STUDIES ON KARYOLOGY AND KARYOMORPHOLOGY OF INDIAN CHAROPHYTA FROM
NORTH WESTERN REGION
CNSSEnTATIOIf SU0M1TT€O IN PAfmAL ftHf fUMiNT OF TNC
iifQUMiaifMTS roN TNC AWARD Of THE oiOfiiE or
iKaKter of ^Po^opl^ IM
BOTANT
ARUNA GAVTAM
DEPAMTMKNT OF BOTANY ALIGABH MUSLIM UNIVEBSITY
ALIGARH (INDIA) 1993
ALIGARH MUSLIM UNIVERSITY DEPARTMENT OF BOTANY. ALIGARH 202 002. INDIA Tol. (0571) 25C76
A. K. M. GHOUSE M.Sc, Ph.D.. F.L.S.. F.A.E.B. Professor & Chairman Dated.
CERTIFICATE
This is to certify that the dissertation entitled,
"Studies on Karyology and KaryomoTphoIogy of Indian
Charoplyta from North-Western Region", is an original work, of
Ms. Aruna Gautam, under my supervision In partial fulfilment
for tlie requirements of the degree of Master of Philosophy in
Botany. The part of this dissertation has not been submitted
elsewhere for any other degree or diploma.
( A.K.M. GIIOUSE )
Research Supervisor
Miss ARUNA GAUTAM Research Scholar
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY,
ALIGARH-202002. INDIA.
ACKNOWLEDGEMENT
I bow In reverence ^o God, whose benign benediction
gave me the required Zeal for the completion of this work.
I deem great pleasure in expressing my debt of
gratitude to my supervisor, Prof. A.K.M. GIIOUSE, proffessor of
Botany, Aligarh Muslim University, ALIGARH, who has been backen of
light to me throughout this endeavour. I am also thankful to my
ex-supervisor. Dr. (Late) Mahmood Khan, Reader in Botany, Aligarh
Muslim University, ALIGARH for his val;uable suggestions and
guidance during the initial stage of this task.
I am highly indebted to Dr. Shabbir Ashraf for his
valuable help in numerous ways and Dr. M.C. Gupta, Lecturer in
Botany, D.S. College, Aligarh, who always encouraged me in
different ways.
I take this opportunity to acknowledge the help and
cooperation rendered by my laboratory colleague Mr. U.S. Pundhir.
I express my Co-ordial thanks to Miss Neeraj Sharma,
for her selfles assistance & Cooperation.
Besides, I am also thankful to all my friends and
relatives, who have been my well-wishers and stood by me through
thick and thin. A piece of love is reserved for my younger
brothers Mr. Aaurag Gautaa & lliaanshu for their Co-operation.
I would miss my duty, if I do not make a mention of my
parents, whose sacrifices and blessings gave me impetus to achieve
this goal.
(ARUNA GAUTAM)
CONTENTS
^•^' Chapter Page No No
o o o o o
O O O O
O o o
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From To
CHAPTER-I
1. INTRODUCTION 1 - 9
CHAPTER-II
2. REVIEW OF LITERATURE 10 - 31
CHAPTER-III
3. MATERIAL AND METHODS 32 - 45
CHAPTER-IV
4. PLAN OF WORK 46 - 47
5. LITERATURE CITED 48 - 78
6. TABLES I - XXIV
CHAPTER - I
INTRODUCTION
Charophyta, commonly referred as "Stoneworts"
consMtute a small, but sharply defined group of
macroscopic, chlorophyllous algae. Their several
dlstinguishig features, viz. reproductive organs,
biflagellated asymmetrical scaly anthrozoides and a
characteristic protonemal type of oospore germination.
Cells of the charophytes are very long, uninucleate and
chlorophyllous. Chlorophyll occur in spherical forms.
Division charophyta has a single order Charales
with single class characeae consisting fossil as well as
modern living forms (Feist and Grambast-Fessard, 1982).
There are only six living genera grouped under three tribes
viz. the charae, the Tolypelleae and the Nitelleae (Sawa and
Frame, 1974; Bhatnagar, 1987). The tribe chareae consists of
four genera, viz. Cbara, LychnothamnuB, Lamprothamnium and
Nitellopsis, the tribe Nitelleae and the tribe Tolypelleae
represent only one genus each Nitella & Tolypella
respectively. Womersley and Ophel (1947) have described a
new genus Protochara from Australia but Mac-Donald and
Hotchkiss (1956) and Wood and Imahori (1965) merged it with
Chara Corallina.
The "Stem" of Charophyta is divided by well
marked, alternating nodes and internodes. It shows an
unllmlfed growth by the continued activity of a dome-shaped
apical cell, which continuously cuts of transverse cells
below. Each of these daughter cells divides into an upper
nodal and lower Internodal cell. The nodal cells divides
by a "halving wall" into two semicircular cells and each of
these cuts off a semi-circle of peripheral cells. Finally
the node have a central pair of cells surrounded by 6-20
peripheral cells (Giesenhagen 1896, 1898; Kuckzewski, 1906;
Sluiter 1910; Sunderlingam, 1954). These two daugher cells
do not divide further in Chara (Sunderlingam, 1954, 1963,
1966), Lychnot-haMnus (Sunderlingam, 1962a) and Nitellopsis
(Giesenhagen, 1897; Bharthan, 1983). In Lamprothamnium
(Giesenhagen 1898; Iwasaki , 1963; Frame and Sawa 1975,
Bharathan and Sunderlingam, 1984a), Nitella (Sunderlingam
1965) and Tolypella (Iwasaki, 1963), the pair of central
cells divide further. The stem nodes of charophytes give
rise to four types of appendages viz. branchlet, cortex,
stipulodes, and axillary branches.
Each of peripheral cells gives rise to a lateral
branchlet with a limited number of nodes and internodes.
Each peripheral cell functioning as an apical cell, cuts
off a series of segment, 6-12 in Chara, 4-6 in
LychnothamauB and LaaprothamnluiB, 3-4 in NitcllopsiB, 2-3
in Tolypella and two In Nltella and then gets converted
int-o a poln^ed and allantold terminal segment , losing
thereby its capacity to divide further (Sunderlingam,
1992). Growth of the branchlets in all the four genera of
the tribe Chareae and genus Tolypella exhibit monopodial
branching, while in Nitella the growth of branchlets are
sympodial. In most of the species of Chara, the internodes
of the main axis and branchlets are covered by a single
layered cortex made up of longitudinally extending rows of
cells. The cortex may be haplostichous, diplostichous or
triplostichous. In Lychnothamnus, the cortex is generally
imperfect, but in Laaprothamnium, Nitellopsis, Nitella
andTolypella, the axis is totally ecorticated.
Among Chareae except Nilrellopsis there is
generally one or two circle of stipulodes at the base of
each whorl of the branchlets. The stipulodes may be well
developed as in Chara zeylanica or rudimentary as in Chara
globularis and Chara vandalurensis. Some charophytes have
single row of stipulodes (monostephanous) while otliers have
two rows of stipulodes (distephanous). Migula (1897)
reported a third row of stipulodes between the upper and
lower row (tristephanous ) in Chara ceratophylla.
