Cytologia

Irradiation

Studies

on

the

I. X-Rays

Karyology

and

Y. S. R. K. Sarma Laboratory

of Algal Banaras

Received

October,

Cytology Hindu

1977

of Charophyta

gamma-rays and

S. B. Singh

and Cytogenetics,

University,

42: 279-290,

Department

Varanasi-221005,

of Botany,

India

18 1975

The studies on the effcts of X-rays and gamma-rays on algae are still in their infancy as compared with those on higher plants. The few contributions that have been made so far show that certain algae possess an extra-ordinarily high degree of resistence. Godward (1954) for the first time employed X-rays to study their effect on algal chromosomes. Since then several workers contributed further to our knowledge on this aspect of algal cytogenetics (Jacobson 1957, Prasad and Godward 1968, Dodge and Godward 1963, Sarma and Khan 1967, Patel 1970 etc.). While most of these studies established that higher doses of X-radiation were required to bring about chromosomal aberrations in algae as compared to higher plants, such aberrations were successfully induced by X-rays at low dose ranges by Howard and Horsley (1960) in Oedogonium cardiacum and by Sarma and Khan (1967) in Nitella flagelliformis. Earlier studies on the effects of gamma-radiation on algal chromosomes are fewer as compared with those on X-rays and include those made by Rayns and Godward (unpub.), Gailey and Tolbert (1958), and Leedale (1958). Their obser vations also show that the algal chromosomes are very resistant to gamma-rays. However, Sarma and Singh (1974) reported that the chromosomes of Nitella flagel liformis were affected in much the same way as those of angiosperms in response to gamma-radiation. In the present study, a concerted attempt has been made to assessing the effects of X-rays and gamma-rays on the karyology of some taxa belonging to Characeae, particularly appreciably

selected as experimental materials, since these taxa are characterised by long chromosomes. The latter feature is of great advantage in making

qualitative and quantitative studies of the effects of irradiation on the chromosomes of these taxa and in helping to determine the levels of sensitivity of different taxa towards the X and gamma-radiations. Materials

and methods

The experimental materials which consisted of three species of Nitella (N. opaca Ag., N. flagelliformis (A. Br.) R. D. W., and N. accuminata (A. Br.) ex. Wallm and four species of Chara (C. fibrosa Ag. ex. Bruz. em. R. D. W., C. globularis var. virgata (Kutz.) R. D. W., C. setosa Klein ex. Wild and C, zeylanica var. diaphana f. oerstediana (A. Br.) R. D. W.) were collected from some fresh water ponds situated at Sarnath and Bawan Pokhara in the vicinity of Varanasi. The selected plants

280

Y. S. R. K. Sarma and S. B. Singh

Cytologia

42

were brought to the laboratory in the same pond water in which they were growing in natural environment and were transferred to biphasic (soil-water) culture medium for further study. For and

irradiation

placed

experiments,

in different

were

then

with

X-rays,

exposed

to X-rays

the X-ray

tube

at a distance

of 15cm.

experiments

with gamma-rays,

gamma-rays radiations different ments

doses

5ml.

and gamma-rays

size were of filtered

as the case

was run at 100Kv,

The exposure

were

4mA

may

with

with

the

and

those

Radiation

of the doses

by increasing

carried

the

be.

materials

were

Laboratory,

with X-rays

were carried

Table

of 1mm

Al

exposed

to 80Co

All the

experi

Department

of

out in the Department

of Medical

and gamma-rays

For irradiation

the both types of experiments. The

of exposure.

Institute

which

For the irradiation

time

Medicine,

selected

water

a filtration

out in the Cytogenetics with gamma-rays

carefully pond

rate was 200 rads per min.

petriplates

obtained

were

B. H. U., while

of Radiotherapy The details

of suitable

containing

at a distance of 60cm from the source. With dose range varied from 100 to 2000 rads in various

with X-rays

Zoology,

antheridia

petridishes

Sciences,

are given in Table

B. H.

U.

1.

1.

