989). Control of embryogenesis and organogenesis in immature wheat embryo callus using increased medium osmolarity and abscisic acid., J. PI. Physiol., 133 : 727-733.
Butenko, R.G. and Dzhardemaliev, K. (1986). Callus forming ability in explants of different organs of winter wheat varieties. So v. Plant Physiol.. 33 : 277-281.
Carman, J.G. (1989). The in Ovulo environment and its relevance to cloning wheat via somatic embryogenesis. In vitro Cell. Deve. Biol., 25(12) : 1155-1162.
Carman, J.G., Jefferson, N.E. and Campbell, W.F. (1987). Induction of embryogenic Triticum aestivum L. calli. I. Quantification of genotype and culture medium effects. PI. Cell Tiss. Org. Cult, 10:101-113.
Carman, J.G., Jefferson, N.E. and Campbell, W.F. (1988). Induction of embryogenic Triticum aestivum L. calli. II. Qualification of organic addenda and other culture variable effects. PI. Cell Tiss. Org. Cult., 12 : 97-110.
Ceoloni (1987). Current methods of chromosome engineering in wheat. EWAC Newlt.,95 : 1-9.
Ceoloni, C , Donin, P. and Ercoli, L. (1993). Chromosome engineering for durum wheat improvement. In Damania, A.B. (ed.). Biodiversity and Wheat Improvement @ ICARDA, A Wiley-Sayce Publ. pp : 39-47.
Chauhan, R.S. and Singh, B.M. (1995). Induction of somaclonal variants from different explants of bread wheat for resistance to Kamal Bunt (Tilletia indica). In Proc. Indian Nat. Sci. Acad. B61, 6 : 479-486.
Chawla, H.S. 1989. Regeneration responses of callus from different explants and changes in isozymes during morphogenesis in wheat. Biol. Plant., 31(2): 121-125.
Cherkaoui, S., Lamsaouri, O., chIyah,A., Chlyah, H. (2000). Durum wheat x maize crosses for haploid wheat production : Influence of parental genotypes and various experimental factors. Plant Breeding, 119(1) : 31-36.
Bibliography iv
Chin, J.C. and Scott, K.J. (1977). Studies on the formation of roots and shoots in wheat callus cultures. Ann. Bot. (London) 41 : 473-481.
Chu, C.C, Sun, C.S., Chen, X., Zhoy, W.X and Du, Z.H. (1984). Somatic embryogenesis and plant regeneration in callus from inflorescences of Hordeum vulgare x Triticum aestivum hybrids. Theor. Appl. Genet., 68 : 375-379.
Chwala, H.S. and Wenzel, G. (1987). Regeneration potential of callus from wheat and barley. Archi fur Zuchtungsforschung, 17(6): 337-343.
Cistue. Ramos, A and Castillo. A.M. (1999). Influence of anther pretreatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars. PI. Cell, Tiss. Org. Cult., 55: 159-166.
Claesson, L., Koitimaki, M. and Von Bothmer, R. (1990). Crossabihty and chromosome pairing in some interspecific Triticum hybrids. Hereditas, 112:49-55.
Collins. G.B. and Grosser, J.W. (1984). Culture of embryos. In Vasil l.K. (ed) : Cell Culture and Somatic Cell Genetics of Plants. Vol. I : Laboratory Procedures and Their Applications. Academic Press, New York, pp 241-257.
Collins G.B.. Taylor, N.L.. and Deverna, J.W. (1993). In vitro approaches to interspecific hybridization and chromosome manipulation in crop plants. In : Gustafsan P. (ed). Gene manipulation in Crop Improvement., pp 323-383.
Comaau, A., Plourde, A., St. Pierae, C.A. and nadeau, P. (1988). Produciton of double haploid wheat lines by wheat x maize hybridization. Genome, 30 : 1,482.
De Buyser, J., Harl-mann, C , Heru-y, Y. and Rode, A. (1988). Variation in long-term wheat somatic tissue culture. Can. J. Bot., 66 : 1891-1895.
Dhaliwal, H.S. and Gill, K.S. (1982). Screening and utilization of wild-wheat germplasm for rust resistance. Wheat Inf Serv., 54 : 39-42.
Dhaliwal, H.S., Singh, H., Gill, K.S. and Randhawa, H.S. (1993). Evaluation and cataloguing of wheat germplasm for disease resistance and quality. In Damania, A.B. (ed). Biodiversity and Wheat Improvement. A Wiley-Sayce Publication, UK, pp 123-140.
Djenadi C. (2000). Obtaining embryos by interspecific crossing between durum wheat and Aegilops. In Royo et al. (ed) : Durum Wheat Improvement in the Mediterranean Region : New Challenges. Proc. Zaragoza, Spain. 40 : 271-273.
Dolezel, J., Novak, F.J. and Luzny, J. (1980). Embryo development and in vitro culture of Allium cepa and its interspecific hybrids. Z. Pflanzenzuecht. 85: 177-184.
bibliography y
Dvorak, J. and Zhang, H.B. (1990). Variation in repeated nucleotide sequences sheds light on the phylogeny of the wheat B and G genomes. Proc. Natl. Acad. Sci., USA, 87 : 9640-9644.
Dyck, P.L. and Kerber, E.R. (1970). Inheritance in hexaploid wheat of adult-plant leaf rust resistance derived from Aegilops squarrosa. Canadian Journal of Genetics and Cytology, 12 : 175-180.
Eapen. S. and Rao, P.S. (1985). Factors controlling callus initiation, growth and plant regeneration in bread wheat (Triricum aestivum L.) Proc. Ind. Acad. Sci.. Plant Sci.. 94(1)-.33-40.
Endo. T.R. (1979). On the Aegilops chromosome having gametocidal action on common wheat. In : (Ramanujam S. ed.) Proc. 5' Int. Wheat Genet. Synip.. New Delhi, 1978 : 306-314.
Endo. T.R. (1988). Induction of chromosome structural changes by a chromosome of Aegilops cylindrica L. in common wheat. Journal of Heredity. 79 : 366-370.
Farooq, M.. IhsanuUah, Rashid, H., Quaraishi. A. and Marwat, K.B. (2001). Comparative tissue culture response of wheat cultivars and evaluation of regenerated plant. PI. Tis. Cuh.. 11(2): 181-186.
Fedak. G. (1986). Hordecale {Hordeiim vulgare x Secale cereale L.). In Bajaj Y.P.S. (ed.). Biotechnology in Agriculture and Forestry 2 : Crops : Spinger Verlag, Berlin, pp 544-555.
Feldman, M. and Sears, E.R. (1981). The wild gene resources of wheat. Sci. Am., 244: 102-112.
Felsenburg, T., Teldman, M. and Galun. E. (1987). Theor Appl. Genet. 74 : 802-810.*
Fennel, S., Bohorova, N. Ginkel, M.Van, Crossa, J. and Hoisington, D. (1996). Plant regeneration from immature embryos of 48 elite CIMMYT bread wheats. Theor Appl. Genet., 92 : 163-169.