Generally a single axillary branch arises at
each node in the genera of tribe Chareae, whereas in
Nitella and Tolypella two axillary branches formed at each
node. In Chareae, the primary internodal cell of the
oldest branchlet is the starting point of axillary branch
while in Hir.ella and Tolypella the adaxial peripheral cell
of the basal node of the oldest branchlet as well as that
of the next oldes branchlet gives rise to axillary
branches.
Gametangia are borne on the upper whorls of the
main axis and on the axillary branches. In some taxa, the
fertile axis and branchlets are reduced to form dense
heads, conspicuously distinguished with vegetative portions
(Wood and Imahori, 1965). In most of the species of
Nitella, these heads are often covered by mucilagenous
substance which may extend over sterile whorls also.
Antheridia are globular and orange coloured, whereas
oogonia ovoid and possess orange or yellowish green colour.
Insertion of gametangia are very much important in
distinguishing of different genera. In Chara the oogonium
is placed above the antheridium in contrast fo
Lamprothaianlum where the oogonium is placed below the
antheridium. In Lychnothamnus, two antheridia flank a
centrally placed oogonium. The antheridium or oogonium is
formed at the branchlet node singly or in pairs in
Nitellopsls. In Nitella, each furcation terminated by
single antheridium with one or more lateral oogonia. In
• • Q • • • • J • •
Tolypella where each node bears generally a lateral
antherldlum and one or more oogonia.
After fertilization, changes takes place in the
Characean oosporangium to produce a thick, multilayered,
organic wall around the oospore. The outermost layer of
this wall is often highly ornamented. A large number of
taxa was undertaken for investigation and assess the range
of form found in the ornamentation layer (Jhon & Moore,
1987; Jhon, Moore & Green, 1989). The ornamentation layer
was far move complex and varied in the Nitelleae than in
the Chareae. In some species of Nitella the ornamentation
or the structure of the ornamentation layer appear to be
more unique within the group (IJ. Knightiae, II.
struthioptila, ^. acuminata). A special ribbon like
structure which is analogous to the flange of Nitella, so
far found only in Chara. This ribbon is a portion of the
thickened, lateral wall of the ensheathing cell and consist
a distinct ornamentation which differs from that of the
fossa surface lying between the striae (Moore & John,
1989).
The characteristic life cycle involves
germination of oospore after meiotic division and its
production of hyaline filaments which develops into
non-chlorophyllous, single celled, the rhlzolds. Though
: : 6 : :
melosls is zygotic the interpolation of a protonemal stage
in life cycle results into a type of characteristic
monoblontic, digenlc, heterowonphic, haplontic life history
(Chapman & Chapman, 1962) not known to occur in any other
division of algae.
In Switzerland, the charophytes are used to keep
off the insects from soils by spreading dried plants.
According to Zaneveld (1940), the charophytes are used as
manure, waterpurifier', as food for aquatic animals and farm
stocks, in manufacture of polishes and mud baths, in sugar
refining and insect control (pal et. al. 1962).
Caballero (1919) described the larvicidal
properties of charophytes but later on Blow (1927, 1931)
and pal (1982) explained that charophytes have no
larvicidal properties. Matheson and Hinmann (1928)
affirmed that this plant checked the breeding of
mosquitoes. Pal (1932) experimenting with £. gymnoptitys,
N. acuiMlnata and JM. Qllgospira showed that lethal effect of
these plants is due to the presence of dragon fly larva
(sub-family Libelluinae) hidden among the branchlets.
Crooker (19A8) has described Charophytes as good water
purigying agents. It is also used as food by water fowls.
Gordow (1943) observed that these plants might not be
direct source of food but promote the growths of epiphytes
and other small herbivorous animals, which in turn are used
as food by fishes. In some European countries these plants
are used as manures. The high incrustation of calcium
prevents the sourness of the soil. Excess growth of these
plants may cause nuisance in reserviors.
The charophyta are used for several
physiological experiments such as studies on permeability
of cytoplasmic membrane. Ion accumulation, bio-electric and
action potentials, internal hydrostatic pressure, light and
streaming action potentials, viscosity and temperature
(c.f. Pal e_t . aj^. 1962).
The charophytes were appreciated as a distinct
group from the time of Valliant (1719). After Valliant
Lebeck. (1798) collected Chara zeylanlca for the first time
from Ceylon (Sri Lanka) (Braunn, 1849). In India Braunn
(1849) gave the first systematic account of Indian
charophyta with the publication of "Characeae Indlae
Orientalla et insularua narls pacifies". Later on Groves
(1924) published "Notes on Indian Charophyta" which gave
impetus for vigorous taxonomic work on charophyta of this
country. Valuable contribution made by Allen ( 1925, 1928,
1933, 1942) from Uttar Pradesh^ Kundu (1938, 1941, 1959)
8
from Bengal, Pal (1929, 1932) from Burma, Dixit (1935,
1940, 1942) from Bombay and Sunderlingam (1959) from South
India, which ultimately paved the way for the publication
of a monograph "Charophyta" by Pal, Kundu, Sunderlingam and
Venkataraman (1962) bringing together all the taxa
described from this region upto that time.
The important landmark in the history of
charophyte is the publication of valuable monograph
entitled "The revision of the Characeae" by Wood and
Imahori (1964) . They have employed a number of criteria
based on their personal observations for forming the
systematica of charophytes. All these above mentioned
studies totally based on morphological observations as the
morphological characters greatly, influenced by the
ecological factors which changed with the change in
ecological parameters. Such morphological characters can
not be considered as taxonomic criteria. During these
taxonomic studies and different classifications, they all
have ignored the karyological characters, and genitic
alternations, which resulted in unwanted merging of various
species into one or creation of the new species. Algal
cytologists or charophycologists took it as a challenge and
decided to classify them on the basis of karyotypic
organisation, karyological characters and chromosome
• • q • •
numbers (Sarma & Khan 1967; Sinha & Verma 1970; Ramjee &
Sarma, 1971; Chatterjee, 1976, 1979, Ramjee & Bhatnagar
1985; Bhatnagar & Johri, 1985).
Karyological studies seems t-o offer a good deal
of evidences for solving the controversial problem of
Algae, viz. systematic position, speciatlon, phylogeny,
inter-relationship and evolution etc. However, Informations
regarding karyology and karyomorphology of Indian
charophytes are in plenty, but these are meagre in
comparison to what remains to be done ? Although the
chromosome numbers of various taxa of Indian charophyta
have been worked out, but still a large number of taxa from
different parts of India particularly from North-We stern
Region (Haryana, Himachal Pradesh, Jammu and Rajasthan)
remain to be investigated. The present work is proposed to
achieve some of the above objectives by way of detailed
examination of various taxa and their karyology and
karyomorphology of Indian Charophyta from North-Western
region.
:• o o O o
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CHAPTER - II
REVIEW OF LITERATURE
Charophytes due to their unique morphological,
anatomical, ecological, phytogeographical , karyological,
karyomorphological and phylogenetlc position have attracted
the attention of the phycologists throughout the world.
Extensive investigations have been carried out on different
aspects of charophytes particularly during the past two
decades. The methods employed by various algologists depend
not only on the type of studies undertaken by them, but due
to the advanced technologies that were within they reach.
