biot 202 tulip
TRANSCRIPT
Table of Content
Abbreviations.................................................................................................................................2
Introduction to Tulip......................................................................................................................4
Scientific Classification of Tulip..................................................................................................4
Horticultural Classification of Tulip............................................................................................4
Uses of Tulip...............................................................................................................................5
Why Tissue Culturing of Tulip is Important?..............................................................................5
Objective........................................................................................................................................5
Literature Review...........................................................................................................................6
Materials and Methods............................................................................................................... 16
Preparation of Stock Solution...................................................................................................16
Media Preparation...................................................................................................................16
Media Optimization................................................................................................................. 17
Procurement of Ex-plant..........................................................................................................17
Sterilization..................................................................................................................................18
Glassware.................................................................................................................................18
Culture Media.......................................................................................................................... 18
Ex-Plant.................................................................................................................................... 18
Laminar Air Flow Cabinet.........................................................................................................18
Sterilization of Hands...............................................................................................................19
Sterilization of Metallurgic Instruments...................................................................................19
Inoculation...................................................................................................................................19
Incubation Conditions..................................................................................................................20
Results and Discussion.................................................................................................................21
Appendix I....................................................................................................................................24
Bibliography.................................................................................................................................26
AbbreviationsM.S: Murashige and Skoog
mg: milligram
mg/l: milligrams per liter
”l: micro liter
NAA: Naphthalene acetic acid
BAP: Benzylaminopurine: 6-benzylaminopurine phosphate
BA: Benzyladenine: 6-benzyl adenine
IBA: Indole butaric acid
TDZ: Thidiazuron
IAA: Indole acetic acid
GA3: Gibberellicacid
2, 4 D: 2,4 Dichloroacetic acid
K: Kinetin: 6-furfural aminopurine
L: liter
M: Molar (Concentration)
mm: millimeter
w/v: percentage âweight in volumeâ ; number of grams of constituent in 100cm3 of solution
s: second
%: percentage
psi: pounds per square inch
2
EDTA: ethylene diamine tetra-acetic acid
U.V: Ultraviolet
2, 4-D: 2, 4 dicholorophenoxy acetic acid
ELS: Embryo like structure
WAP: weeks after pollination
c.v. : Cultivar
3
Introduction to TulipTulip is a perennial ornamental flowering plant and is considered as one of the most famous
ornamental flowering plants of the world. The popularity and importance can be understood by
the fact that it is considered now as an ornamental crop because Holland, also known as tulip
Capital of the world, exports nearly 2 billion tulip bulbs every year. The tulip plant is associated
with spring and is incredibly popular during spring time. Although, Holland has become the
âtulip capitalâ now, but they are native of Middle East and Central Asia. They were bought to
Europe in the 16th century, where they became so popular that it became a symbol of opulence.
Scientific Classification of TulipTulip belongs to the Liliaceae family and Tulipa genus. Its common name in Pakistan is
Gul-e-Lala . ۧÙÙ ÚŻÙ If we were to give a complete classification of tulip, it would look something
like this:
Kingdom: Plantae
Phylum: Angiospermae
Class: Monocotyledonae
Order: Liliales
Family: Liliaecae
Genus: Tulipa
Species: gesneriana (1/109 species)
There are about 109 known species of Tulipa and various cultivars for each species with
gesneriana having the most cultivars.
Horticultural Classification of TulipBesides the scientific classification, the Royal General Bulbgrowerâs Association has also
developed a classification, based on flower morphology and plant size, for organizing numerous
cultivars or varieties of tulips. So, in this classification tulips are divided into 15 groups.
4
One other Horticulture Classification is done on the basis of the flowering season of tulip which
is:
i. Early Flowering Tulips- Those tulips which flower in early spring.
ii. Mid-Season Flowering Tulips- It includes those divisions of tulips which blossom 2-3 weeks
after spring.
iii. Late Season Flowering Tulips- This includes those tulips which flower late during spring.
Uses of Tulip
As written above, the tulip plant can be a major export ornamental crop. So, by growing tulip,
foreign exchange is earned in countries like Holland.
Other than that, there has been evidence that during World War II, people of Holland got short
of food supplies and so ate tulip bulbs, petals and stems. One of the source describes tulip bulb
as a replacement for onion (If one is that desperate). The sepals of tulips have been reported as
edible parts.
Why Tissue Culturing of Tulip is Important?