Franzone, P.M., Rios, R.D., Procopiuk,A.M.. Diaz, D.G., Robredo, C.G. and Aguinaga, A. (1998). Genetic analysis of in vitro regeneration in wheat {Triticum aestivum L.). J. Genet. Breed., 52(3): 195-201.
Friebe, B., Jiang, J.M., Raupp, W.J., Mcintosh, R.A. and Gill, B.S. (1996). Characterization of wheat-alien translocations conferring resistance to diseases and pests : current status, Euphytica, 91 : 59-87.
Gale, M.D. and Miller, T.E. (1987). The introduction of alien genetic variation in wheat improvement. In : Lupron (ed). Wheat Breeding : Its Scientific Basis- Chapman and Hall, London, 173-210.
Bibliography v i
Galiba, G. and Yamada, Y. (1988). A noval method for increasing the frequency of somatic embryogenesis in wheat tissue cultures by NaCl and KCl supplementation. Plant. Cell. Rep., 7 : 55-58.
Gamborg, O.L., Miller,R.A. and Ojima, K. (1968). Nutritional requirements of suspension cultures of soyabean root cells. Exp. Cell Res., 50 : 151-158.
Ganeva, G., Karadimova, M., Bocheva, N., Zozikova, E., Djambova, G. and Bochev, B. (1995). Effects of Hordeum vulgare L. 2H and of second homoeologous Triticum aestiviim L. group chromosome on in vitro plant response to exogenous auxins. Cer. Res. Commn., 23 (1-2): 35-43.
Gaponeko, A.K., Muntyan, M.A.. Malikova. N.l. and Sozinov, A.A. (1985b). Regeneration of plants of the wheat, Triticum aestivum L. in vitro. Tsitologiya-i-Genetika, 19(5): 335-342.
Gaponenko, A.K., Malikova, N.I.. Okhrimenko. G.N. and Sozinov, A.A. (1985a). Producing somaclonal lines in cereals {Triticum aestivum L. and Hordeum vulgare L.). Doklady Akademii Nauk, SSSR, 283(6) : 1471-1475.
Gerechter-Amitai, Z.K., Van Silfliout. C.H., Grana, A. and Kleitman, F. (1989). Yr 15 - A new gene for resistance to Puccinia Striiformis in Triticum dicoccoides sel. G-25, Euphytica, 43 : 187-190.
Gill, B.S., Browder, L.E., Hatchelt, J.H., Harvely, T.L., Martin, T.J., Raupp, W.J., Sharma, H.C. and Waines, J.G. (1983). Disease and insect resistance in wild wheats. Proc. 6"' Int. Wheat Genet. Symp. Kyoto, Japan, pp 785-792.
Gill, B.S., Waines, J.G. and Sharma, H.C. (1981). Endosperm abortion and the production of viable Aegilops squarrosa x Triticum boeticum hybrids by embryo culture. Plant Sci. Lett. 23 : 181-187.
Gosch-Wackerle, G., Avivi, L. and Galun, E. (1979). Induction, culture and differentiation of callus from immatvire rachis, seeds and embryos of Triticum. Z. Pflanzenphysiol., 91 : 267-278.
Gyarfas, J. (1978). Transference of disease resistance from Triticum timopheevii to Triticum aestivum. M.Sc. Ag. Thesis, University of Sydney, Australia.
Haccius, B. (1978). Question of unicellular origin of non-zygotic embryos in callus cultures. Phytomorphology, 28 : 74-81.
Hanson, H., Borlaug, N.E. and Anderson, R.G. (1982). Wheat in the third world. West view Press, Boulder, Colorado.
Hare, R.A. and Mcintosh, R.A. (1979). Genetic and cytogenetic studies of durable adult-plant resistances in 'Hope' and related cultivars to wheat rusts. Zeitschrift fur Pfanzenzuchtung, 83 : 350-367.
Bibliography vii
Harlen, J.R. (1976). Genetic resources in wild relatives of crops. Crop Sci., 16 : - » n n ^ *» *»
He D.G. (1989). Embryo and protoplast culture of wheat {Triticum spp.) Ph.D. Thesis. The Queensland University.
He, D.G., Tanner, G. and Scott, K.J. (1986). Somatic embryogenesis and morphogenesis in callus derived from the epiblast of immature embryos of wheat {Triticum aestivum) Plant Sci.. 45 : 119-124.
He. D.G.. Yang, Y.M. and Scott, K.J. (1989). The effect of macroelements in the induction of embryogenic callus from immature embryos of wheat {Triticum aestivum L.) Plant Sci., 65.
He. D.G., Yang, Y.M., Dahler, G. and Scott. K.J. (1988). A comparison of epiblast callus and scutellum callus induction in wheat : The effect of embryo age. genotype and medium. Plant Sci.. 57 : 225-233.
He. D.G.. Yang. Y.M., Scott, K.J. (1991). Zinc deficiency and the formation of white structures in immature embryo cultures of wheat {Triticum aestivum L.)
Henry. Y. and De Buyser, J. (1990). Wheat Anther Culture. In, Bajaj Y.P.S. (ed.) Biotechnology in Agriculture and Forestry, Vol. 13 , Wheat. Springer Verlag, Berlin, Heidelberg, pp. 285-350.
Heun, M. and Friebe, B (1989). Introgression of powdery mildew resistance from rye into wheat. Phytopathology, 80 : 242-245.
Heyser. J.W.. Nabros, MU, Mackinnon, C, Dykes, T.A. Dmott, D.C. Kantzmann, D.C. and Mujeeb-Kazi. A. (1985). Long term, high frequency plant regeneration and the induction of somatic embryogenesis in callus cultures of wheat {Triticum aestivum L.). Z. Pflanzenziicht, 94 : 218-233.
Higashi, K., Kamada, Hiroshi, and Harada, H. (1996). The effects of redcued nitrogenous compounds suggests that glutamine synthetase activity is involved in the development of somatic embryos in carrot. PI. Cell, Tiss. Org. Cult., 45 : 109-114.
Higgins, P. and Mathias, R.J. (1987). The effect of the 4B chromosomes of hexaploid wheat on the growth and regeneration of callus cultures. Theor. Appl. Genet., 74 : 439-444.
Huber, M., Hohn, R. and Hess, D. (2002). High transformation frequencies obtained from a commercial wheat {Triticum aestivum L. cv 'Combi') by microbombartmetn of immature embryos followed by GFP screening combined with PPT selection. Mole. Breed., 10 (1-2): 19-30.
Hunsinger, H. and Schatuz, K. (1987). The influence of dicamba on somatic embryogenesis and the frequency of plant regeneration from cultured
Bibliography \,[\\
immature embryos of wheat (Triticum aestivum L.). Plant Breed., 98 : 119-123.