The work done by various charophycologists is
reviewed here :
2.1 TAXONOMY :- In early phase, most of the work on
charophytes are based on taxonomic and
systematic studies. The first systematic account of Indian
charophyta was given by Braunn (1849). He described 11
species in his paper. Hate (1909) described two taxa
from Bombay island. Groves (1919) has given notes on
LychnothamnuB and in 1922 he showed the presence of
Nitellopals obtusa from Northern India. The publication of
"Notes of Indian Charophyta" provides a resume of our
knowledge of this group upto 1924.
• • 11 • •
Allen has worked on charophytes of various places in
Uttar Pradesh Viz. Gonda (1925), Saharanpur (1928), Agra
(1933), Bareilly (1936), Banaras (1961), Groves and Allen
(1927) described some Indian species of charophytes. Kunda
(1929, 193A, 1935,1938, 1942, 1959) worked on charophytes of
Bengal & gave systematic account. Kundu (1934) described
Nitella RrovcBsi from Assam. Dixit (1931, 1935, 1936, 1940,
1942), Mukherjee (1934), Agarkar & Kundu (1937) also reported
many forms of charophytes from Bombay, Kashmir and Bengal,
lyenger (19958) described terrestrial form of Nitella viz.
Nitella terrestris from Madras. Sunderlingam (1959)
contributed nine species of Nitella, 10 species of Chara from
South India, of which a new species of Chara was also
described, viz. Chara vandalurensis sp. nov. Sunderlingam.
In 1962 he reported Lychnotha»nu8 from South India. Goyal
(1960) investigated charophytic flora of Jodhpur and its
environs. Chaudhary & Sinha (1962) and Sinha & Chaudhary
(1962) made valuable contribution from Bihar.
Pal e_t £1,. (1962) have given a detailed monographic
account of 18 species of Fossil charophytes, 33 texa of
Nitella, 3 taxa of Tolypella, 38 taxa of Chara and one taxon
each of Nitellopsis, Lychnothamnus, and Lamprothamnium.
However, they have given a little description of two taxa of
Protochara which were later merged with Chara corallina by
Wood & Imahori (1965).
12
Agarkar (1963) enumerated 9 species from Gwalior,
Vaidya & Gonzalves (1963) from Western-India, Rao & Rao
(1969) from Chltthor districts, Andhra Pradesh. Sarma & Khan
(1967) described two new species of charophytes from India,
Sinha & Choudhary (1968) gave the comparative account of
Southern-Eastern United states and Indian flora. Among 92
taxa, 67 species, 13 varieties and 12 form of Chara and
Nitella found in these two regions, 27 of them are unallied
and have no similarities among them. Choudhary (1969),
Choudhary (1970, 1971), Bharati & Cheenaveeraiah (1971) gave
systematic enumeration of Indian Charophytes. Chatterjee
(1975) introduced a new species of Chara from West Bengal
viz. C_. socotrensis f. nuda. , Sunderlingam & Bharathan (1978)
and Patel & Jawale (1979) reported Lychnothamnus barbatus L.
from Gujrat and Madras respectively.
Exaushtive study of charophyta taxonomy in India has
been done by Khan (1980), Mukherjee & Noor (1980), Sinha &
Srivastava (1980), Subramaniam (1981), Goswami (1981), Patel
& Jawale (1982), Labh & Verma (1983), Subramaniam (1983),
Bharthan & Sunderlingam (1984), Anand & Langan (1988) ,
Pandey e_t_. al_ (1988), Khan (1989) and Chaturvedi & Iqbal
(1991) . In abroad Imahori (1950, 1953, 1963) and Wood
(1962, 1963, 1965) from the Asia and Australia respectively
established valuable contribution. Wood & Imahori (1965)
: : 13 : :
published an Invaluable monograph & icnograph on charaphyta.
All the work was based on their personal observations.
2.2 MORPHOLOGY :- Giesenhagen (1896, 1897, 1898) and
Sunderlingam (1954, 1962, 1963, 1966)
were the pioneers in the field of developmental morphology of
charophytes. Iwasaki (1963) described comparative morphology
of the reproductive structures of the Chara, Nitella,
Tolypella, Nitellopsis and Lamprothamnium.
The antheridium of Characeae has been studied in
depth by Groves (1931), Sunderlingam & Francis (1958) and
Caceres (1975). So far in four taxa (C_ zeylanica, Nitella
terreatris N. stuarti & N_ cardobensis) quadrlosculate
antheridia have been reported. Kaushik & Bhattacharya (1971)
studied the structure and development of the sex organs in
Tolypella nidlfica (0. Mull.) A. Br. and Maier (1973)
improved their misinterpretations. Sawa & Frame (1974) have
given account on the oogonla and oospores of Tolypella with
reference to sterile oogonial cells. Comparative anatomy of
Tolypella and La«pro»:ha«niuM and its approach to the taxonomy
was given by Frame & Sawa (1975, 1976).
Moore & Jhon (1989) gave an account of structure and
ornamentation of the outer wall of the fertilised
oosporangium in charophyta. Jhon e_t al (1990) give the
preliminary observation of Chara oosporangium wall.
:: 14 : :
The anatomical characters like the number of central
cells at the axial nodey in different genera, the arrangement
of cells in the basal node of the branchlet in different taxa
have been studied in detail (daily, 1980; Bharthan &
sunderlingham , 198A; Bharthan, 1987) and their importance in
the taxonomy and phylogeny of characeae has been emphasized.
2.3 CULTURE MEDIA :- Chu (1942) was a pioneer worker,
who established culture media
having resemblance to these in which algae grow naturally.
His successful medium is comparable in composition and degree
of dilution to rhe water of a eutrophic lake. Chu medium Is
fresh water of a eutrophic lake. Chu medium is fresh water
culture medium.
Culture media in charophytes have been carried out
elsewhere in world by different authors (Imahori, 1963;;
Forsberg, 1965; smith, 1966; starling et aJL. , 1984) for
experimental, taxonomlcal and physiological investigations.
Andrews ej: al. (1984); Sokal ej; al. (1986); Okazaki and
Tokita (1988) used various media under controlled conditions.
Biphasic medium is widely used for culture studies in
charophyta. This culture contains sterilized pond water and
fine sterilized clay. The culture were maintained at 15-25°
C temperature and exposed to 12 hours of light and dark
periods with an intensity of 1500 lux (Bharthan, 1990).
: : 15 : :
2.4 BIOCHAMICAL AND PHYSIOLOGICAL STUDIES :- Since 1961
only very
few investigations have been carried out among the members of
charophyta in relation to their biochemistry and physiology.
Analysis of organic constituents, particularly the amino
acids of charophytes have been carried out by Vaidya (1961);
Jacob & Venkatarman (1962). Through x-ray diffraction
analysis, the nature of the cell wall of Chara was
investigated by Krishnamurthi (1962).
Hoist and Yopp (1976) have reported the occurrence
of polyphenol oxidase from Nitella acuminata and also studied
isoenzymes complement by thin layer gel plate method
employing Saphadex G. 100 and the activity of the enzyme was
measured by spectrophotometric method.