Although Tulip is easily grown plant, but the bulb of the tulip plant requires a cold storage
period during the winter in order for it to grow during spring season, a requirement which
makes it unfeasible to be grown in tropical areas. So, by propagating tulip in vitro, somaclonal
variation can be induced and plant can be given a direction in order to grow them without cold-
storage period.
Secondly, the tulip plants grow from a bulb which are exported in a large quantity which
requires large containers for transport and hence more storage space. But by making somatic
embryo culture, synthetic seeds of tulips can also be formed which can be stored in a small
space and easily transported.
Objective
5
To develop a protocol with the best media formulation for in vitro callus formation in
Tulipa gesneriana.
Literature Review
A lot of tissue culture research has been done on Tulipa and related genera of the Liliacae
Family. But some of the work is presented below.
Alderson et al., (1986) micropropagated tulip and studied bulbing of shoots in culture. The
adventitious shoots arising from the outer layer of cells of floral stem explants of tulip cultivar
Merry Widow were cultured on MS medium containing 1 mg/l NAA and BAP, resulting in
initiation of a bulb primordium at their base after incubation at 20°C for 12 to 16 weeks. It was
observed that development of this primordium was enhanced when sub-culturing was done
with media containing 1 mg/l NAA with or without 0.1 mg/l BAP. Incubation at 4°C for 12 weeks
followed by a period at 25°C on media containing 6% sucrose also enhanced bulb formation at
the base of shoots.It was also seen that bulbing of shoots was enhanced in cultures incubated
in light with a low red/far-red ratio (3.4) and after treatment with ethylene at 1 and 10 ppm
applied as a gas to the culture vessels. Shoot cultures treated at 12 weeks of age showed the
greatest response. Roots were induced on bulbs produced in vitro by manipulation of plant
growth regulators and sucrose in the culture medium.
Nischiuchi (1990) performed an experiment on organogenesis in the scale tissue culture of
tulip. It was observed that organogenetic activity in scale tissue cultures of tulip bulbs in vitro
was increased when the mother plants were treated with foliar application of growth regulators
during the growing period after flowering. It was also seen that in ABA-treated bulb scales,
adventitious shoot formation in vitro showed the best results with three cultivars, "Apeldoorn",
"Oxford's Elite", and "Oxford". Contrary to expectations there was no stimulatory effect of
adventitious shoot formation on BA-treated bulb scales. The cultivar "Red Matador" showed
acceleration of shoot formation in NAA-treated and GA-treated bulb scale cultures, but there
was no stimulation in the other three cultivars
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Baker et al., (1990) studied and compared the precultural treatments and cultural condition on
in vitro response of tulip. Temperature and duration of bulb storage, type of explant, surface
disinfestation method, medium, and culture temperature were compared for effects on in vitro
response of explants from âApeldoornâ bulbs. It was observed that explants obtained from scale
tissue turned brown whereas those excised from the floral axis developed leaf-like structures. It
was also recorded that bulbs stored for either 56 or 63 days had superior mean visual ratings
than those bulbs which were stored for 36 days. When media compositions were compared, it
was noted that explants placed on MS medium containing NAA + BA (0.001 g/l each) had higher
mean visual rating and produced organized structures in 1 month, without callus formation. On
the other hand, the explants placed on a MS medium containing 2, 4-D and kinetin (0.001 g/l
each) produced organized structures via callus. When the leaf like structures were placed at 5°C
then 24°C, bulbs were induced. The organized structures could also be recultured to produce
increased numbers of leaf-like structures.
Custers et al., (1992) studied seedling formation and bulblet production from immature ovula
of tulip (Tulipa gesneriana L.). It was recorded that the percentage germination, on a modified
Murashige and Skoog (MS) medium, was increased by prolonging the 5°C chilling period, given
at the beginning of the culture, from 8 to 12 weeks. Culture that was kept in continuous
darkness was superior to the one placed in 16 h light in producing good quality stolon and
formation of the bulblet from the seedlings. When the stolons were prevented from entering
into the medium and growing thick and turgid by using a thin layer of medium, approximately 6
mm thick, there was an improved bulblet formation. Improvement in formation of bulblet was
also observed when the growing temperature was lowered from 24 to 12â15°C. The optimal
concentration of sucrose in the growing medium was 6% (w/v). Under the improved conditions,
up to 90% bulblet formation was obtained, with an average dry weight of approximately 50 mg.
It was noted that transfer to soil became possible only if bulblets were used, but these
7
frequently developed abnormal or scierotic. The percentage of second-year bulblet production
after transplantation was 12â30%, depending on the genotype used.