Huskins, C.L. (1931). J. Genet.. 25 : 113-124.*
Hussain, M.T. and Islam, A. 1993. In vitro plant regeneration in triticale and wheat. Bangladesh J. Scientific and Industrial Res., 28(4): 17-24.
Immonen, A.S.T. (1996). Influence of media and growth regulators on somatic embryogenesis and plant regeneration for production of primary triticales. PI. Cell Tiss. Org. Cuh., 44 : 45-52.
Immonen, A.S.T. (1996). Influence of media and growth regulators on somatic embryogenesis and plant regeneration for production of primary triticales. PI. Cell, Tiss. Org. Cult., 44 : 45-52.
Inagaki, M. (1986). Callus induction and plant regeneration from immature haploid embryos of wheat. Jpn. J. Breed. 36 : 49-53.
Inagaki, M. and Mujeeb-Kazi, A. (1995). Comparison of polyhaploid production frequencies in crosses of hexaploid wheat with maize, pearl millet and sorghum. Breed. Sci., 161 :157-161.
Inagaki. M.N. (1998). Production of wheat haploids using wide crosses and its application to breeding programs. Pro. 9" Int. Genet. Symp., Saskatoon, Saskatchewan, Canada, 2-7 August, 1998.
Inagaki, M.N. and Tahir, M. (1990a). Effect of silver nitrate on callus induction and plant regeneration in wheat. Rahis. 19 : 19-20.
Inagaki, M.N. and Tahir, M. (1990b). Comparison of haploid production frequencies in wheat varieties crossed with Hordeum bulbosum L. and maize. Japan J. Breed.40 : 209-216.
Inagaki, Nagamine, T. and Mujeeb Kazi A. (1987), Use of pollen storage and detached tiller culture in wheat polyhaploid production through wide crosses. Creal Res. Commun., 25 ( I ) : 7-13
Ivanov, P. Zhivko, A., Venetzyia, M. and Ludmila, N. (1998). Culture selected somaclonal variation in fine Triticum aestivum L. genotypes. Euphytica, 104: 167-172.
J'Aiti, P., Benlhabib, O., Sharma, H.C., El-Jaafari, S and El-Hadrami, I. (1999). Genotypic variation in anther culture and effect of ovary coculture in durum wheat. PI. Cell, Tiss. Org. Cult., 59 (1): 71-76.
Jauhar, P.P. (1991) Hybrid between durum wheat and diploid Thinopyrum bessarabicum. Genome, 34 : 283-287.
Jauhar, P.P. (1991). Hybrid between durum wheat and diploid Thinopyrum bessarabicum. Genome, 34(2): 283-287
Bibliography ix
Jauhar. P.P. (1993). Alien gene transfer and genetic enrichment of bread wheat. In : Damania AB (ed.) Biodiversity and wheat improvement. John Wiley & Sons, Chichester, U.K., pp 103-119.
Jauhar. P.P. (1993). Alien gene transfer and genetic enrichment of bread wheat. In : Damania, A.B. (ed). Biodiversity and wheat improvement. John Wiley and Sons, Chichester, pp 103-119.
Jauhar. P.P. (1995). Meiosis and fertility of Fi hybrids between hexaploid bread wheat and decaploid tall wheat grass {Thinopyrum ponticum). Theor. Appl. Genet., 90:865-871.
Jauhar. P.P. and Joppa (1996). Chromosome, pairing as a tool in genome analysis, merits and limitations. In Jauher (ed) Methods of Genome Analysis in Plants, CRC Press, Boca Raton. FL, pp 9-37.
Jauhar. P.P. and Peterson, T.S. (1996). Thinopyrum and Lophopyrum as sources of genes for wheat improvement. Cereal Res. Commun., 24 : 15-21.
Jauhar. P.P.. Almouslem, A.B., Peterson, T.S. and Joppa, L.R. (1999) Inter and intragenomic chromosome pairing in haploids of durum wheat. The American Genetic Association, 90 : 437-445.
Jia, J.F. and Zhao, X.S. (1993). In vitro selection of wheat cell lines tolerant to NaCl and study on their biochemical characteristics. In, Li, Z.S. and Xin, Z.Y. (eds.) Proc. 9" Int. wheat Genet. Symp., Beijing, China. Vol. -2 T071-1072.
Jiang. J., Friebe, B. and Gill, B.S. (1994). Recent advances in alien gene transfers in wheat. Euphytica, 73 : 199-212.
Kaleilau, E.K.. Sears, R.G. and Gill, B.S. (1989). Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.) Theor. Appl. Genet. 78 : 625-632.
Karaca. O. and Burun, B. (1999). Callus development in embryo cultures of wheats. Turkish J. Agric. For., 23(2): 269-274.
Karadimova, M., Dorosiov, L., Gianeva, G., Bochev, B. and Dorossiev, L. (1985). Regeneration of platns from callus tissue of Triticum aestivum L. Genetika-i-Selektsiya, 18(2): 157-162.
Karan. L.C., Nick, L.L. and Ravindra, N.C. (2000). An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars. PI. Cell Tiss. Org. Cult., 32 : 77-82.
Kema, G.H.J, and Lange, W. (1992). Resistance in spelt wheat to yellow rust II. Monosomic analysis of the Iranian accession, 415. Euphytica, 63 : 219-224.
Kibirige-Sebunya I and Knott, D.R. (1983). Transfer of stem rust resistance to wheat from an Agropyron chromosome having a gametocidal effect. Can. J. Genet. Cytol., 25 : 215-221.
Kihara, K. (1924). Cytologische and genetische studien bei wichtigen Getreidearten mit besonderer Rucksicht auf das verhalten der Chromosomen und die Sterilitat in den Bastarden. Mem. Coll. Sci. Kyoto. Imp. Univ. Serv.. Bl : 1-200.
Kihara, K. (1994). Die Entdeckung der DD-Analysa-toren beim weizen. Agric. Hortic (Tokyo) 19:889-890.
Kim, N.S., Armstrong, K. and Knott, D.R. (1993). Molecular detection of Lophopyrum chromation in wheat -Lophopyrum recombinants and their use in the physical mapping of chromosome 7D. Theo. Appl. Genet., 85 : 561-567.
Kiniber, G. (1993). Genomic relationships in Triticum and the availability of alien germplasm. In : Damania A.B. (ed.). Biodiversity and Wheat Improvement, A Wiley-Sayce Publication. ICARDA, pp 9-16.
Kimber, G. and Feldman, M. (1987). Wild wheat : an introduction. Coll. Agric. Univ. Missouri. Col. Spec. Rep. 353.
Knott, D.R. (1962). The inheritance of rust resistance IX. The inheritance of resistance to races 15B and 56 of stem rust in the wheat variety Khapstein. Canadian J. of PI. Sci., 42 : 415-419.
Koba, T. and Shimada, T. (1993). Crossability of common wheat with Aegilops squarrosa. Wheat Information Service, 77 : 7-12.