After irradiation the irradiated materials were fixed at different intervals of time (12, 36, 60 and 84 hr following irradiation) in Carnoy's fixative (3 parts of absolute alcohol and 1 part of glacial acetic acid). The fixed materials were cytolo gically examined by employing Godward's iron alum acetocarmine method (God ward 1948) throughout the course of this investigation. Observations

The chromosome numbers recorded in the present work of the taxa viz., N. opaca (n=6), N. flagelliformis (n=9), N. acuminata (n=18), C. fibrosa (n=14), C. globularis var. virgata (n=14), C. setosa (n=28) and C. zeylanica var. diaphora (n=42) are confirmatory of the findings of Hotchkiss (1963, 1966), Guerlesquin (1964, 1967), Khan and Sarma (1967) and Chatterjee (1972). The karyological

effect on the interphase

and dividing

filaments as a result The most frequently

of irradiation were qualitatively observed effects commonly met

varying

stickiness

laggards,

degrees unequal

were

separation

and

clumping

of daughter

nuclei

and quantitatively with in most of the

of chromosomes,

chromosomes

of spermatogenous

anaphase

to the two

poles,

studied. taxa to bridges, grouping

1977

Irradiation

of chromosomes, nuclei

and For

gamma-rays at metaphase breakage

granulation

occasional the

purpose

and

formation

vacuolization

effects. of X-rays

of interphase

281

nuclei,

degenerating

of micronuclei.

of quantitative

estimates

of different taxa, the effects under and anaphase stages on the basis

at metaphase

of cytological highest dose

Studies on the Karyology of Charophyta I

and anaphase,

of the

effectiveness

of X-rays

and

each of the treatment were estimated of affected cells showing chromosome

and anaphase

bridges

as the main

indicators

The percentage of affected cells at lowest effective dose and and gamma-rays administered in all the taxa are given in

Table 2. Controls were main tained in all the cases. Since the percentages of affected cells with X-rays and gamma-rays at any given dose in a particular taxon were differing only slight ly (cf. Table 2), the results of all the experiments with gamma radiation for all the taxa in vestigated are presented through histograms (Figs. H1 H7).

282

Y. S. R. K. Sarma and S. B. Singh

Cytologia

42

Figs. H1-H7. Hi, histogram showing percentage of affected cells in Nitella opaca subjected to gamma radiation. H2, histogram showing percentage of affected cells in Nitella ftagelliformis subjected to gamma radiation. H3, histogram showing percentage of affected cells in Nitella ac uminata subjected to gamma radiation. H4, histogram showing percentage of affected cells in Chara fibrosa subjected to gamma radiation. H5, histogram showing percentage of affected cells in Chara globularis subjected to gamma radiation. H6, histogram showing percentage of affected cells in Chara setosa subjected to gamma radiation. H7, histogram showing percentage of affected cells in Chara zeylanica subjected to gamma radiation.

Chromosomal effects were recorded even with low loses of 100 rads in case of N. opaca and N. flagelliformis, while no chromosomal abnormalities were visible upto 200 rads in N. acuminata and upto 500 rads in C. fibrosa, C. globularis, C. setosa and C. zeylanica. The most frequently observed effects after irradiation with X-rays and gamma-rays at doses of 100 and 200 rads in N. opaca and N. flagelli formis, 200 and 400 rads in N. acuminata, and 500 to 1000 rads in all the four species of Chara (C. fibrosa, C. globularis, C. setosa and C. zeylanica) were stickiness and clumping of chromosomes at metaphase as well as anaphase (Fig. 1). The number of chromosome fragments per cell also varied from species to species at different doses of X-rays and gamma-rays. For instance, in N. opaca and N. Figs.

1-13.

rads)

in

1500. cell

Effects

each 1,

case N.

showing

showing

at

shows

a

sticky

chromosome

lated

of nuclei

(2000-G). chromosome

(300-X). of

and

into chromosome

C.

12, zeylanica

breakage, left

middle showing

cell

N.

flagelliformis

minute chromosome

C. fibrosa three cell unequal

11,

anaphase cells

at

shows separation

of

C.

anaphase,

C.

cell

daughter

the

right

chromosome

cell

chromosomes

of

early

(2000-G).

each

showing

up

grouping of

different

showing

chromosome

fragments

N.

other

stages

showing

filament

at

7,

strands,

anaphase

antheridial

on of

breakage

(300-X).

made

formed

of lower

opaca-Anaphase

globularis

opaca •~

fragmentation

continuous

N.

N.

Figs.

metaphase

chromosome

presumably

bridge

2,

breakage,

showing 9,

10,

fibrosa

formed of

long

All

opaca

breakage

cells

sizes,

heavy

showing

two

two

N.