Grant and Proctor (1980) through horizontal slab
gel electrophoresis identified gene duplication via
polyploidy in charophyta. Electrophoretic analysis of twelve
species of Chara was carried out by them and they have also
identified the phenotypes of phosphoglucose isomerase,
glutamate oxaloacetate, transminase and glutamate
dehydrogenase. Gemayl (1988) carriedout biochemical
systematic studies on several species of Chara by using the
technique of electrophoresis and tested 14 enzymatic systems
(8 oxidoreductases, 2 transferases, 3 hydrolases and 1
:: 16 : :
Isomerase) occurring in fhem. Godleswski (1980) st:udied
D.N.A. polymerase in antheridial filaments during r.he cell
cycle (S + G2 + M + C type).
Bhatnagar & Sazena (1991) isolated Glycolate oxidase
(GOD) and super oxide dimutage (SOD) by employing modified
techniques at different evolutionary level.
2.5 MUTAGENIC STDDIKS :- Thallophytes, particularly the
algal plants have been
subjected to the treatments of various chemical mutagens and
nuclear radiations the treatments of various chemical
mutagens and nuclear radiations by many workers all over the
world. The effect of chemicals have largely been studied by
various workers to solve taxonomic problems.
The use of physical and chemical mutagens greatly
promoted the studies on algal genetics through the production
of mutants (Sarma L Singh 1974, 1977). Irradiation studies
on eukaryotic algae particularly with reference to their
karyology are very scanty and have been reviewed by Godward
(1962) and Vidyawati & Sathaiah (1984).
The Charophytes have feebly been subjected to
various chemicals in the past (Turner, 1970; Chatterjee &
Sharma, 1972; Sarma & Tripathi, 1976 a,b, & c).
Labh & Verma (1979) Induced morphological variations
:: 17 : :
in Chara species with E.M.S. (1981), Bhat.nagar & Johrl
(1985); Bhatnagar & Kirti (1988); Bhafnagar BT al. (1989)
made notable contributions in this field. Bhatnagar & Johrl
(1985) for the first time studied the applicability of
Triacantanol on Chara braunii and observed chromosomal
aberrations. Bhatnagar (1990) studied EMS action and noted
14 types karyological aberrations.
Sarma & Khan (1967) studied X-ray effect on
choromosomes of N. f lagillif ormis. After that Sarma 6e Singh
(1974); Sharma & Tripathi (1976 a,b) made val-uable
contributions.
Though several charophycologists from India and
abroad investigated the frequency and nature of various
induced aberrations alongwith mitotic index, but none of them
have tried firmly to make their application in solving the
speciation problem, evolutionary and phylogenetic
controversies.
2.6 ECOLOGICAL AND ENVIROHMENTAL FACTORS :- The charophytes
have been
studied mainly from morphological, systematical and
karyological point of view, while little attention has been
paid to their ecology and water environment.
Anderson & Lommasson (1958) for the first time
studied the effect of temperature on the growth of Chara
: : 18 : :
zeylanlca. After then Gonzalves & Vaidya (I960)", Forsberg
(1965), Vaidya (1967) made valuable contribution in the
ecology and environmental conditions of Chara, in which they
grow. Langangen (19A7) gave the account of Norwegian
charophyta in relation to their ecology. Bhatnagar (1988)
has found that various ecological attributes influence the
general morphology and ploidy level of Indian charophyta.
2.7 PHYLOGENY, INTERRELATIONSHIPS AND EVOLUTION :- It is
evident,
that charophyta as a distinct division at par with
cholorophyta possesses a number of conspicuous morphological
features similar to the higher plants. On the basis of its
similarities with higher plants, and dissimilarities with
other members of chlorophyceae , Groves & Bullock-Webster
(1920) considered it as a distinct phylum. Round (1963)
emphasized affinities between Bryophyta and charophyta, are
clearly reflected in the presence of complex protection of
egg cell, the formation of sterile wall around spermatozoids
mother cell, the structure of spermatozoids, the protonemal
germination and differentiated plant body (thallus).
Haplontic life cylce of charophyta is entirely dissimilar to
the haplobiontic life cycle of Bryophyta. Vaidya (1963)
expressed his view "when all characters of charophytes are
weighed, its separation into a distinct division seems to be
19
more logical". Thus treatment of this group as Division
charophyta at par with chlorophyta suggested by Papenfuss
(1955), Desikacharya & Sunderlingam (1962) and Silva (1962)
is more justified among algae and not to be excluded from
algae.
The probable chaetophoralean origin of charophyta as
proposed by Desikacharya & Sunderlingam (1962) are totally
discarded by Sharma (196A) and Sarma e_t al. (1970), while
analysing the phylogeny and interrelationships of charophyta
on the basis of cytological evidences. These two groups have
possess vast differences in their chromosomal morphology.
Chromosomes of charophyta are generally much larger in size
and characterised by median, submedlan, terminal and
subterminal centromeres. It seems that morphological
similarities between these two groups as emphasized by
Desikacharya & Sunderlingam (1962) are due to parallel
evolution which is very common in algal classes (Fritsch,
1935). The view of Sarma ££ al. (1970) have been later on
supported by Bhatnagar & Saxena (1990, 1991) on the basis of
chemical studies. According to them the presence of
Glycolate oxidase and SOD marks the biochemicals affinity of
this group to the land plants.
Womersley and Ophel (19A7) and Kasaki (196A)
suggested their scheme for evolution of charophyta on gross
: : 20 : :
morphological grounds like cortication, branchlet furcation,
number of corona cells which are still the valid taxonomic
criteria. Since then, Desikacharya & Sunderlingam (1962),
Wood & Imahori (1965), Guerlesquin (1967), Sharma et al.
(1970), Bhatnagar (1987, 1988) presented their views on the
evolution within charophyta. Very recently khan (1982), Khan
& Sarma (1987) discussed pattern of evolution and phylogeny
of charophyta.
2.8 SPECIATION :- Speciation in charophytes appears to
have occured through gradual
divergence of geographically separate populations, geographic
allopatric speciation and / or sympatric speciation. It is
apparent from the breeding pattern of allopatric and
sympatric population (Grant and Proctor 1972; Proctor, 1975).
Khan & Sarma (1984) noted that the interbreeding pattern
within a species promotes local differentiation. Khan &
Sarma (1985) contributed in the field of charophyta with
particular reference to their karyology viz. on ploidy level.
2.9 PHYTOGEOGRAPHICAL STUDIES :- Phyrogeographical
distribution of 22
taxa from Japan including Chara braunii Gm., C . corallina
Willd. C^. benthaaii A. Br., C. gymnopltys A Br., £- vulgaris
etc. were determined by Imahori (1954, 1955, 1957).
Patterson (1964) has provided notes on the ecology of
: 21
Tolypella gloaerata (Desv.) Leonh. alongwith physico-chemical
properties of the water. He stated that irregular
appearance of the Charophyte mainly depends upon weather
conditions and show two peaks of germination in one year,
i.e. in summer and spring. Guerlesquin and Vaquer (1980)
surveyed the Charophytes flora of Camarquc and collected 10
taxa alongwith principal physical and chemical
characteristics which make it possible to establish the
conditions of their development in the paddy fields.
Corilllan & Reviers (1985) described the impact of
environmental factors and climatic conditions caused some
morphological modifications of Tolypella antractica (A.Br.).
Guerlesquin e_t al. (1987) conducted morphological and
ecological investigations on Lamprothamnium succinatum A.Br.