Koba. T., Handa, T. and Shimada, T. (1991). Efficient production of wheat, barley hybrids and preferential elimination of barley chromosomes. Theor. Appl. Genet., 81 : 285-292.
Kolmer, J.A. (1997). Virulence in Puccinia recondita f. sp. tritici isolates from Canada to genes for adult. 78 ; 600-602.
Kothari, S.L., Varshney, A. and Varshney, A. (1998). Morphogenesis in long-term maintained immature embryo-derived callus of wheat {Triticum aestivum L.) - histological evidence for both somatic embryogenesis and organogenesis. J. PI. Biochem. Biotech., 7(2): 93-98.
Kruse, A. (1967). Intergeneric hybrids between Hordeum vulgare L. ssp. distichum (v Pallas 2n=14) and Secale cereale L. (V Petkus 2n=I4). In : Royal Veterinary and Agricultural College Year Book 1967, Copenhagen Den., pp 82-92.
Kruse, A. (1969). Intergeneric hybrids between Triticum aestivum L. (v. Koga 11 12n=42) and Avena sativa L. (v sat 2n=42) with pseudogamous seed
formation. In : Royal Veterinary and Agricultural College Year Book 1967, Copenhagen Den., pp 188-200.
Kruse, A. (1973). Hordeum x Triticum hybrids. Hereditas, 73 : 157-161.
Laibach, F. (1925). Das Taubwerden von Bastardsmen und die Kunstaiche Aufzucht fruh absterbendet Bastardembryonen, Z. Bot., 17 : 417-459.
Lange, C.E., Federizzi, L.C., Carvalho, F.I.F.. Tavares, M.J.C., Dornelles, A.L.C. and Handel, C.L. (1995). Genetic analysis of somatic embryogenesis and plant regeneration of wheat {Trilicum aestivum L.). J. Genet. Breed., 49 : 195-200.
Lange, W. and Balkema-Boomstra, A.G. (1988). The use of wild species in breeding barley and wheat, with special reference to the progenitors of cultivated species. In. Jorna, M.L. and Slootmaker, L.A.J, (eds). Cereal Breeding Related to Integrated Cereal Production. Pudoc, Wegeninger, 157-178.
Langridge, P., Lazzeri, P. and Lorz, H. (1991). A segment of rye chromosome 1 enhances growth and embryogenesis of calli derived from immature embryos of wheat. Plant Cell Rep., 10(3): 148-151.
Lapitan, N.L.V., Sears, R.G. and Gill, B.S. (1984). Translocations and other karyotypic structural changes in wheat x rye hybrids regenerated from tissue culture. Theor. Appl. Genet., 68 : 547-554.
Lapitan, N.L.V., Sears, R.G., Rayburn, A.L. and Gill, B.S. (1986). Wheat-rye translocations : Detection of chromosome breakpoints by in situ hybridization with a biotin-labeled DNA probe. J. Heredity, 77 : 415-19.
Larkin, P.J., Ryan, S.A., Brettell, R.I.S. and Scowcroft, W.R. (1984). Heritable somaclonal variation in wheat. Theor. Appl. Genet., 67 : 443-456.
Laurie, D.A. and Bennett, M.D. (1986). Wheat x maize hybridization. Can. J. Genet. Cytol., 28 : 313-316.
Laurie, D.A., O'Donoughue, L.S. and Bennett, M.D. (1990). Wheat x maize and other wide sexual hybrids : their potential for genetic manipulation and crop improvement. In : Gustafson, J.P. (ed). Gene Manipulation in Plant Improvement-II. Proc. 19"" Stadl. Genet. Symp. Columbia, pp 95-126.
Lazar, M.D. (1981). Studies of somatic cell culture of wheats {Triticum spp.) Agron. Abs., 73: 65.
Lazer, M.D., Chen, T.H.H., Scoles, G.J. and Kartha, K.K. (1987). Immature embryo and anther culture of chromosome addition lines of rye in Chinese Spring wheat. Plant Sci., 51(1): 77-81.
Bibliography xii
Lazer, M.D., Collins, G.B. and Vian, W.E. (1983). Genetic and environmental effects on the growth and differentiation of wheat somatic cell cultures. J. Hered., 74(5): 353-357.
Lea, P.J., Hughes, J.S. and Miflin, B.J. (1978). Glutamine and asparagine-dependent protein system in maturing legume cotyledons cultured in vitro. J. Exp. Hot., 30 : 529-537.
Lein, A. (1943). The genetical basis of the crossability between wheat and rye. Z. Indukt. Abstamn. Vererbungl. 81 : 28-59.
Liang, Z.Q., Gao, M.W. and Chang, X.Y. (1988). Study of in vitro technique from immature embryo culture of wheat breeding. Acta Agronomica Sinica, 14(2): 137-142.
Liu, G.Z., Gao, M.W., Cheng, X.Y., Liang, Z.Q. and Hu, T.C. (1993). Increasing variation in common wheat through culture of immature embryos and mutagenesis. Jr. Hebei-Agric. Univ., 16(2) : 46-50.
Lu, B.R. and Von Bothmer, R. (199o) Production and cytogenetic analysis of the intergeneric hybrids between nine Elymns species and common wheat (Triticum aestivum L.). Euphytica, 58 : 81-95.
Luig N.H. and Watson, LA. (1967). Vernstein - a Triticum aestivum derivative with Vernal emnier type stem rust resistance. Crop Science, 1 : 31-33.
Macer, R.C.F. (1966). The formal and monosomic genetic analysis of stripe rust (Puccinia striiformis) resistance in wheat. Proceedings of the 2"'' International Wheat Genetics Symposium Lund. Sweeden 1963 (MocKey J. ed.): Hereditas Suppliment, 2 : 127-142.
Machii, H., Mizuho, H., Hirabayashi, H., Li, H. and Hagio, T. (1998). Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures. PI. Cell, Tiss. Cult., 53 : 67-74.
Maddock, S.E. (1985). Cell culture, somatic embryogenesis and plant regeneration in wheat, barley, oats, rye and triticale. In Bright, S.W.J, and Jones, M.G.K. (eds). Cereal Tissue and Cell Culture. Nijhoff/Junk. Dordrecht Boston Lancaster, pp 131-174.
Maddock, S.E., Lancaster, V.A., Risiott, R. and Franklin, J. (1983). Plant regeneration for cultured immature embryos and inflorescences of 25 cuhivars of wheat {Triticum aestivum). J. Exp. Bot., 34 : 915-926.
Magnusion, I. and Bornman, L.H. (1985). Anatomical observations of somatic embryogenesis from scutellar tissue of immature zygotic embryos of Triticum aestivum. Physiol. Plant, 63 : 137-145.
Bibliography \,\\\
Maheshwari, P. and Rangaswamy, N.S. (1965). Embryology in relation to physiology and genetics. In.: Preston R.D (ed), Advances in Botanical Research. Vol.2. Academic Press, New York, pp 219-321.