(in

G).

chromosome

(500-G).

showing

bridge

opaca

cell

formed

different

(2000-X).

3,

chromosome

(200-X). of

N.

administered by

(500-G),

showing

upper

fragments

doses

gamma-rays

(200-X). 4,

and

The

and

chromosomes

cells

showing

upper

(micronuclei)

fragments

(2000-G). 13,

dicentric

clumps

groups

8,

entirely

X

(300-X). two

metaphase

formation,

bridge

breakage

opaca

bridge

composed

chromosomes

N.

anaphase

C. fibrosa

chromosome

6,

showing

groups

numbers

oriented

of

at

formation 5,

taxa. by

clumping

laggards

ring

(500-X).

charophyte

denoted

and

and

and

metaphase

on

(X-rays

stickiness

breakage

(1000-X).

probably

gamma-rays

brackets

showing

transversely

cells

and in

breakage

at

metaphase

daughter

of

opaca

chromosome

opaca-two cell

X-rays

chromosome

chromosomes cell

of

is indicated

granu fragments

anaphase and

showing cell

on

the

1977

Irradiation

Studies on the Karyology of Charpphyta I

283

284

Y. S. R. K. Sarma and S. B. Singh

Cytologia

42

flagelliformis at 100 and 200 rads of X-rays, generally two to three fragments were observed and two to three occasionally four fragments with gamma-rays, the number of fragments becoming numerous at higher levels of irradiation (Figs. 3, 4 and 6). With N. acuminata, at low doses of 200 and 400 rads, chromosome fragments were very rare but as the dose level increased the number of fragments also increased progressively. The two species of Chara viz., C. fibrosa and C. globularis irradiated with X rays and gamma-rays show more or less similar types of chromosomal aberrations. The number of chromosome fragments per cell at a dose of 1000 rads were more than two (Fig. 5) in both the taxa but at higher dose of 2000 rads the number of chromosome fragments increased to more than five. Similarly, in C. setosa and C. zeylanica irradiated with gamma-rays none to one chromosome fragments were recorded at doses of 500 and 1000 rads whereas at higher doses of 1000 and 2000 rads more than two fragments were recorded. It may, therefore, be concluded that the quantitative estimates revealed that there is a linear increase in the frequency of breakage of chromosomes with increase in dose in all the taxa irradiated with X-ravs and gamma-ravs. Anaphase bridges were also observed very frequently in all the taxa of Nitella and Chara whether irradiated with X-rays or gamma-rays. In shape, size and con figuration, anaphase bridges presented a considerable variation at different doses. At low doses of 100 and 200 rads in N. opaca and N. flagelliformis, 200 to 400 rads in N. acuminata and 500 rads to 1000 rads in species of Chara, sticky bridges were more common and rarely dicentric bridges were also recorded in N. opaca (Fig. 9). But slightly higher doses of 300 and 400 rads in N. opaca and N. flagelliformis most of the bridges were formed of two to four long continuous strands with or without fragments (Fig. 7) whereas at higher dose of 500 rads chromosome bridges were made up of mostly fragments (Fig. 8). Similarly at higher doses of 800 and 1000 rads in N. acuminata and 1500 to 2000 rads in Chara the bridges were constituted of long continuous strands and/or with chromosome fragments only (Fig. 12 and 13). Ring chromosomes were also recorded though infrequent, in N. opaca and Nsflagelliformis at doses of 300 and 400 rads of X-rays and gamma-rays at metaphase as well as at anaphase (Fig. 3). Apart from the above abnormalities, laggards (Fig. 2), unusual grouping of chromosomes at metaphase and anaphase (Fig. 10), unequal separation of daughter chromosomes to the two poles (Fig. 13) and oc casional formation of micronuclei (Fig. 10) were also noticed in all the taxa. How ever, persistence of post treatment effects of higher doses in N. opaca, N. flagelli formis (500 rads) N. accuminata (1000 rads) and in C. fibrosa, C. globularis, C. setosa and C. zeylanica (2000 rads) were in evidence even after 84hr which were reflected by the interphase nuclei showing granulation (Fig. 11) and vacuolization. At high dose levels, dividing cells were comparatively few and most of the nuclei were degenerating.

Discussion

The effects of X-rays on algae have been studied by several workers (cf. God

Table

3.