Hulsher et al., (1992) devised a technique for micropropagating tulip shoots and bulbs. For
generating shoots, stem explants and axillary buds were cultured on media with a-
naphthaleneacetic acid (NAA), N6-[2-isopentenyl] adenine (2iP) and 6-benzylaminopurine (BAP)
or Zeatine. Shoots with a meristem were selected. Propagation of these shoots was possible by
cutting them longitudinally. The micropropagated single shoots formed bulbs after a cold
treatment on a basal medium with 70 g/l sucrose. These propagated bulbs were planted in soil.
They emerged for 30â100% depending on the weight of the bulbs. After the growing season
new bulbs were harvested from almost every sprouted.
Wilmink et al., (1995) formed adventitious shoots on explants taken from young floral stems of
tulip (Tulipa gesneriana L.). Direct regeneration of shoots was recorded without an
intermediate callus phase. It was observed that more shoots developed on explants taken from
bulbs which were dry-stored than those bulbs stored on ice. At the basis of the shoots a
meristem was formed that developed into a new bulb.8 cultivars were compared for their
ability to form the bulb meristem, and was highest in Lucky Strike and Monte Carlo.
Adventitious shoot formation was initiated in the first two subepidermal cell layers and a
number of cells of the original explant contributed to the shoot formation. An optimal selection
system was established in order to avoid the formation of chimeric transformed shoots in
future transformation. For selection purposes, it was seen that the aminoglycoside antibiotics
were not very effective in inhibiting shoot formation as tulip tissue showed a high tolerance for
kanamycin. On the other hand, G418 and hygromycin induced severe necrosis of the explants at
low concentrations. In contrast, the herbicides phosphinothricin and glyphosate were very
effective and offer good perspectives to be used for selection of transformed shoots.
Hulscher and Krijgsheld (1995) proposed and compared two methods of micropropagation of
tulip. In the first method, adventitious shoot was regenerated from stem slices and in the
second method; shoot was regenerated from axillary buds of the mother bulb. It was observed
that regeneration from stem slices was very variable. Although many shoot-like structures were
8
formed, only a small percentage of these shoot-like structures proved to be real shoots with
meristems that could be used for further propagation. Comparatively, it was seen that shoot
development was better in the shoots regenerated from axillary buds than the regeneration of
shoots on stem slices. Furthermore, the rate of propagation of shoots from axillary buds proved
to be higher than the rates of propagation of shoots from stem slices. These two factors make
the axillary bud system more useful than stem slices to start micropropagation of tulip.
Gude and Dijkema (1996) proposed a method for somatic embryogenesis is tulip which would
be a rapid in vitro propagation alternative to the micropropagation through adventitious shoot
formation in stem explant which has a low propagation factor, is laborious and expensive. The
long term aim of the study was to develop a propagation method, consisting of a cell
suspension phase, and a regeneration phase, during which the cell clusters regenerate through
somatic embryogenesis, a system which would enable the rapid production of large numbers of
bulblets at low costs. For the suspension culture, friable callus was obtained from bulb scale
and stem explants cultured on a nutrient medium containing the auxins 2, 4-D or Picloram at a
concentration range from 0.5â50 ÎŒM. Undifferentiated and âmeristematicâ type of callus on
bulb scale explants was induced. The meristematic type seemed the most suitable to start a
liquid culture. It was seen in liquid medium that the meristematic nodules divided into smaller
units, which could be released from the explant by shaking. The stem explants, cultured on
medium with 5 or 50 ÎŒM 2, 4-D or Picloram resulted in formation of somatic embryos. It was
also concluded from the results that embryo-tissue from the somatic embryos can be cultured
for the production of embryogenic callus.
Tribulato et al., (1997) described a protocol for somatic embryogenesis and plant regeneration
in Lilium longiflorm. For this purpose, friable callus was obtained from styles and flower
pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige
and Skoog (MS) media containing either 2 ”M dicamba or 2 ”M picloram. Friable callus was used
to establish cell suspension cultures by suspending the callus of L. longiflorum Snow Queen in
liquid medium containing 2 ”M dicamba. Through a purification process, a fine fast-growing cell
suspension was obtained. This suspension was composed of a homogenous population of small
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dense cells, which tended to organize into embryo like structures (ELS). In the liquid culture
containing the auxin dicamba, the ELS underwent continuous callus formation. When it was
transferred to solidified hormone-free MS medium, the ELS germinated, forming complete
plantlets. When histological analysis was done, the results showed that in the ELS both shoot
and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact
somatic embryos.