Malhotra, P.K., Singh, B., Gupta, V.K., Bains, N.S. and Dhaliwal, H.S. (1999). Response to callus induction and rapid plant regeneration in elite wheat varieties of Punjab. Crop. Improv., 26(2): 141-145.
Marais, G.F. and Marais, A.S. (1994). The derivation of compensating translocation involving homoeologous group 3 chromosome of whet and rye. Euphytica, 79 : 75-80.
Mathias, R.J. (1990). Factors affecting the establishment of callus cultures in wheat. In : Bajaj. Y.P.S. (ed.). Biotechnology in Agriculture and Forestry. Vol. 13. Wheat. Springer and Varlag, Berlin, Heidelberg, 24-45.
Mathias, R.J. and Atkinson. E. (1987). In vitro expression of genes affecting whole plant phenotype-the effect of Rlit/GAi alleles on the callus culture response of wheat {Triticum aestivum L. em. Thell.). Theor. Appl. Genet.. 75 : 474-479.
Mathias, R.J. and Boyd, L.A. (1986). Cefotraxime stimulates callus growth, embryogenesis and regeneration in hexaploid wheat {Triticum aestivum L. em. Thell). Plant Sci.. 46 : 217-223.
Mathias, R.J. and Fukui, K. (1986). The effect of specific chromosome and cytoplasm substitutions on the tissue culture response of wheat {Triticum aestivumlL. em. Thell) callus. Theor. Appl. Genet., 71 : 797-800.
Mathias, R.J., and Simpson, E.S. (1986). The interaction of genotype and culture medium on the tissue culture response of wheat. {T. aestivum L. em. Thell) callus. PI. Cell. Tiss. Org. CuU., 7 : 31-37.
Mathias, R.J.. Fukui, K. and Law, C.N. (1986). Cytoplasmic effects on tissue culture response of wheat {Triticum aestivum) callus. Theor. Appl. Genet.. 72 : 70-74.
Mathias, R.J., Higgins, P., Atkinson, E., Miller, T.E. (1988). Genetic control of wheat {Triticum aestivum) tissue culture response. In : Koebner, R.M.D. (ed). Proc. Of the VII Int. Wheat Genet. Symp., Cambridge, U.K, pp 7.
McFadden, E.S. and Sears, E. (1944). The artificial synthesis of Triticum spelta. Rec. Genet. Soc. Am., 13 : 26-21.
Mcintosh, R.A. and Gyarfas, J. (1971). Triticum timopheevi as a source of resistance to wheat stem rust. Zeitschrift fur plfanzenzuchtung, 66 : 240-248.
Mcintosh, R.A., Dyck, P.L. and Green, G.J. (1977). Inheritance of reaction to stem rust and leaf rust in the wheat cultivar. Etoile de Choisy, Can. J. Genet. Cytol., 16:571-577.
Bibliography x i v
Mcintosh, R.A., Miller, T.E. and Capman, V. 1982. Cytogenetical studies in wheat XII. Lr 28 for resistance to Puccinia recondita and Sr 34 for resistance to P. Graninis tritici. Zeitschrift fur Pflanzenzuchtung, 89 : 295-306.
Merker, A. 1992. The Triticeae in cereal breeding. Hereditas, 116:277-280.
Mettin, D., Bwtlher, W.D. and Weinrich, M. (1978). Studies on the nature and the possible origin of the spontaneously translocated 1B-1R chromosome in wheat. Wheat Information Service. 47 : 12-16.
Mok, D.W.S., Mok, M.C. and Rabak-Oarihanta, A. (1978). Inter specific hybridization of Phaseolous vulgaris with P. lunatus and P. acutifolius. Theor. Appl. Genet., 52 : 209-215.
Monika, Dalai, Lakshmi, K.V.S.V., Khanna. Chopra, R., Bharti,S. (1999). Ear culture as a technique to overcome hybrid necrosis in wheat. PI. Cell. Tiss. Org. Cult., 59 (2): 151-154.
Monnier. M. (1978). Culture of zygotic embryos. IN : Thorpe, T.A. (ed.) Frontiers of Plant Tissue Culture. Univ. Calgary Press, Calgary, pp 277-286.
Morris, P.C, Maddock, S.E., Jones, M.G.K. and Bowles, D.J. (1986). Lectin levels in tissues of cultured immature wheat embryos. Plant Cell Rep., 5 : 460-463.
Moseman, J.G., Nevo, E., Gerechter-Amitai, Z.K., El-Moshidy, M.A. and Johary. D. (1985). Resistance of Triticum dicoccoides collected in Israel to infection with Puccinia recondite tritici. Crop. Sci., 25 : 262-265.
Mujeeb-Kazi A. and Kimber, G. (1985). The production, cytology and practicality of wide hybrids in the Triticeae. Cereal Commun., 13 : 111-124.
Mujeeb-Kazi and Astedu, R. (1990). Wide hybridization-potential of alien genetic transfers for Triticum aestivum Improvement. Bioteechnology in Agriculture and Forestry : Wheat (ed) Y.P.S. Bajaj. 112-128.
Mujeeb-Kazi, A. and Asiedu, R. (1990). Wide hybridization -Potential of alien genetic transfer for Triticum aestivum improvement. In : Bajaj, Y.P.S. (ed.), Biotechnology in Agriculture and Forestry, Vol. 13 : Wheat, Springer-Verlag, Berlin, pp: 111-127.
Mujeeb-Kazi, A. and Asiedu, R. (1995). The potential of wide hybridization in wheat improvement. Annals of Bio., 11(1): 1-15.
Mujeeb-Kazi, A., Roldan, S., Sun, D.Y., Sitch, L.A. and Farooq, S. (1987). Production and cytogenetic analysis of hybrids between Triticum aestivum and some caespitose Agropyron species. Genome, 29 : 537-553.
XV
Murashige, T. and Scoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15 : 473-497.
Nikova, V.M., Zagorska, N.A. (1990). Overcoming hybrid incompatibility between Nieotiana Africana Merxm. and N. tabacum and development of cytoplasmically male sterile tobacco forms. PI. Cell, Tiss. Org. Cult., 23 : 71-75.
O'Donoughue, L.S., Bennelt, M.D. (1994). Comparative response of tetraploid wheats pollinated with Zea mays L. and Hordeum bulbosum L.. Theor. Appl. Genet. 87 : 673-680.
O'Hara. J.F. and Street, H.E. (1978). Wheat callus culture : The initiation, growth and organogenesis of callus derived from various explant sources. Ann. Bot. (London), 42 : 1029-1038.
Ogihara, Y. and Tsunewaki, K. (1988). Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis. Theor Appl. Genet. 76 : 321-332.
Ou, G., Wang, W.C. and Nguyen, H.T. (1989). Inheritance of somatic embryogenesis and organ regeneration from immature embryo cultures of winter wheat. Theo. Appl. Genet., 78(1): 137-142.
Ozias-Akins, P. and Vasil, I.K. (1982). Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (Wheat) : Evidence for somatic embryogenesis, Protoplasma, 110: 95-105.