The scarcity of water and dissolved gases adversely affects
the growth and development of sex organs in charophytes (Pal
at. al. 1962). Correlation between the ecological attributes
and their chromosome number of the charophyta have been
described by Bhatnagar (1988). Imahori (1962) studied that
despersal factors influenced the migration of charophytes.
Other Important attempts to understand geographical
distribution of charophytes have been studied by Kelner
(1944), Imahori & Suga (1958), Wood & Imahori (1959), Griffin
& Proctor (1961), Imahori & choe (1963), Corlllion &
:: 22 : :
Guerlesquln (1971, 1983), Langangen (1974), Khan & Sarma
(1981), Khan (1991) and others.
Charophytes occur in all t-he continents except
Antarctica representing AAO taxa out of which 125 are known
to occur from Indian region. After the analysis of available
data, it has observed that out of total 440 taxa, 274 are
endemic 151 restricted, 7 cosmopolitan and 8 are
sub-cosmopolitan. The seven cosmopolitan taxa are C.
braunli," C. globularis, C. vulgaris, £. contrarla, V.
gracilis, F[. hyalina and Tolypella glomerata, C. zeylanica,
C. flexills,yv. acualnala, H. opaca, ^. mucronata, JM.
oligospira, JM. tenuissima and 2i' batrachosperma are
sub-cosmopolitan (Pal e_t a_l. 1962; Khan and Sarma 1989).
2.10 CONCEPT OF BASIC CHROMOSOME NUMBERS :- Wood and
Imahori (1965)
have categorised the tribe Nitelleae into three subgenera
viz. Nitella, Tleffallenia and Hyella. The subgenus Nitella
characterised by the basic chromosome number X = 6 except ^,
mlrabllls (n = 9), H^. situratli, 2i« acuminata, whereas,
subgenera Tieffallenia & Hyella are characterised by X " 9.
The basic chromosome number for the genus Chara was
first suggested by Moutschen, Dahmen & Gillet (1956) as <<7>>
is supported by Bhattacharya (1972), Gillet (1959)
: : 23 ::
Guerlesquin (1961, 1967); Hotchkiss (1958, 1964), Khan &
Sarma (1967), Sarma (1973), Sinha & Verma (1976).
The concept: of basic chromosome numbers X " 7 for
the genus Chara received a concrete support by the reports of
n • 7 in Chara braunii (Sarma & Khan 1965), and £. vulgaris
(Sinha & Verma 1969), called it a natural polyploidization.
Gillet (1959) suggested x = 6 as the basic
chromosome number for the genus Nitella. Guerlesquin (1961,
1967) has however suggested <<6>> and <<7>> as the basic
chromosome number for this genus. Hotchkiss (1963, 1964)
supported the views of Gillet (1959) and proposed additional
basic chromosome number X = 9 for artho-and anarthrodactylous
forms of Nitella. Later on Sarma & Khan (1964) suggested X •
3 as the basic chromosome number for the genus Nitella on the
basis of karyotype analysis of n. nirabilis (n=6). Bhatnagar
(1983) also supported the view of X = 3 as the basic
chromosome number for this genus. Guerlesquin (1969, 1963,
1967); Bhattacharya (1972) suggested the basic chromosome
number for the genus TolypeHa is x = 5. But Hotchkiss
(1966) revised the basic chromosome number for Tolypella as x
•• 11, which has been largely supported by Sarma & Ramjee
(1969); Sawa (1973, 1974); Ramjee & Bhatnagar (1978).
Simultaneously, a chromosome count of n=8 by Sawa (1973,
1974) appeared as exception to the series of x = 11. This
2A
chromosome number has also been considered as a new base
chromosome number for the genus Tolypella. Thus t:he three
basic chromosome numbers viz. x " 5, 8, & 11 have been
suggested for the gemls Tolypella.
Here it is clear that the base chromosome number of
tribe chreae is x~7, tribe Nitelleae is x'"3 & tribe
Tolypelleae is x"5, 8 & 11 have been ascertaied the
hypothetical scheme of their origin as pronounced by Sawa
(197A).
2.11 KARYOLOGICAL STDDIES :- Much progress has been made
in the field of karyology
of charophytes from India particularly during the last three
decades. Sunderlingam (19^7) was the first worker, who
reported chromosome number in Chara zeylanica (n-28) for the
first time in India. Later on, a considerable attention was
paid by 5arma & Khan (1964, 1965a, 1965b, 1965c, & 1966),
Khan & Sarma (1967a, b, (, c), Sarma & Ramjee (1967, 1969,
1967) & Ramjee & Sarma (1971) on Karyology and
Karyomorphology of Indian charophyta & provided a great
impetus. Noor (1969), Sinha & Verma (1970), Ahmad & Sinha
(1973), Bharati & Cheenaveeraiah (1974) Cheenaveeraich &
Bharti (1974), Chatterjee (1976, 1979) Noor & Mukherjee
(1977), Khan & Sarma (1980), Labh & Verma (1985), Ray &
Chatterjee (1987, 1989) Bhatnagar & Johri (1991) & others
: 25 :
have made sincere attempt to count chromosome numbers &
analyse karyomorphologlcally. Chromosome numbers reported
from various parts of India have been summarised in table -
I, II, III, IV, & V.
On the basis of available data Sarma & Ramjee
(19A2) & Bhatnagar (1983) discussed significance of
chromosome numbers in charophyta. Euplold chromosome numbers
i.e. n-7 1A,28,35, 42, 49, 56 & 70 for the genus Chara; n-6,
9, 12, 15, 18, 21, 24, 27, 36 & 48 for the genus Nitella;
n-10, 15, 20, 25 & 50 for the genus Tolypella; n = 14, 28, 35
for the genus Lychnotha«DU8, & n " 28 for the Nitellopsis
have been recorded. On the basis of above euplold series the
concept of basic chromosome numbers x = 7 for tribe chareae,
X - 3 for tribe Nitelleae & x " 5, 8, 11 for tribe
Tolypelleae have been justified. Although some aneuploid
chromosome numbers of charophytes usually from Europe and
America, n " 12, Corillion ^ - aO.. (1959); n=16. Oehlkers
(1916); n - 18, Lindenbein (1927); n - 24, Lindenbein (1927);
n - 26, Geitler (1949) & n = 32 Gillet (1959) for the tribe
Chareae and n = 14, Sato (1959), Imahori & Kato (1961); n
-16, Gillet (1959); n - 17, Lindenbein (1927); n - 28,
Imahori & Kato (1961) & n = 34, Karling (1926) for the tribe
Nitelleae have been reported. However, some aneuploid
chromosome numbers have also been reported from some Indian
26
forms of charophytes n = 37; Cheenaveeraeah & Bharati (1974);
11-8, Noor & Mukherjee (1975) n = 27, Prasad & Verma (1985); n
= 48, Bhatnagar & Johri (1986) in tribe Chareae and n = 29;
Bhatnagar & Johri (1986); n = 8, Subramaniam et: al. (1991) in
tribe Nitelleae. Occurrence of aneuploid chromosome numbers
in contrast to euplolds, it seems that these aneuploidy are
either due to counting error or may be due to faulty
techniques (Khan & Sarma, 1987) .Because the availability of
the conclusive evidences concerning the behaviour of the
meiotic chromosomes as no success has yet been achieved in
meiotic studies.