Famelaer et al., (1997) studied how cold treatment of seeds, obtained from crosses between
cultivars of T. gesneriana L., affects the developmental stage of embryos, which in turn
influences the frequency of callus induction and the development of different callus types. For
the research, cold-treated, mature embryos and basal segments of in vitro-derived bulblets,
were used as explants for the initiation of regenerative callus on medium with 2,4-
dichlorophenoxyacetic acid. The bulblets were initiated on flower-stalk segments from cold-
stored bulbs of T. gesneriana âChristmas Marvel.â When the regenerative callus was analyzed
histologically, the regeneration of bulb-like structures was recorded.
Suh (1997) analyzed and studied the stem elongation and flowering response of Tulipa cultivars
as influenced by cold treatment duration, plant growth regulator application, and light quality
during tulip forcing. It was observed that the flowering percentage of âApeldoornâ and âGolden
Apeldoornâ was more than 90%, regardless of low temperature and GA3 treatments. It was also
seen that flowering in both cultivars was accelerated by gibberellins (GA3 and GA4+7) or
Promalin injected into bulb-scale tissue and the flower bud after bulbs had been stored at 5°C
for 6â12 weeks. The results also showed that the total stem length was shorter after injection
of bulbs and flower buds with GA than in control plants. When compared with âApeldoornâ, it
was seen that the flower stem length was slightly reduced when GA3, GA4+7 or Promalin were
injected into bulb-scale tissue. When light quality was tested, it was found that blue and far-red
light, as well as darkness increased the length of the 1st and 2nd internode in âApeldoornâ tulips
when compared with control plants or other light treatments. Growth of the last internode was
stimulated by darkness and by red and blue light treatments.
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van Rossum et al., (1998) investigated the effect of different O2 levels on regeneration of
adventitious organs in various systems, viz., shoot regeneration from tulip bulb scale and stalk
explants, bulblet regeneration from lily scale explants and root regeneration from apple stem
slices. The explants were exposed to 2% O2 (low O2), 100 % O2 (high O2) or ambient air (20 %
O2). It was noted that the culture under continuous high O2 conditions had a moderate adverse
effect in all systems. It was also observed that the continuous culture at a low level of O2 had
strong inhibitory effects in tulip and apple, but was promotive in lily. The results also showed
that when low O2 was applied to tulip stalks only concurrent with the wound reaction, it had no
or even an inhibitory effect. When High O2 was applied directly to apple stem slices after
cutting, there was a slight inhibitory effect on formation of adventitious roots. Once the
regenerated organs were formed, low O2 levels inhibited growth (in lily and tulip), and high O2
levels were stimulating in tulip and inhibitory in lily on organ growth. The results showed that
the alledgedly toxic effect of O2 does not exist or is very small.
Van Creij et al., (2000) studied the effect of several media components on the germination
percentage of ovules in intraspecific T. gesneriana L. crosses. Two embryo rescue techniques
were used; one was viz. ovary-slice culture followed by ovule culture and the other was direct
ovule culture. By the addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at
5 weeks after pollination (WAP), showed significant improvement in the germination
percentage as compared to 5% sucrose. The germination percentage did not differ between
both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with
ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained
with media containing the full or half of the concentrations of micronutrients and
macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule
culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between
ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or
0.1 mg/l) had no effect on the germination percentage. The use of liquid-shaken culture
resulted in germination percentages which were similar to those on agar-solidified media.
Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-
slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose.
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The total concentration of carbohydrates decreased with 19%â48% in the media for ovary-slice
culture, whereas the total concentration of carbohydrates did not decrease remarkably in
media for ovule culture.
Bach and Ptak (2002) found a new method to micropropagating tulip from ovary tissue. The
object of this study was the possibility of improving the multiplication rate of micropropagated
tulips (Tulipa gesneriana L.). Explants of ovary were isolated from chilled (12 weeks) and
unchilled bulbs of âApeldoornâ and âRed Matadorâ cultivars and were exposed to various
concentrations of Picloram and BAP. Plants were generated through somatic embryogenesis via
an intermediary callus phase. Callus was observed to be formed on all medium types but the
embryogenic callus was formed only on the media containing a higher concentration of
Picloram than BAP. Embryos were induced in high frequency in explants isolated from chilled
bulbs. Efficiency of embryogenesis depended on the genotype. âApeldoornâ cv. produced more
somatic embryos then the âRed Matadorâ cv. (16.7 and 9.7 embryos per one explant,
respectively). Cytological analysis did not reveal any changes in chromosome number of
regenerated explants.