Ozias-Akins, P. and Vasil, I.K. (1983a). Callus induction and growth from the mature embryo of Triticum aestivum (wheat). Protoplasm, 115: 104-113.
Ozias-Akins, P. and Vasil, I.K. (1983b). Proliferation of 2"** plant regeneration from epiblast of Triticum aestivum (Wheat : Gramineae) embryo. Am. J. Hot., 70: 1092-1097.
Ozias-Akins, P. and Vasil, I.K. (1983c). Improved efficiency and normalization of somatic embryogenesis in Triticum aestivum (wheat). Protoplasma, 117:40-44.
Ozias-Akins, P., Vasil, I.K. (1982). Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (Wheat) : Evidence for somatic embryogenesis. Protoplasma, 110: 95-105.
Ozygen, M., Turet, M., Altiinok, S. and Sancak, C. (1998). Efficient callus induction and plant regeneration from mature embryo culture of winter wheat {Triticum aestivum L.) genotypes. PI. Cell Rep., 18 (3-4) : 331-335.
Papenfuss, J.M. and Carman, J.G. (1987). Enhanced regeneration from wheat callus cultures using dicamba and kinetin. Crop Sci., 27 : 588-593.
Bibliography xv\
Pathak, G.N. (1940). Studies in the cytology of cereals. J.Genet., 39 : 437-467.
Pluknett, D.L., Smith, N.J.H., Williams, J.T., Anishetty, N.M (1987). Gene banks and the world's food. Univ. Press, Princeton, N.J.
Porceddu, E., Ceoloni, C , Lafiandra, D., Tanzarella, O.A. and Scarascia Mugnozza, G.T. 1988. Genetic resources and plant breeding : Problems and Prospects. In: Proc. 7th Int. Wheat Genet. Symp., Cambridge, 1 : 7-22.
Purnhauser, L., Medgysey, P., Czako, M.. Dix, P.J. and Martin, L. (1987). Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumaginfolia via tissue cultures using the ethylene inhibitor AgNo.v Plant Cell Rep., 6 : 1-4.
Quareshi, J.A. Kartha, K.K., Abrams, S.S. and Sheinhauer, L. (1989). Modulation of somatic embryogenesis in early and late stage embryos of wheat {Triticum aestivum L.) under the influence of (±) abscisic acid and its analogs. PI. Cell Tiss. Org. Cult., 18 : 55-69.
Raghavan, V. (1976). Experimental Embryogenesis in Vascular Plants. Academic Press, New Delhi.
Raghavan, V. (1986). Variability through wide crosses and embryo rescue. In, Vasil, T.K. (ed). Cell Culture and Somatic Cell Genetics of Plants, Vol. 3 : Plant Regeneratin and Genetic Variation, pp 613-633.
Raghavan, V. and Srivastava, P.S. (1982). Embryo Culture. In : Johri, B.M. (ed.) Experimetnal Embryology of Vascular Plants, Springer Verlag, Berlin, 195-230.
Rashid, H. and Qurashi, A. (1989). High frequency embryogenic callus induction and its regeneration in three wheat cultivars. In : Mujeeb Kazi A and Sitch, C.A. (eds). Review of Advances in Plant Biotechnology 1985-88, 2"'' Int. Symp. Genet. Manipualtion in Crops. Mexico-D.F., Mexico and Manila, Phillipines, CIMMYT and IRRI. Pp. 205-215.
Redway, F.A., Vasil, V., Lu, D. and Vasil, I.K. (1990). Identification of callus types for long term maintenance and regeneration from commercial cultivars of wheat {T. aestivum L.). Theor. ApU. Genet., 79 : 609-617.
Riera-Lizarazu, O. and Mujeeb-Kazi, A. (1993). Polyhploid production in the a triticeae : wheat x Tripsacum crosses. Crop Sci., 33 : 973-976.
Riley, R., and Chapman, V. (1967). The inheritance in wheat of crossability with rye. Genet. Res., 9 : 259-267.
Riley, R., Chapman, V. and Johnson, R. (1968a). Introduction of yellow rust resistance of Aegilops comosa into wheat by genetically induced homoeologous recombination. Nature, 217 : 383-384.
Bihliographyxvn
Riley, R., Chapman, V. and Johnson, R. (1968b). The incorporation of alien disease resistance in wheat by genetic interference with the regulation of meiotic chromosome synapsis. Genetical Research, Cambridge, 12 : 199-219.
Rimpau, W. (1891). Kreuzungsprodukte landwirtschaftlicher kulturpflanzen. Landwirtsch Jahrb., 20 : 335-371.
Rode, A., Hartmann, C, lejeune, B., Benslimane, A., Falconet, D., Quetier. F., Henry, Y., Buyser, J.de. (1986). Unusal mitochondrial DNA variation in a non regenerating cell culture of Triticum aestivum. International symp. On PI. Mol. Biol., D8.
Rowland, G.G., Kerber, E.R. (1974). Telocentric mapping in hexaploid wheat of genes for leaf rust resistance and other characters derived from Aegilops squarrosa. Can. J. Genet. Cytol.. 16 : 137-144.
Sarkar, P. and Stebbins, G.L. (1956). Morphological evidence concerning the origin of the B genome in wheat. Am. i. Bot., 43 : 297-304.
Scott, K.J., He, D.G. and Yang, Y.M. (1990). Somatic embryogenesis in wheat. In, Bajaj, Y.P.S. (ed). Biotechnology in Agriculture and Forestry, Vol. 13 : Wheat Springer and Varlag, pp. 46-67.
Sear, E.R. (1961). Identification of the wheat chromosome carrying leaf rsut resistance from Aegilops unbellulata. Wheat Information Service, 12 ; 12-13.
Sear, E.R. (1972). Reduced promimal crossing-over-in telocentric chromosomes of wheat. Genetics Iberia., 24 : 233-239.Sears, E.R. 1973. Agropyron-wheat transfers induced by homoelogous pairing. In : Poc. 4" Int. Wheat Genet. Symp., (Columbia, Missouri, USA (Sears E.R. and Sears, LMS eds.).: 191-199.
Sears, E.R. (1954). The aeuploids of common wheat. Mo. Agric. Expt. Stn. Res. Bull., 572: 1-58.
Sears, E.R. (1956). The transfer of leaf rust resistnace from Aegilops umbellulata to wheat. Brookhaven Symposium, Biol., 9 : 1-22.
Sears, E.R. (1966). NuUisomic tetrasomic combination in hexaploid wheat. In Riley, R. and Lewis, K.R. (eds.). Chromosome Manipulations and plant Genetics. Edinburgh and London, UK : Oliver and Boyd Publ. Heredity (Suppl.) 20 : 29-45.
Sears,. E.R. (1972). Chromosome engineering in wheat. Stadler Genet. Symp. Univ. of Missouri, Columbia, 4 : 23-38.