Table effective

percentage

of affected

of irradiation

number, materials

radiation

taxa

and percentage

at

used

of affected

and gamma-rays

in various

length

employed

taxa

with X-rays charophyte

chromosome

used in various

of gamma

as experimental

doses

chromosome

effective

between

dose

cells as a result

dose and at highest

cells at lowest and highest

relationship

lowest

Showing

Showing

2.

286

Y. S. R. K. Sarma and S. B. Singh

Cytologia

42

ward 1962) who have suggested that in general algae are more resistant in comparison to higher plants. On the other hand, Sarma and Khan (1967) in Nitella flagelli formis, Howard and Horsley (1960) in Oedogonium cardiacum, reported that the organisms showed more or less same sensitivity as in higher plants. Dodge and Godward (1963) .reported that Prorocentrum micans, a member of Dinophyceae, was more resistant as compared to the cells of higher plants but less resistant in comparison to other algae. The present findings on N. opaca, N. flagelliformis and N. acuminata also agree with those of Sarma and Khan (1967) who studied earlier the effects of X-rays on N. flagelliformis and pointed out that this alga is more sensitive to X-rays in comparison to other algae investigated by other workers (cf. Godward 1962). It may be mentioned here that the species of N. opaca and Ns flagelliformis show similar radiosensitivity levels as reported by Sarma and Khan (1967). Likewise, the general effects observed in Cafibrosa and C. globularis agree with those of Chatter jee (1972) in C. braunii irradiated at doses ranging from 500 to 2000 rads. The results obtained with N. opaca, N. flagelliformis and N. acuminata are also comparable to the reports of Sax (1938, 1940) in Tradescantia pollen tube chromosomes, who reported 4.7% of total chromosomal abnormalities at a dose of 200 rads, whereas Ostergren, Morris and Wakonig (1958) reported 100% of abnormal cells at a low dose of 300 rads in Hyacinthus orientalis but in H. dalmaticus only 24.7% of abnormal cells were observed at the same dose level. Similar observation was made by Swanson (1940) who also reported 5.58% of chromosomal abnormali ties at a low dose of 240 rads in pollen tube chromosomes of Tradescantia. Thus observations made on the three taxa of Nitella particularly N. opaca and N. flagelli formis are comparable with the cases of flowering plants cited above with reference to their level of sensitivity, where the percentages of affected cells ranged approxi mately between 17% to 96% depending upon dose level (100-500 rads). On the other hand, results obtained on C. fibrosa and C. globularis agree with those of Sharma and Chatterji (1962) in five species of higher plants viz., Vicia faba, V. sativa, Haemanthus multiflorus, Aloe perfoliata and Cipura palmdosa who re ported chromosomal and other abnormalities at doses of 500 and 1000 rads. The earlier work on algae with gamma-rays is practically insignificant. The limited work on Eudorina elegans by Rayns and Godward (unpub.), Chlorella pyrenoidosa by Galley and Tolbert (1958), and on Euglena gracilis by Leedale (1958) showed that these organisms are very resistant to gamma-rays. No details con cerning the chromosomal abnormalities in those studies have been given. From the data presented in Table 2 it is quite obvious that the percentages of affected cells agree in treatments of X-rays and gamma-rays at same dose level of radiation on the given taxon. However, slight differences of 1.00 to 4.00% exist in the frequency of affected cells (cf. Table 2) between the two types of radiation. The results obtained for N. opaca, N. flagelliformis and N. acuminata agree with the reports of Kollar (1953), and Swanson (1955) in Tradescantia species with reference to the microspores following gamma-irradiation. Chromosomal and chromatid breakage and other abnormalities were achieved at very low doses of 50 to 200 rads (Swanson 1955). Acentric fragments and dicentric bridges were