Podwyszynsky and Sochacki (2003) conducted a research and described the effects of TDZ and
Paclobutrazol on the primary regeneration on tulip flower stalk explants of six cultivars and
subsequent shoot multiplication. The explants used were flower stalk slices which were excised
from cooled and subsequently forced bulbs. The explants were incubated for two months in
darkness on medium containing NAA and cytokinins, 2iP and BAP, as control, or TDZ (0.5-4
mg/l) and paclobutrazol (0.05-0.4 mg/l). After two months, the regenerating explants were
subcultured on a medium with TDZ and NAA which were applied at low concentrations. The
results showed different regeneration capabilities which depended on cultivar and growth
regulators. It was seen that the percentage of explants forming leaf-like structures ranged, on
the control medium, from 80% in 'Blue Parrot' and 'Prominence' to below 30% in 'Apeldoorn'
and 'Mirjoran'. It was also noted that TDZ applied at optimum for each cultivar concentration
greatly increased the regeneration potential up to 70-100%. When Paclobutrazol was added to
the TDZ-containing medium, the explant responded significantly resulting in a high number of
12
leaf-like structures formed per explant. The structures developed gradually into characteristic
forms: the growing up cotyledonary leaf, the probable root primordium formed at its base, the
growing downwards stolon and the shoot meristem developed finely on its tip. It was suggested
by the study that such primary regeneration may have a nature of somatic embryogenesis.
After that, the adventitious shoots developed and formed clusters, which were divided into 2-3
smaller ones every two months. It was recorded that the growth regulators, used at initial stage
markedly influenced subsequent shoot multiplication. As a result, the most intensive shoot
formation was noted with TDZ at concentrations of 0.5-2 mg/l and paclobutrazol of 0.05-0.1
mg/l.
Ghaffoor et al., (2004) conducted a study on in vitro response of tulips to various growth
regulators. Different explant sources of three cultivars (Beauty of Apeldoorn, Page Polka and
Toronto) of tulip were used for study for their responses to different growth regulators on MS
medium. It was noted that there was maximum shoot formation in culture media with NAA (2
mg/l) and BAP (1mg/l). It was also seen that bulblets formation was better on the medium that
contained 2 mg L-1 NAA combined with 0.5 mg L-1 BAP.
Minas (2007) proposed a method for in vitro Micropropagation of Akama tulip via adventitious
organogenesis from bulb slices. For sterilization, five bulbs of Akama tulip (Tulipa cipria
akamantis) were sliced and soaked in 20% commercial bleach solution with a few drops of
Tween-20 for 10 min and were then washed 3 times in distil sterile water. The explants
obtained from bulb scale slices were placed on MS medium resulting in production of micro-
embryonic shoots. The growth medium was composed of MS basic salts and vitamins,
supplemented with 3% sucrose, 4.5 mg/l BAP, 0.009 mg/l IBA, and 55.7 mg/l ascorbic acid and
solidified with 2.5 g L-1 phytagel. Cultures were incubated in a growth room for two weeks in
the dark and constant temperature of 15±2°C after which they were transferred to a growth
room with 16 hours light of 600 lux provided by white and red light fluorescent lambs (50-50%)
and 8 hours dark at constant temperature of 15±2°C. Proliferation was on average 4-fold per 2
weeks and rooted in vitro, in the same medium, micro-plants were produced.
13
Ptak and Bach (2007) conducted a study for developing a protocol for in vitro somatic
embryogenesis in tulip flower stem culture. Somatic embryogenesis was initiated on flower
stem explants isolated from âApeldoornâ bulbs during their low-temperature treatment. Bulbs
had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured
with exogenous auxins 2, 4-dichlorophenoxyacetic acid (2, 4-D), 4-amino-3,5,6-trichloropicolinic
acid (Picloram), α-naphthaleneacetic acid (NAA) at 1â100 ÎŒM and cytokinins: benzyladenine
(BA) and zeatin (ZEA) at 0.5â50 ÎŒM. It was observed that increasing the concentrations of auxin
caused an intensive enlargement of the explant parenchyma, which changed into homogenous
colorless callus and the vein bundles on the same media developed into yellowish, nodular
callus. The induction of nodular embryogenic callus was more efficient with Picloram than 2,4-
D, whereas the latter stimulated formation of colorless callus. It was also noted that callus
formation was best in the explant taken from the base of the lower part of the flower stem
isolated from bulbs chilled for 12 weeks. The results showed that the highest number of
somatic embryos was produced on medium with 25 ÎŒM Picloram and 0.5 ÎŒM BA. Adventitious
roots were developed in the presence of 2, 4-D. Globular embryos developed into torpedo
stage embryos under the influence of BA (5 ÎŒM) and NAA (0.5 ÎŒM).