Sears, E.R. (1973) Agropyron-wheat transfers induced by homoeologous pairing. Proceedings of the 4"' Int. Wheat Genet. Symp. Columbia, Missouri, USA (Sears ER andSears LMS eds): 191-199.
BibliogrophxVm
Sears, R. G. and Deckard, E.L. (1982). Tissue culture variability in wheat : Callus induction and plant regeneration, Crop Sci., 22 : 546-550.
Seko, Y. and Nishimura, M. (1996). Effect of C02 and light on survival and growth of rice regenerants grown in vitro on sugar-free medium. Plant Cell Tissu. Org. Cult., 46 : 257-264.
Sharma, H.C. and Gill, B.S. (1983). Current status of wide hybridization in wheat. Euphytica, 32 : 17-31.
Sharma, K.D., Singh, B.M. and Chauhan, R.S. (1998). Variation in Karnal bunt and powdery mildew resistance among somaclones and double haploids of bread wheat cv. Sonalika. Indian-Phytopathology, 51 (4): 324-328.
Shimada, J., Sasakuma, J. and Tsunewaki, K. (1969). In vitro culture of wheat tissues 1. Callus formation, organ redifferentiation and single cell culture. Can. J. Genet. Cytol., 11 : 294-304.
Shimada, T. (1978). Plant regeneration from the callus induced from wheat embryos. Jpn. J. Genet., 53 : 371-374.
Shrivastava, S., Singh, N. and Chawala, H.S. (2000). Role of growth regulators and glutamine for enhancing regeneration response in wheat (Triticum aestivum L.). J. Genet. Breed., 54(1): 71-75.
Snape. J.W., Chapman, V., Moss, J., Blanchard, C.E. and Miller, T.E. (1979). The crossability of wheat varieties with Hordeum bulbosum. Heredity, 42 : 291-298.
Srivastava, S. and Chawla, H.S. (2001). Synergistic effect of growth regulators and glutamine on regeneration response in high yielding cultivars of wheat (Triticum aestivum L). Indian J. Genet., 61 (1): 12-15.
Stebbins, G.L. Jr. (1950). Variation and evolution in plants, Columbia Univ. Press, New York.
Stewart, J.M. (1981). In vitro fertilization and embryo rescue. Env. Exp. Bot., 21 ; 301-315.
Symillides, Y., Henry, Y. and de Buyzer, J. (1995). Analysis of Chinese Spring regenerants obtained from short and long term wheat somatic embryogenesis. Euphytica, 82(3): 263-268.
Taghvaii, M., Majidi-Heravan, E., Sarmadnia, G., Mazaheri, D. and Shakb, M.A. (1998). Salt tolerance of calluses of six wheat cultivars obtained from tissue culture on different media. Seed and Plant, 13(4): 60-68.
Taira, T. And Larter, E.N. 1978. Factors influencing development of wheat-rye hybrid embryos in vitro. Crop Sci., 18 : 343-350.
Bibliography xix
Tanner, D.G. and Falk, D.E. (1981). The interaction of genetically controlled cossibility in wheat and rye. Can. J. Genet. Cytol., 23 : 27-32.
Tanzarella, O.A. and Greco, B. (1985). Clonal propagation of Triticum durum Desf. From immature embryos and shoot base explants. Euphytica, 34(2) : 273-277.
Ter-Kuile, N., Nabors, M. and Mujeeb-Kazi. A. (1988). Callus culture induced amphiploids of Triticum aestivum and T. turgidum x Aegilops variabilus Fi hybrids : production, cytogenetics and practical significance. 80' Annu. Meet. Am. Soc, Agron. Agric. Abstr. Pp : 82.
Thiele, A., Schumann, E., Peil, A. and Weber, W.E. (2002). Eyespot resistance in wheat x Aegilops kotschyi back cross lines. Plant Breed.. 29-35.
Tomerlin, J.R., El-Morshidy, M.A., Moseman, J.G., Baenziger, P.S. and Kimber, G. (1984). Resistance to Eyrsiphe graminis f sp. tritici, puccina recondite f. sp. tritici and Septoria nodorum in wild Triticum species. Plant Dis., 68 : 10-13.
Tuberosa, R., Ravaglia, S. and Lucchese, C. (1988). Callus induction and plant regeneration in Italian cultivars of bread wheat. Agricoltura Mediterranea, 118(4): 361-365.
Valkoun, J., Dostal, J. and Kucerova, D. (1990). Triticum x Aegilops hybrids through embryo culture. In : Bajaj, Y.P.S. (ed). Biotechnology in Agriculture and Forestry, Vol. 13 : Wheat : Springer-Vorlag Pub., 152-166.
Vasil, I.K. (1987). Developing cell and tissue culture system for improvement of cereal and grass crops. J. Plant Physiol. 128 : 193-218.
Vasil, I.K., 1990. The realities and challenges of plant biotechnology. Bio/technology, 8:296-301.
Vasil, V. and Vasil, I.K. (1982). Characterisation of an embryogenic cell suspension culture derived from inflorescences of Pennisetum americanum (pearl millet, Gramineae).Am. J. Bot., 69 : 1441-1449.
Verma, V., Bains, N.S., Mangat, G.S. Nanda, G.S., Gosal, S.S. and Kuldeep Singh (1999). Maize genotypes show striking differences for induction and regeneration of haploid wheat embryos in the wheat x maize system. Crop. Sci. : 3 9 : 1722-1727.
Villareal, R.L., Mujeeb-Kazi, A. and Pena, R.J. (1999) Agronomic performance and quality characteristics of tissue culture derived lines of spring wheat {Triticum aestivum L.) cultivar Pavon. Cereal Res. Com., 27(1-2): 41-48.
Waines, J.G., Rafi, M.M. and Ehdaie, B. (1993). Yield components and transpiration efficiency in wild wheats. In Damania, A.B. (ed). ,
Bibliography xx
Biodiversity and Wheat Improvement. A Wiley-Sayce Publication, UK, pp 173-186.
Wang, L.S., Wang, Y.F., Zhang, D.H. and Qiu, R. (1993). Selection of salt tolerant callus variant of wheat. In : Li, Z.S. and Xin, Z.Y. (eds). Proc. VIII Int. Wheat Genet. Symp. Beijing, China, Vol. 2, pp 1073-1076.
Ward, K.R. and Jordan, M.C. (2001). Callus formation and paint regeneration from immature and mature embryos of rye (Secale cereale L.) In vitro Cell Dev. Biol.-Plant, 37 : 361-368.
Warham, E.J., Mujeeb-Kazi, A. and Rosas, V. 1986. Karnal bunt (Tilletia indica) resistance screening ofAegilops species and their practical utilization for Triticum aestivum improvement. Can. J. Plant Path., 8 : 65-70.
Wernicke. W. and Milkovits, L. (1984). Developmental gradients in wheat leaves-response of leaf segments in different genotypes cultured in vitro. J. Plant Physiol., 115 : 49-58.