1977

Irradiation

Studies on the Karyology of Charophyta

I

287

observed by Mikaeisen (1967) in barley root meristem at doses of 200 to 400 rads. On the other hand, the results obtained in the present work for C. fibrosa, C. globularis, C. setosa and C. zeylanica are comparable to the reports of Amer and Mikael (1972) on Vicia faba root tip. They reported chromosome and chromatid breakage at different doses ranging from 500 to 10,000 rads and concluded that chromosomal abnormalities rapidly increased with the increase in the dose of gamma radiation. Similar observations were made by Kalloo (1972) with dry seeds of Pisum at high dose of 10, 20 and 40K rads and Shaikh and Godward (1972) in Lathyrus sativus, Vicia sativus and Vicia ervilia at doses of above 5K rads of 60Co gamma-rays. Marshak (1937) in his study with heterogenous materials viz., Lycopersicum esculantum, Mus musculus, Pisum sativum, Allium cepa and Vicia faba, suggested that the frequency of X-ray induced chromosomal abnormalities per cell increased with increase in chromosome size. Recent studies on growth inhibition and sterility also support direct correlation between the sensitivity of radiations and the size of the chromosomes (Sparrow and Christensen 1953). The data recorded on algal species in the present study also establishes a correlation between the size of chromo somes and the level of radiosensitivity. For example, Nsflagelliformis having on an average the largest chromosome size showed the maximum radiosensitivity as com pared to the other allied taxa which have comparatively smaller size of chromo somes (cf. Table 3). This may be explained on the basis that larger the chromosome, more is the energy absorption which in turn may have induced greater number of chromosomal aberrations. Data on the influence of chromosome number on the radiosensitivity of the plants are not too plentiful. However, Sparrow and his colleagues (Sparrow 1967) suggested that the species with lower chromosome numbers are more sensitive than the species with higher chromosome number. Sparrow further suggested that the reason for the chromosome number influencing the radiation response may not be related to difference in the amount of genetic information lost per damaging event, but rather to the fact that chromosome size tends to decrease as chromosome number increases. The present study also supports the conclusions of Sparrow (1965). It may be seen that N. opaca and N. flagelliformis having n=6 and n=9 chromosomes respectively are most radiosensitive, while C. zeylanica with n=42 is the most resistant of all (cf. Table 3). These correlations between the size as well as number of chromosomes and the radioresponse can be examined on the basis of deletions. A single deletion in a nucleus with few chromosome number is expected to cause a considerably greater genetic loss on an average than a single deletion in a nucleus having its information dispersed over a large number of chromosomes. For instance, N. opaca having the least chromosome number (Table 3) seems to be more radiosensitive as compared to all other allied species except N. flagelliformis (n=9) which has a higher chromosome number.

However, the slightly higher level of radiosensitivity in N. flagelliformishaving 9 chromosomes in comparison to N. opaca with only 6 chromosomes may be ac counted on the established fact that larger the size of chromosomes greater will be

288

Y. S. R. K. Sarma and S. B. Singh

the

radioresponse.

(4.24ƒÊ)

is

expected

chromosome length of

flagelliformis absorb

aberrations of

3.8ƒÊ

more

N. to

4.18ƒÊ among

as

(Table the

radiosensitive

species

to

Similarly, of

to

opaca

having

in

the

on

an

damage

an

with

(Table

size

greater

globularis

included others

chromosome causing

N.

C.

Chara

compared

larger

energy

compared

3).

as

with greater

Cytologia

average

largest

present

in

average terms

of

chromosome

chromosome

study

42

size

appeared

to

be

3).

On the basis of their levels of radiosensitivity, it may be concluded that a) the taxa belonging to Nitella are more radiosensitive as compared to those of Chara b) that amongst the taxa of Nitella studied here, N. ftagelliformis and N. opaca are more radiosensitive as compared to N. acuminata and c) amongst the taxa of Chara, C. globularis and Cafibrosa are more sensitive to radiations as compared to C. setosa and C. zeylanica. When all the taxa used here as test materials of both Nitella and Chara are taken together, N. opaca and Ns flagelliformis with low chromo some numbers and long chromosomes are the least radioresistant and C. zeylanica with the highest chromosome numbers and with shorter chromosomes, is the most resistant to radiations. Further amongst algae, members of Charophyceae seemed to be less resistant in comparison to others studied so far and approach more closely the flowering plants in this respect. Thus, on the basis of the present study, a dis tinctive status for charophytes under a separate division Charophyta also seems to be justified. Summary

The present study deals with the effects of X-rays, gamma-rays on the karyology of some selected taxa of charophyta, antheridia having being exposed to radiations. Species of Nitella viz., N. opaca (n=6), N. flagelliformis (n=9) and N. acuminata (n=18) and four of Chara viz., C. fzbrosa (n=14), C. globularis var. virgata

(n=14),C.setosa(n=28)andC.zeylanica var.diaphora f. oerstidiana (n=42)were employed

as experimental

materials

in various

irradiation

experiments.