Xin-Ying et al., (2007) performed an experiment in look for a method of micropropagation and
also for establishing a base for research of molecular level. The effect of six factors on tissue
culture of bulb scales of four tulip cultivars was studied. The results showed that bulbs sterilized
with 0.1%HgCl2 for ten minutes and 5% NaClO for twenty minutes proved to be best for
sterilization. Induction of callus of cv.'Parade' was observed to be best in MS + NAA 1.0 mg/l
+BA 1.0mg/l. The rate of callus induction was up to 100%. The best culture media for shoot
regeneration of cv.'Parade' and cv.'Merry Widow' were MS+NAA 2.0mg/l + BA 0.5mg/l and
MS+NAA 0.5mg/l + BA 1.0mg/l respectively. The overall rate of shoot regeneration was 15.4%.
It was also observed that the middle-layer scales of bulb were easier to induce callus, with the
rate of 8.0% and inner-layer scales of bulb proved better for direct shoot induction, with the
rate of 26.7%. Adding 50-200mg/l ascorbic acid in culture medium proved advantageous
regeneration of shoot but was disadvantageous in callus induction. The rate of shoot
14
regeneration was up to 12.5% and 10.3% when bulb scales were incubated in darkness for five
days and fifteen days after inoculating respectively.
Podwyszynsky and Sochacki (2010) formulated a new tulip micropropagation method based on
the cyclic shoot multiplication in presence of the Thidiazuron (TDZ), which enables the
production of virus-free stock plants, speeds up breeding, and provides new genotypes for the
market. In their novel protocol, cyclic shoot multiplication can be performed for 2-3 years by
using TDZ instead of other cytokinins, as 6-benzylaminopurine (BAP) and N6-(-
isopentyl)adenine (2iP). This protocol makes it possible to produce 500-2,000 microbulbs from
one healthy plant. Six main stages of tulip micropropagation were studied. Stage 1 is the
selection of true-to-type and virus-free plants, confirmed by ELISA. Initially, explants were
obtained from fragments of flower stems isolated from bulbs. Shoot multiplication was based
on the regeneration of adventitious shoots, which were sub-cultured after every 8 weeks. In the
third stage, the specially prepared shoots were induced by low temperature treatment to at
20ËC on a sucrose-rich medium to form bulbs. After that, the bulbs were dried for 6 weeks and
rooted in vivo. It was concluded that to reduce the risk of mutations, the number of
multiplication subcultures should be limited to 5-10 cycles. Also, virus indexing should be
repeated 3-4 times, at the initial stage and also during shoot multiplication.
15
Materials and Methods
Preparation of Stock SolutionThe Medium used for this project was Murashige and Skoog (MS) media, for which the
following stock solutions were prepared.
1. Macronutrients (20X)
Macronutrients were prepared at 20X i.e. at 20times the strength of the normal solution,
according to the composition stated in Appendix 1A. The stock solution was poured in
brown bottle for storage and later use.
2. Micronutrients (100X)
The stock solution for Micronutrients was prepared at 100X i.e. at 100 times the strength of
the normal solution, prepared according to the composition stated in Appendix 1B. After
preparation, it poured in brown bottle for storage and later use.
3. Iron EDTA (200X)
Stock solution of Iron EDTA was prepared at 200X, according to the composition stated in
Appendix 1C, and was stored in brown bottle for later use.
4. Vitamins (200X)
Stock Solution for Vitamins was also prepared at 200X, according to the composition stated
in Appendix 1D. The stock solution was poured in a brown bottle for storage and later use.
16
Media PreparationFor preparing 250ml MS media, following procedure was implied.
1. First I took a 250ml clean glass beaker and rinsed it with distilled water.
2. Then added about 80-100ml water in it.
3. After that, I added 12.5ml Macronutrients, 2.5ml Micronutrients, 1.25ml Iron EDTA and
0.25ml Vitamins, each with a separate clean pipette.