The chromosome morphology has been considered as one
of the important criteria In the cytotaxonomlcal studies. In
India studies based on the chromosome morphology the
karyotypes of several species of charophytes have been
prepared by Sarma & Khan (1965); Sarma (1968); Sarma & Ramjee
(1971); Sinha & Ahmad (1974), Chatterjee (1976, 1979); Ray &
Chatterjee (1988), have taken into consideration the TF%
values when analysing chromosomes (TFX = ratio in percentage
of the total sum of short arm lengths to the total length of
the chromosomes).
Based on Karyological studies the taxonomlc validity
of C. wallichii. vandalurensis, N. opaca, N.
f lagellif orBJs, h^. globularis, & Ji. annandalel have been
established by Sarma & Khan (1967); Sinha & Ahmad (1974) and
:: 27 : :
Chatterjee (1974).
2.12 CYTOGEOGRAPHY AND CYTOSYSTEMATICS :- Cyt--otaxonomlc
dat-.a contiributed
by various workers on the charophyta have proved t-o be of
immense value in the cytogeographic and cytosystematic
assesment of the group.
Khan & Sarma (1984) gave a persual of cytological
literature, that reveals so far about 383 different
individuals of charophytes populations related to 168
(38.79%) taxa of all the six genera have been investigated
from various zones. The reported chromosome numbers for 76
(40.00%) taxa of Chara, 3 (37.50%) taxa of Lamprothamnium;
76(35.65%) taxa of Nitella and 9(60.00%) taxa of Tolypella.
exclusively, proves the occurrence of ploidy in the
charophyta with basic chromosome number x=7 in chareae, x"3
in Nitella and x"3 & 5 In Tolypella.
Generally, most of the monoecious taxa have
chromosome numbers twice that of the morphologically similar
related dioecious taxa, based on which Wood & Imahorl (1965)
expresed the opinion that the dioecious taxa represents
genetlic variance of the monoecious ones. The opinion of
these workers is untanable as already demonstrated by
Corrillion & Guerlesquln (1959), Sarma & Khan (1964, 1965),
Sawa (1965), Procotor (1971).
: : 28 ::
All the known chromosome numbers of charophytes are
not distributed evenly in every zones (North America, South
America, Africa, Europe, Asia, India, Pacific, Australia).
According to Khan & Sarma (1984) from the distribution
frequency of a known chromosome number for genera indicating
the zones in which they occur, it is evident that the
chromosome numbers n"i4, 28, 42 in Chara and n=12, 18 in
Nitella have maximum distribution frequencies of 87.50% to
100.00% and 62.50% to 87.50% respectively. Perhaps they
represent more successful adaptable genome in comparison to
less frequent (50.00%) chromosome numbers, like n « 56 in
Chara and n - 6,9,48 in Nitella.
2.13 POLYPLOID COMPLEX :- Polyploidy is the more common
in charophytes. The
polyploid complex of charophyta contains even and odd
ploidylevel of chromosomes ranging from 3x to lOx in the
chareae and upto 16x in the Nitelleae.
The polyploidization has succeeded in the dioecious
taxa but ro a very limited extend, while in the monoecious
taxa polyploids of higher order n = 70 in, Chara globularis,
Chara geylaaica; n " 70 in LaMprothaanium populasum; n = 48
in Nitella f urcata, and H. pseudoflaballata and n * 50 in
Tolypella sallna are recorded (Khan & Sarma (1985)).
:: 29 : :
On fhe basis of euploid series of Chromosome numbers
recorded in charophytes, basic number x=7 for Chara, x = 3
for Nitella. X"5 for Tolypella (Sarma & Khan; 1964) have been
suggested. The suggested basic numbers x " 7 and x • 3 also
appear to be basic chromosome numbers for the Tribe Chareae,
Nitelleae respectively. The lowest chromosome number in
living chareae (n"l4) and Nitelleae (n=6) may be considered
as secondary basic chromosome numbers.
According to Khan & Sarma (1985) the frequency of
polyploidy with reference to primary and secondary basic
numbers is 8A.12Z and 49.73% in charophyta; 70.71% and 25.96%
in chareae and 96.44Z and 71.57% in Nitelleae respectively.
The highest frequency is recorded for monoecious and lowest
for dioecious taxa.
Occurrence of aneuploidy in charophyta may be on
account of either technical or counting errors as chromosome
numbers in related taxa are found to be very sticky and
fairly large (Khan & Sarma, 1967) or may be the result of
non-disjunction which led to increased or decreased
chromosome number before game-togenesis in the antherldial
filaments, (c.f. Moutschen, Dahmen & Gillet, 1956).
Polyloidy in charophytes caused by various ways :-
: : JO : :
A) POLYPLOIDY AND GENE DUPLICATION :- The electrophoresis
analysis of genetic
variation in natural population of charophytes (Grant. &
Proctor, 1980) indicate that all the extent forms have
undergone atleast one polyploidization or more likely, are
derived from the ancestors which did so. The
polyploidization led to the duplication of the functional
genes in their nuclei due to which are likely to tolerate
the loss of one or more chromosome pairs and led to the
modified series of polyploidy viz. n •• 8 & 11 in Tolypella
(Khan & Sarma; 1985).
B) POLYPLOIDY AND pH :- The existence of two
karyological races in Nitella
opaca,one with n"6 growing in sub-neutral or slightly
alkaline water while the other with n = 12 in acidic
environment. Indicates some correlation of polyploidy with
the pH (Guerlesquin, 1967).
C) POLYPLOIDY AND SALINITY :- Some charophytes, mostly
dioecious and a few
monoecious, growing in coastal saline ponds, are found to
have usually high chromosome numbers, e.g. Chara hornemannil
(n=28 D) Tolypella salina (N = 50 M) (Khan & Sarma, 1968).
:: 31 : :
D) POLYPLOIDY AHD ALTITDDB, LATITUDE. LONGITUDE;-
Some Indian charophytes collected from an
altitude 1800 m (Kashmir) and 2,330 m (Assam) as well as
from Plains (100m) show ployploid races within a species
complex which are restricted to high altitude. However, in
C. vulgaris the situation is just reverse and the taxa
revealed low chromosome number (n*14) at high altitude and
high number (n"36) in plains. This relation of ploidy with
altitude becomes evident to some extent (Ramjee & Sarma,
1971; Sarma 1968; Khan, 1991).
Sarma & Khan (1984), Khan (1991) studied the
chromosome number in relation to latitude and longitude.
According to them highest chromosome number n=48 in Nitella
f areata and fl. pseudoflabellata are found to be restricted
conicidently along the N. 30°-A5° latitude, E 110° Longitude
and S 30''-45° latitude, E-110°, Longitude respectively. The
chromosome number n " 70 in Chara Robularis and C^. zeylanica
is restricted along W-100 Longitude.
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CHAPTER - III
MATERIALS AND METHODS
3.1 COLLECTION :- The algal materials investigated in
the present work will be collected
from rice fields, ditches, temporary and permanent ponds or
reservlors, canals and rivers chiefly in certain localities
of Indian subcontinent specially from Rajasthan, Haryana,
Himachal Pradesh and Jammu provinces. The material will be
collected in the form of living specimens in polythene bags.
