ikakter of ^po^opl^ - ir.amu.ac.inir.amu.ac.in/7208/1/ds 2154.pdf · material and methods 32 - 45...

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

DS2154

• M i l 'J_»'A >1_ML »MM L'

l> V vr V • «

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

o o

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

LITERATDRE CITED

AGARKAR, D.S., 1963 : Systematic studies of the Charophytes

of Gwalior, Madhya Pradesh. The vlkram, 7: 43-51.

AGARKAR, D.S., 1967: A note on Chara vandalurensie Sund.

from Gwalior (M.P.). Science & Culture, 33: 59-60

AGARKAR, S.P. & KUNDU,B.C., 1937 : Charophytes of Bengal.

J. Dept. Science, Calcutta Univ., 1 : 1-23.

AHMAD, S. & SINHA, J.P., 1973 : Chromosome numbers In some

taxa of Chara Linn, from Bihar. Proc. Ind. Sci.

Cong. Asaoc. : 302.

ALLEN, G.O., 1925 : Notes on Charophytes from Gonda, U.P.

J. Boabay Nat. Hist. Soc, 30 : 589-599.

ALLEN, G.O., 1928 : Notes on charophytes from saharanpur,

U.P. J.Ind. Bot, S o c , 7 : 49-61.

ALLEN, G.O., 1933 : Charophyte notes from Agra, U.P. J. Ind.

Bot. S o c , 12 : 17-19.

ALLEN, G.O., 1936 : Charophyte notes from Bareilly, U.P.

J. Ind. Bot. Soc, 15 : 51-54.

ALLEN, G.O., 1961 : Charophyte notes from Banaras, U.P.

J. Ind. Bot. Soc. 40 : 234-242.

ANAND, V.K. & LANCER, G., 1988. Occurrence of charophytes

in Jammu region. Phykos, 27 : 25-27.

: : A9 : :

ANDERSON, R.G. & LOMMASSON, R.C., 1958 : Some effects of

temperature on the growth of Chara zeylanica Willd.

J. Butler DaiverBlty Botanical Studies, 13 :113-120

ANDREW, S. & IMAHORI, K. , 1984 : Culture of Chara hlsplda

Br. PhycoloRla. 19 : 589-599.

BANDHOPADHYA, S. & CHATTERJEE, P., 1989 : Chara fibrosa var.

fibrosa f. keukensls, A new record for India and

its chromosome number. Phykos, 28 : 178-180.

BHARATHAN, S., 1983 : Development morphology of Nltellopsls

obtusa (Desv.) Groves. Proc. Ind. Acad. Scl., 29 :

373-379.

BHARATHAN, S., 1987 : The basal node of the branchlet in the

taxonomy of Characeae. J. Ind. Bot. Soc., 66: 345-349

BHARATHAN, S. & SUNDERLINGAM, V.S., 1984a.: Taxonomic

validity of Chara vandalurensis Sund. Phykos, 23:

15-20.

BHARATHAN, S. & SUNDERLINGAM, V.S., 1984b. : Developmental

morphology of LaaprothamniuM papulosum (Wallr.) J.

Groves & LaMprothamniua succinatum (A.Br.) RDW.

J. Ind. Bot. Soc. 63 : 97-107.

BHARATI, S.G. & CHEENAVEERAIAH, M.S., 1971 : Lychnothamnus

bidarii Gm. et• Sp. nov., a new taxon in

charophyta. Proc, Ind. Scl. Cong. Assoc. : 387.

:: 50 : :

BHARATI, S.G. & CHEENAVEE RAIAH, M.S., 1974 : Cytotaxonomical

studies on Chara zeylanlca complex. Inter. Symp. on

taxoaoay of Algae, MADRAS : pp. 3.

BHARATI, S.G. & CHEENAVEERAIAH, M.S., 1983 : Lycbnothamnus

barbatus var. Rlgantli nov. ( A new variety).

Cytologia. 48 : 843-846.

BHATNAGAR, S.K., 1981 : Cytotaxonomy of charophytes of

Rohllkhand division and effect of different

chemicals on selected taxa. Ph.D. Thesis, Rohllkhand

Dniversity, Bareilly, India.

BHATNAGAR, S.K., 1983: The concept of basic chromosome

numbers In Charophyta, A review.

Cryptogaalc-AlRoloRlc (France), 4 : 111-116.

BHATNAGAR, S.K., 1987 : Phylogeny and Interrelationships of

the genus Tolypella, A multldlsciplinary outlook.

Phykos, 26 : 140-148.

BHATNAGAR, S.K., 1988a : The first cytotaxonomlc report In

Chara braunii f. novJBexlcana (Div. Charophyta).

Curr. Sci». 57 : 88-90.

BHATNAGAR, S.K., 1988b : Nltella hyallna f. hyallna (D.C.)

Ag . RDW, A new chromosome count and Its cytotaxonomy

curr. Sci.. 57 : 343-344.

:: 51 : :

BHATNAGAR, S.K., 1988c : A karyological approach to phylogeny

and Interrelationship of charophyta. Cytologja, 53:

723-729.

BHATNAGAR, S.K. 1988d : Cytotaxonomy and polyploidy in

relation to various ecological attributes among

Indian charophyta. Geobios, 7 : 143-146.

BHATNAGAR, S.K., 1989a : Polyploidy in relation to sexual

dimorphism in charophyta. Cytologia, 54 : 1-3.

BHATNAGAR, S.K.,1989b ; Cytological prospectives of charophyta

I. Chara ftlobularis complex Phykos, 28 : 156-165.

BHATNAGAR, S.K., 1989c : Cytological prospectives of

charophyta II. Nitella furcata complex. Phykos. 28:

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