4. After that, I added 7.5g sugar (sucrose) in it and mix the sugar on the magnetic stirrer.
5. When sugar was completely mixed, PGRs (which and how much stated in the âMedia
Compositionâ section) were added to the solution.
6. The solution was given a slight stir and poured in a 250ml measuring cylinder after which
distilled water was poured in measuring cylinder to make it 250ml.
7. The liquid media was then transferred to a 1 liter glass beaker (to preventthe media from
boiling and overflowing) in which 2.5g (1% w/v) was added.
8. After that, the beaker was kept in microwave oven for 4-5 minutes to boil the mixture in
order to dissolve agar.
9. Lastly, after the dissolution of agar, beaker was taken out of the microwave oven and
culture media was poured into the culture tubes, which were then sealed with
polypropylene sheets using rubber band.
Media OptimizationThe growth media was optimized with different PGRs and increasing and decreasing the
nutrients in the culture media. The following media formulations were made for this project.
1. MS + 10mg/l 2,4-D
2. MS + 0.5uM 2,4-D + 5uM BAP
3. MS + 1mg/l NAA + 1mg/l BAP
4. MS + 1mg/l 2,4-D + 1mg/l Kin
5. MS + 8mg/l BAP
6. MS + 2mg/l Kin + 4mg/l IBA + 60g/l sugar + 0.5ml Vitamins
17
Procurement of Ex-plantEx-plants used for the purpose of this project were tulip bulbs, leaves and tulip shoot. The ex-
plants were provided to us by our professor Dr. Kanwar Shoaib, which were stored for later use
according to their condition stated as follows:
i. Leaves and stem were submerged in plenty of water in beaker to prevent desiccation and
kept in a refrigerator at 4-5 C,
ii. Bulbs were also placed in the refrigerator at 4-5 C without removing their outer, brown
covering.
Sterilization
GlasswareGlassware which includes beakers, pipettes, petri-plates, glass bottles, etc. was sterilized by implying the
dry heat method. All the glassware was washed, dried and finally wrapped in brown paper. Then the
glassware was kept in an oven at 160 oC for 2 hours.
Culture MediaAfter preparation and pouring of growth culture media in the culture tube, it was sterilized by placing
the culture tubes in an autoclave at 121 oC at 15psi for 15mins. This makes the culture media absolute
germ free, even the microspores and extremophiles do not survive at this temperature and pressure.
Ex-PlantThe procedure for sterilization of ex-plant is written below.
First, I added about 2-3 spoons of liquid detergent and 1-2 drops of Tween-20 in a beaker containing
150-200 ml water. Afterwards, I put my ex-plant (leaf or bulb) in this mixture and scrubbed it gently with
the tips of the finger. In case of stem, the stem was scrubbed quite rigorously with finger tips. After
washing for about 2 minutes, I drained the water and then washed the beaker and the ex-plant, 3-4
times with distilled water very thoroughly to remove the detergents. After washing the ex-plant, I
sterilized the ex-plant in laminar flow cabinet by putting it in sterilized petri-plates containing Mercuric
Chloride, HgCl2 (0.1%-1%). The ex-plants were dipped in mercuric chloride for only 30secs to 1minutes
18
maximum. Then the ex-plant was washed 3-4 times with Autoclave Water in the laminar flow cabinet.
After that, the ex-plant was suspended in autoclave water in petri-plate till inoculation.
Laminar Air Flow CabinetPrior to sterilizing the ex-plant and inoculation, the laminar air flow cabinet was sterilized. For that, the
base and the sidewalls of the air flow cabinet (Fig. 1) were first thoroughly swapped with cotton dipped
in ethanol. Then UV lamp was switched ON for 30-40 minutes.
Fig. 1: Laminar Air Flow Cabinet
Sterilization of HandsBefore performing inoculation, hands and forearms were first washed with soap and then sprayed with
ethanol to disinfect them of microorganisms.
Sterilization of Metallurgic InstrumentsThe metal instruments like forceps and scalpels were kept in a beaker containing alcohol and were flame
sterilized before using.
Inoculation
19
The ex-plant was inoculated under germ-free, aseptic environment (created by following the above
stated sterilization protocol). Before inoculation, the UV light was turned off (UV radiations are
carcinogenic) and the fluorescent light and air flow was switched ON. The ultra-filtered, pressurized air
flows towards the person using airflow cabinet, thus preventing micro-organisms from entering.