X-ray

and

gamma-ray doses ranged from 100 to 2000 rads. The materials in each case, after exposure to required amount of radiation, were transferred to fresh culture medium and were examined cytologically controls were maintained. The radiations somes some

qualitative to a greater

at metaphase and chromatid

changes

that

at varying were

or lesser extent

periods

observed were:

gards, unequal grouping of chromosomes, and rarely, formation of micronuclei.

in all the

stickiness

and anaphase, chromosome breaks at metaphase and

of time. taxa

In with

separation,

ring

experiments both

and of clumping

erosion, chromatid anaphase, anaphase

unequal

all

types

of

of chromo

gaps, chromo bridges, lag chromosomes

The quantitative estimates of affected cells, based on percentages of those show ing chromosome breakage at metaphase and anaphase and cells showing anaphase bridges, showed that levels of radiosensitivityof different taxa differed. The chromo somes of N. opaca having the lowest chromosome number (n=6) and of N. flagelli formis (n=9) with longest chromosomes seem to be more sensitive in comparison to other taxa, while C. zeylanica, with highest chromosome number in the series

1977

Irradiation

Studies on the Karyology of Charophyta

I

289

(n=42) as also with shortest chromosomes, was found to be more radioresistant of all the taxa investigated. However, charophyte taxa in the present study were shown to be more sensitive to radiations as compared with many other algal taxa belonging to other groups investigated earlier. The irradiation studies lend further support to the now more widely held view that Charophyta constitute a very dis tinctive group amongst algae. The generally held view that algae in general are more resistant to radiations in comparison to higher plants does not seem to apply to the members of Charophyta. On the basis of the present study it has been shown that the chromosomes of N. opaca and N. flagelliformis are as sensitive as to radi ations as those of higher plants. The results obtained in the study were adequately discussed. Acknowledgements

the

The authors

wish to express

Department

of

Sciences

and

to Dr.

their

gratitude

Radiotherapy

and

T. Sharma,

Department

to Professor

Radiation

Medicine,

of Zoology

G. C. Pant,

Head

Institute

Medical

for their

of

generous

help

of in

providing facilities of irradiation. Thanks are also due to the Head of the Depart ment of Botany for providing laboratory facilities. The financial assistance provided to the second

author

by the

authorities

of

Banaras

Hindu

University

is gratefully

acknowledged.

Literature

cited

Chatterjee, P. 1972. Ph. D. Thesis, Calcutta. Dodge, J. D. and Godward, M. B. E. 1963. Some effects of X-rays on the nucleus of a Dinoflagel late. Rad. Bot. 3: 99-104. Gailey, F. B, and Tolbert, N. E. 1958. Effect of ionizing radiation on the development of photo synthesis in etiolated wheat leaves. Arch. Biochem. Biophys. 76: 188-195. Godward, M. B. E. 1948. The iron alum acetocarmine method for algae. Nature (London) 191: 203. 1954. - Irradiation of Spirogyra chromosomes. Heredity 8: 293 (Abs). 1962. Invisible radiation in "Physiology and Biochemistry of Algae" - ed. Lewin, R. A., Academic Press, New York, London. Guerlesquin, M. 1967. Recherches caryotypiques et. cytotaxonomiques chez les charophycees d'Europe Occidentale et d'Afrique du Nord. Ph. D. Thesis. Hotchkiss, A. T. 1963. A report of chromosome number in the genus Lychnothamus (Rupr). Leorh. and comparison with other charophyte genera. Proc. Linn. Soc. N. S. W. 138: 368-372. 1966. - A new revised base chromosome number for the genus Tolypella. Bull. Torrey Bot. Club. 93: 426-432. Howard, A. and Horsley, R. H. 1960. Filamentous green algae for radiobiological study. Int. J. Rad. Biology 2: 319-330. Jacobson, B. S. 1957. Evidence for recovery from X-rays damage in Chlamydomonas. Rad. Res. 7: 394-407. Kalloo. 1972. Chromosomal alterations in mitotic and meiotic system as influenced by gamma rays in Pisum. Cytologia 37: 643-651. Khan, M. and Sarma, Y. S. R. K. 1967. Some observations on the cytology of Indian Charophyta. Phykos 6: 62-74.

290

Y. S. R. K. Sarma and S. B. Singh

Cytologia

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