The materials will be collected from different places and
brought to the laboratory in the same water, in which they
will be growing in the natural environment.
3.2 PRESERVATIOM i- For systematic study, the
appropriate portion of thalli will
be preserved in the form of a dried herbarium specimens, and
fixed and preserved in AX formalin for systematic
identification and karyomorphological studies. Some
features such as plant height and colour will be recorded
from living materials. All materials will be identified by
the monograph of Wood & Imahori (1965) and Pal et^. al.
(1962).
3.3 KARYOLOGICAL FIXATION :- Karyological observations
will be made from young
antheridial filaments of charophytes. Various portions of
:: 33 : :
young vegetative portions and apices will be used for the
nuclear division (Khan, 1965). in the present investigation
for karyological studies, the young antherldlal filaments
will be fixed in a mixture of 3 part absolute alcohol and 1
part glacial acetic acid (Carnoy's solution) between 7 p.m.
and 10 p.m. with the 15 minutes interval. Within 3 hours 12
antherldlal filaments will be fixed for profile divisions.
Because prolific division figures are generally obtained
between 7 p.m. and 10 p.m. in all charophycean material
(Khan, 1964).
3.4 CLIMATIC AND ECOLOGICAL PARAMETERS :- Various climatic
and ecological
attributes like pH, depth of water etc. were recorded.
Ecological parameters will be recorded from the water
sample, which was collected from collection site. Recorded
climatic and ecological parameters for investigation will be
as below :-
1. Altitude
11. Latitude
ill. Longitude
Iv. Frozen relative humidity
V. Depth of water
vi. Temperature of atmosphere
vll. Temperature of wftter
vili. Specific conductivity of water x 20.10
:: 3A : :
ix. Acidity or pH of water
X. DOD
xi. Turbidity of water
3.5 KARYOLOGICAL STDDIES :- Karyological investigations
will be made by using
GODWARD'S iron-alum acetocarmine squash technique (1948).
This method will be employed throughout the present
investigation successfully for karyological studies.
3.5.1 PRETREATMENT :- Mordanting in 4% iron-alum
solution for 3-5 minutes will be
found helpful for securing well stained preparations. For
preparing 4Z iron-alum solution 4 gm. Ammonium ferric
sulphate (NH^Fe(SO^)2•I2H2O) dissolved in 100 ml. distilled
water at room temperature.
3.5.2 STUDY OF MITOSIS :- The mitosis will be studied
from young antheridial
filaments. After mordanting, the antheridial tips will be
washed through simple water 2-3 times. Acetocarmine squash
of antheridial filaments will be made with 2% acetocarmine
solution. Repeated gentle heating and thumb pressing will
be applied for chromosomal spreading and proper staining.
All the observations will be made from the temporary slides,
which will be made permanent by using normal butyl schedule
• • IS • •
(Bhaduri and Ghose, 1954). The chromosome numbers will be
determined from premetaphase, mevaphase and anaphase plate.
3.5.3 MICROPHOTOGRAPHY :- All the microphotographs
will taken on "Prior"
microphotographic microscope with the aid of lOx compound
eye-pieces and apochromatic oil immersion objectives
(100/1.25/160). Minolta camera will be improvised and roll
films (Agfa-copex) as well as cut films (DK-5) will be used
for taking microphotographs. An illford tricolour green for
filter enhanced/ the contrast and details effectively. All
micrphotographs are of equal magnification i.e. X2000.
3.5.4 CAMERA LUCIDA DRAWING :- For karyotype analysis
camera lucida drawing
of suitable division phase from temporary preparations will
be drawn on drawing paper with "Olympus compound
microscope". It is drawn with the aid of lOX compound eye
pieces and apochromatic oil immersion objectives (100/1.30).
measurements for karyonorphologlcal studies will be taken
with the help of occular scale.
3.6 CHARACTERS TO BE STUDIED :- For the determination
of various qualitative
and quantitative characters to be studied for
cytosystematics, karyomorphological and karyological
investigation :
: : 36 : :
3.6.1 PRINCIPLES EMPLOYED FOR SYSTEMATIC INVESTIGATIONS
OF DIFFERENT TAXA :- For systamattic study of
charophytes some morphological
features have been found useful. These features will be
studied for both families of charophytes (i. Nitalleae and
11. Chareae) .
3.6.1.1 SOME IMPORTANT FEATURES FOR THE SYSTEMATIC
IDENTIFICATION OF NITALLEAE :-
—Plant height
- Dioecious / Monoecious
- Homoeoclemous/ Heteroclemous
- Branchlet furcation
- Shape of dactyls
acute
confluent
allantoid
acuminate
mucronate
- Number of cells per dactyl
- Dactyls abbreviated
elongated
- Gametanglal position
at branchlet node
at branchlet base
:: 37 : :
lateral
apical
- Gametangia stalked
Sessile
- Number of gaoetangla at a place
antheridium
oogonium
- Coronula tiers equal
unequal
- Oospore wall oranmentation
- Antheridium octosculate / totraosculate
3.6.1.2 SOME KIMPORTANT FEATURES FOR THE SYSTEMATIC
IDENTIFICATION OF Chareae :-
- Plant height
- Monoecious / Dioecious
- Diameter of axis
- Axial cortex absent / present
haplostichous
di. plostichous
triplostichous
- Stipulodes Haplostephanous/diplostephanous
rudimentry/elongated
- Branchlet number
38
• Branchlet cortex
haplostichous
diplosMchous
r. riplostichous
• Relative position of stipulodes and branchlet
alternate
opposite
- Branchlet diameter
• Branchlet length
• Basal branchlet segment phloeopodous
discoloured
gymnopodous
- Bract cells rudlmentry/elongated
- Brancteol number
length
- Spine cell present /absent
- Gametangial position on branchlet node
branchlet base
- Number of Gametangla at each node
antheridium
oogonium
• Gametangla conjoint/sejoint
- Length of oogonium
• breadth of oogonium
-AntherldluB octosculate/tetraosculate
:: 39 : :
- Diameter of anrheridium
- Length of oospore
- Breadth of oospore
3.6.2 PRINCIPLES EMPLOYED IH KARYOMORPHOLOGICAL STUDY OF
VARIODS TAXA :-
The karyomorphological features, which have been
found to be useful In the present investigation, are as
given below :-
- Dividing cell length
width
position In filament
- Size of Interphase nucleus
- Nucleolus number
Size
- Metaphase plate length
Width
position in cell
- Chromosome number
- Number of chromocentre
3.6.3 PRINCIPLES EMPLOYED IN KARYOTYPE STUDY OF VARIOUS
TAXA :-
The karyological features which have been found
to be useful in the present study besides the chromosome
number, on the basis of which comparison of karyotype of
different taxa are made, are as given below -
AO : :
a Length of chromosomes
b Thickness of chromosomes
c Position of chromocentre
3.6.3.1 LENGTH OF CHROMOSOMES :- On the basis of the
size of chromosomes
at meta phase, the chromosomes are grouped arbitrarily into
A groups as suggested by the Sarma (1959).