Sneeze guard was also worn in order to prevent contamination of ex-plant and culture medium by either
talking or sneezing. Even in the presence of sneeze guard during the inoculation procedure, talking with
any group member was avoided or kept to minimum
After that, the scalpel dipped in alcohol was given a slight jerk to remove alcohol and was kept on flame
to sterilize it. Then the petri-plate containing ex-plant was opened and â1 cm pieces of ex-plant were
made using the sterilized scalpel. After cutting ex-plant, the polypropylene sheet sealing the culture
tubes was removed and the neck of the tube was kept on flame. Then using forceps (also flame
sterilized), the ex-plant was placed on the culture media in the culture tube i.e. the ex-plant was
inoculated. As soon as the inoculation was done, the culture tube was sealed using the same
polypropylene sheet which was taken off before.
Incubation ConditionsAfter performing inoculation, the culture tubes were kept in a culture room (Fig. 2). The conditions in
the culture room were:
i. Temperature: 26 ± 2ËC
ii. Light Intensity: 4000 lux. Light provided was cool fluorescent white light.
iii. Dark condition was provided by putting the culture tubes in a box.
20
Fig. 2 Culture Room showing racks with culture tubes, fluorescent light and boxes for providing dark treatment
Results and DiscussionThe main observation regarding the tulip is that it is very recalcitrant towards callogenesis and
embryogenesis. During the 2-3 months of working, only 2 test tubes showed some callogenesis and one
tube showed caulogenesis.
The MS + 10mg/l media, inoculated with leaf ex-plant were kept in dark and light. The leaf ex-plants in
all the MS + 10mg/l tubes kept in light showed complete dedifferentiation in 2 weeks and became clear
white (Fig. 3), so much so that they were hard to distinguish from the white color of the media. One of
the leaf explant showed some swelling but it was not very noticeable.
21
Fig. 3: Dedifferentiated leaf ex-plant which has become white
On the other hand, the ex-plant placed in dark was de-differentiating but very slowly. During
the course of two months of incubating in dark, the color of the leaf changed only slightly
turning pale green.
The ex-plants cultured on the MS + 0.5uM 2, 4-D + 5uM BAP media, showed some hyaline
colored callus as shown in Fig. 4. Stem was also regenerated on the same bulb explant (Fig. 5)
22
Fig. 4: Ex plant showing hyaline callus formation
Fig. 5: Microscopic image showing caulogenesis in Tulip Bulb
As there was not very evident callogenesis and regeneration in the above two media, and no
activity in any of the other media, it was thought that the PGRâs in the media might be diluted
23
thus showing slow or no activity. So the concentration of the PGR was increased 2-3 times the
normal concentration. As a result, the ex-plant showed callogenesis in about 2-3 weeks (Fig. 6).
Fig. 6: Bulb Explant showing callus formation in two weeks
The rest of the 4 media showed no visible signs of activity. The culture tubes containing the
stem ex-plant was contaminated with microorganisms that resulted in breakage of the solid
media in the tubes. Some of the bulb explants turned brown after a period of 2-3 weeks after
inoculation and incubation.
The results stated above can bring us to a conclusion that for regeneration and callogenesis of
tulip, an increase in the concentration of the PGR results in rapid activity in the explants of
ârecalcitrantâ tulip.
24
Appendix IMurashige and Skoog (1962) Medium Preparation
Stock MS Media(mg/L)
A Macronutrients 20X(mg/L) (50cm3 SS gives)
N
H4N
33,000 1,650
K
N
39,800 1,900
CaCl2.2H2O 8,800 440
MgSO4.7H2O 7,400 370
K
H2P
3,400 170
B Micronutrients 100X(mg/L) (10cm3 SS gives)
MnSO4.4H2O 2,230 2.3
ZnSO4.4H2O 860 8.6
H
3B
620 6.2
K
I
83 0.83
Na2MoO2.2H2O 25 0.25
CuSO4.5H2O 2.5 0.025
CoCl2.6H2O 2.5 0.025
25
C Iron EDTA 200X(mg/100cm3) (5cm3 SS gives)
N
a2ED
672 37.6
FeSO4.7H2O 556 27.6
D Vitamins 200X (5 cm3 SS gives)
Glycine 20 0.2
Nicotinic Acid 5 0.05
Pyridoxine HCl 5 0.05
Thymine HCl 1 0.01
Sugar(Sucrose) = 30g/L
Agar = 10g/L
PH should be 5.8+ 0.1
26
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