Wernicke, W., Potrykus, I. and Thomas, E. (1982). Morphogenesis from cultured leaf tissues of Sorghum hicolor - the morphogenic pathways. Protoplasma, 111 : 53-62.
Williams, E.G. and Delautour. G. (1981). Produciton of tetraploid hybrids between Ornithopus pinnatus and Ornithopus sativus using embryo culture. N.Z.J. Bot., 19 : 23-30.
Williams, E.G. and Maheswari. G. (1986). Somatic embryogenesis : Factors influencing coordinated behaviour of cells as an embryogenic group. Ann. Bot. (London), 57 : 443-462.
Williams, E.G., Delautour, G. (1980). The use of embryo culture with transplanted nurse endosperm for the production of interspecific hybrids in pasture legumes. Bot. Gaz., 141 : 252-257.
Williams, W.M., Williams, E.G. (1983). Use of embryo culture with nurse endosperm for interspecific hybridization in pasture legumes. In : Smith, A. (ed.) Proc. Fourteenth Int. Grassland Congress, pp. 163-165,
Wilson, A.S. (1876). On wheat and rye hybrids. Trans Proc. Bot. Soc. Edinburgh, 12 : 286-288.
Yamamoto, K. and Miuri, H. (1995). Influence of the semidwarf genes, Rhtl and Rht2, upon embryoid induction from anther culture of wheat. Wheat Information Service, 81 : 6-12.
Zair, I., Chlyah, B. and Chlyah, H. (1995). Effect of two biological extracts on somatic embryogenesis and on the retention of regeneration capacity of Triticum aestivum callus. Can. J. Bot., 73(3) : 498-504.
Pii>liography xxi
Zamora, A.B. and Scott, K.T. (1983). Callus formation eind plant regeneration from wheat leaves. Plant Sci. Lett., 29 : 183-189.
Zeller, F.J. (1973). IB/IR wheat-rye chromosome substitutions and translocations. In Sears, E.R. and Sears, L.M.S. (eds). Proc. 4"" International Wheat Genetics Symposium. Columbia Missouri, USA, University of Missouri, 209-229.
Zhang, J., Friebe, B., Raupp,w.J., Harrison, S.A. and Gill, B.S. (1966). Wheat embryogenesis and haploid production inwheat x maize hybrids. Euphytica, 90:315-324.
Zhang. L. (1986). A new method to increase callus induction and plant regeneration from immature embryos of wheat. Scientica Agriculture-Sinica, 2 : 93.
ANNEXUKBS
Annexure I
COMPOSITION (mg/1) OF DIFFERENT MEDIA USED
I n g r e d i e n t s
NH4NO3
KNO3
CaCl2. 2H2O
M g S 0 4 . 7H2O
(NH4)2S04
KH2PO4
NaH2P04 .H20
IKI
H3BO3
M n S 0 4 . 4 H 2 0
i M n S 0 4 . H 2 0
' Z n S 0 4 . 2H2O
Na2Mo04. 2H2O
CUSO4, 5H2O
C0CI2.6H2O
F e S 0 4 . 7 H 2 0
Na2EDTA
I n o s i t o l
N ico t in i c ac id
P v r i d o x i n e - H C l
T h i a m i n e - l i c e
Glyc ine
S u c r o s e
PH
MS
1650
1900
4 4 0
3 7 0
170
0 . 8 3
6.2
2 2 . 3
8.6
0 . 2 5
0 . 0 2 5
0 . 0 2 5
2 7 . 8
3 7 . 3
100
0 .5
0 .5
0 .1
2 .0
3 %
5.8
B 5
2 5 0 0
150
2 5 0
134
150
0 . 7 5
3 .0
10 .0
2 .0
0 . 2 5
0 . 0 2 5
0 . 0 2 5
2 7 . 8
3 7 . 3
100
1.0
1.0
10 .0
3 %
5 .5
Annexure II
STOCK SOLUTION PREPERTIONS FOR MS BASED MEDIUM
INORGANIC SALTS:
C a t a l o g u e
MS 1 (10 x)
MS 2 (10 x)
MS 3 (1000 X
MS 4 (lOOx)
MS 5 (100 x)
MS 6 lOOx)
VITAMINS
C a t a l o g u e
Vi (lOOx)
V2 (lOOOx)
V3 (lOOOx)
V4 (lOOOx)
Vs (lOOOx)
1_
I n g r e d i e n t s
C a C b 2H2O
N H 4 N 0 3
K N 0 3
KI
C0CI2. 6H2O
K H 2 P 0 4
Na2Mo04. 2H2O
H3BO3
MgS04
M n S 0 4
Z n S 0 4
CUSO4
Na2EDTA
FeS04
5 0 0 m l
2 .2g
8 .25
9 .5g
4 1 5 m g
1 2 . 5 m g
8.5g
1 2 . 5 m g
aiOmg
18 .5g
8 4 5 m g
4 3 0 m g
1.25mg.
1.865g
1.390g
I n g r e d i e n t s
I n o s i t o l
N ico t in i c ac id
P v r i d o x i n e - H C l
T h i a m i n e - H C l
Glyc ine
100ml
I g
5 0 m g
5 0 m g
lOmg
2 0 0 m g
1 0 0 0 m
4 .4g
16 .5g
19.Og
8 3 0 m g
2 5 m g
17.Og
2 5 m g
6 2 0 m g
37.Og
1.69g
8 6 0 m g
2 . 5 m g
3 . 7 3 g
2 . 7 8 g
2 0 0 m l
2 g
lOOmg
lOOmg
2 0 m g
4 0 0 m g
I S t o r e d
At
10°C
10°C
10°C
10°C
10°C
10°C
S t o r e d a t
<40"C
<40°C
<40°C
<40°C
<40°C
Annexure III
WORKING DETAILS OF GROWTH REGULATIONS
H o r m o n e
2 ,4 -D
lAA
NAA
Kine t i c
BA(BAP)
CA3
S o l v e n t
E t o n /
IN .NaOH
E t o n /
IN .NaOH
IN.NaOH
l N , N a O H
l N , N a O H
E t o n
D i l u t a n t
T c W a t e r
Tc w a t e r
Tc w a t e r
Tc w a t e r
Tc w a t e r
Tc w a t e r
S t e r i l i z a t i o n
CA
C A / F
CA
C A / F
C A / F
C A / F
S t o c k
s o l u t i o n
1 m g / m l
1 m g / m l
1 m g / m l
1 m g / m l
1 m g / m l
1 m g / m l
Liquid
s t o r a g e
0 - 5 0 ° C
-0°C
0-5°C
-0°C
0 -5 .0 °C
0-5°C
CA = C o a u t o c l a v a b l e w i t h o t h e r m e d i a c o m p o n e n t s
C A / F = C o a u t o c l a v a b l e w i th o t h e r m e d i a c o m p o n e n t s ,
h o w e v e r s o m e l o s s of ac t iv i ty m a y o c c u r .