- large size 5-10 li (L)
- Medium size 3-5 ii (M)
- Small size 1-3 li (S)*
- Minute size .25-1 Jb (Mi)
* abbreviations used in the karyotype formulae
3.6.3.2 BREADTH OF CHROMOSOMES :- Due to marked
differences in the
breadth of chromosomes of different taxa, they are
classified into three groups again arbitrarily -
i. Thick (more than 1 L wide)
ii. Moderately thick (1 ix wide)
iii. Slender (less than 1 ii wide)
3.6.3.3 POSITION OF CENTROMETERS :- Since in most cases a
well defined centrom
eres has not been observed either at metaphase or anaphase,
the cetromeric position has been assessed only by the nature
and extent of bending and shapes of anaphase chromosomes.
The scheme proposed by Levan et. al. (196A) as to the
: : 41 ::
terminology regarding the position of centromeres has been
adopted to a considerable extent. Chromosomes are
classified into the following 4 categories, assuming the
total length of chromosomes, to be composed of 10 units.
- Chromosomes with median centromeres (m)
Chromosomes with sub-median centromeres (Sm)
Chromosomes with terminal centromeres (St)
- Chromosomes with terminal centromeres (t)
The term "terminal" centromeres is employed here
from a strictly descriptive and practical point of view,
without any prejudice to the view held by several
cytologists that theoretically there are no terminal
centromeres in normal chromosomes.
3.7 KARYOTYPE PORMOLA :- In order to facilitate
comparison of two karyotypes of
different chromosome numbers, formulae employing the
notations given above, are given to characterise each
karyotype under cytologlcal descriptions of individual
species given by Khan (1968) e.g.
Chromosome number, n " 14, thick.
Karyotype formula :
* dm + ISm)^ + dm + 6Sm + It)^ + (3Sm + 1 St)„* h n o
Where,
L " long sized chromosomes
: : 42 : :
M •* Median sized chromosomes
S *• Small sized chromosomes
m " Chromosome with median centromere
Sm •• Chromosome with submedlan centromere
t. " chromosome with terminal centromere
St • chromosomes with subterminal centromere
Which means that out of 14 chromosomes, two are long, eight
are medium sized and 4 are small. Of the two long
chromosomes, one has median centromeres and another a
submedlan centromeres; of the eight medium sized chromosomes
one has a median centromeres, six have submedlan centromeres
and one terminal centromere, of the four small chromosomes,
three have submedlan centromeres, and one have subterminal
centromeres.
3.8 STATISTICAL ANALYSIS :- Taxonomical distinction of
taxa at hand has been
discussed in the light of conclusions drawn from on the
basis of karyotype data like chromosome number, total
chromatin, length, TFZ and coefficient of variation.
3.8.1 TOTAL FORM PERCENT (TFZ) :- Chromosome preparations
for studying karyotypic
details were prepared from actively growing cells of
antheridial filaments. Chromosome morphology was designated
according to Levan e^. a .' (1964) on the basis of
:: 4 3 : :
centromeric Index value. Total form percent (TF%3 was
calculated according to Huzlwara (1962) and Kapoor & Love
(1970) and the grouping of chromosome according to length as
was done by Khan & Sarma (1967).
i e TFX " Total sum of short arm length ^ J QQ Total sum of chromosome length
TF% =• Ratio in percentage of total sum of short arm lengths to the total length of chromosomes.
3.8.2 COEPFICIEHT VARIATION (C.V.) :- Coefficient
variation will be
calculated from the standard deviation and mean length of
chromosome.
„ _ Standard deviation ^ 2 Q Q Mean length of chromosome
3.9 CDLTDRE STUDIES :- Charophytes were grown under
controlled conditions using
various media by different authors (Imahori, 1963; Forsberg,
1965; Smith, 1966; Andrews et_ £L1. 1984) for experimental,
taxonomical and physiological studies.
For meiotlc, cytogenetic and hybridization
studies pure culture of Imahori and Iwasasl, K. (1963) was
widely used.
: : AA : :
3.9.1 CONSTITDEMTS OF CDLTORE :-
0 A B
1. KNO- 5xl0~^ M 0.000505 gms/lirre 0.05 gm/100 c.c.
2. Gael 2.5xlO'^M 0.00275 gms/litre 0.275 gm/100 c.c.
3. Ca(NO-), 2.5xl0~^M O.OOAl gms/lltre O.Al gm/100 c.c.
A. K2HPO, 2xl0~^ 0.0272 gm/litre 2.7 gm/100 c.c.
5. MgSO. 10"^ 0.00015 gms/litre 0.015 gm/100 c.c.
6. Casela hydrolysate
7. Sodium hypochlorire
8. CH^COGH
9. Agar
3..9.2 PREPARATION OF CULTURE :- Stock solution of column
".B'wlll be added 1 c.c.
each from every item no. 1-5 and volume will be made 1000
c.c. by adding rest 995 c.c. of double distilled water.
Above mixture will provide the requirement specified in
original column 0.
3.9.3 PROCEDURE :- Oospore chilled at 0°C for 10 days
were sterilized in 1% sodium
hypochlorite solution for 5 minutes followed by washing in
20 times water. If oospore are calcified, decalcification
has been done in 3Z acetic acid than chilled. So, chilled
and sterilized oospores are inoculated in each test tube
(120 mm X 27 mm) containing 0.5 gm fine meshed clay and 50
ml. water autoclaved for its germination.
: : A5 ::
Culture will be carried out in a sterilized
cabinet at a temperature of 25° C illuminated intermit-ently
(12 hrs. light, 12 hrs. dark) at a light intensity of above
1500 lux from flurascent lamp.
After gerfflination for 8-10 days, germinated
oospores are transferred in an organic liquid media 0.01% of
casein hydrolysate will be added for promoting adult plant.
Simultaneously addition of G.A. (20 ppm) and kinatin (1 ppm)
promotes the growth of adult plant and not- of protonema.
o o o o o o o o o
o o o
o o
CHAPTER - IV
PLAN OF WORK
Ir is clear from the brief review of lit-.erature
that the work on karyology and karyomorphology of Indian
charophyta is quite meagre and a lot of it is left
untouched. Although the chromosome numbers of several taxa
have been worked out but still a large number of taxa
particularly from North - Western region (Haryana, Jammu,
Himachal Pradesh and Rajasthan) remain to be investigated.
Hence, in order to achieve the proposed above objective
study will greatly help in the elucidation of
karyosystematics, evolutionary and phytogeographic trends
among charophyta. The detailed plan of work is as under :-
i. Floristic survey of charophyta from unexplored or
less explored regions with reference to some
climatic and ecological parameters.
ii. Determination of their vegetative and fruiting
periods in relation to climatic factors like rain
fall, temperature etc.
iil. Identification of collected specimens upto
species level with the help of standard monograph
and flora.
iv. Fixation and preservation of taxa in the field
with appropriate fixatives and preservatives.
47
V .
vi.
vii.
vlii
Ix
Studies on karyology and karyomorphology i.e.
determination of chromosome number of taxa and
other nuclear characters like size of nucleus and
nucleolus, size of dividing cell etc. employing
modern cytological techniques.
Elucidation of karyotype pattern.
Correlation of cytological data with
a) Taxonomic criteria
b) Geographical distribution of species.
Calculation of form percent (TF%) value and
coefficient of variation (C.V.) for karyotype
study.
Utilization of successful cultures for meiotic,
cytogenetic and hybridization studies.
o o o o o
o o o o
o o o
o o
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• • S7 • •
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