CYTOLOGICAL STUDIES IN CAESULIA AXILLARIS ROXB

CYTOLOGICAL STUDIES IN CAESULIA AXILLARIS ROXB.

BY K. V. JITENDRA MorIh~q* (Research Scholar in Botany, St. Berchmans' College, Changanacherry)

Received November 8, 1963

[Communicated by Prof. T. S. Raghavan, M.A., Ph.D. (Lond), F.A.SC.]

I. INTRODUCTION

THE family Compositae has been widely investigated by Cytologists and Cytogeneticists. Crepis, Dahlia and Chrysanthemum have come in i'or intensive studies (Babcock et al., 1930, 1934; Lawrence, 1929; Shimotomai, 1933). Cytologically, this family which is the biggest among dicotyledons seems to be characterised by comparatively low chromosome numbers. The plant with the lowest chromosome number, Crepis capillaris, with a haploid number of 3, is a member of this family. Recently, a still lower number has also been recorded, i.e., n=2 in Haplopappus gl, acilis (Jackson, 1961) which is also a member of Compositae.

The genus Caesulia Roxb., a member of the tribe Inulae, does not seem to have been investigated cytologically so far. A recent note (Deshpande, 1960) gives a short account of the morphology of the endosperm in Caesulia axillaris. Another by Ramayya (1962) gives a short discription of the capitulum. In neither of these is there any treatment of the cytological aspect, nor has the chromosome number been recorded. Caesulia is a mono- typic genus and it was thought desirable to fill the lacuna of knowledge in its cytology. The present investigation pertains to the somatic and meiotic counts and some details connected with the chromosomal behaviour in meiosis and an interpretation thereof.

II. MATERIAL AND METHODS

The material for the present investigation was collected from the marshy fields on the northern frills of the Western Ghats. The technique employed was an adaptation of the Haematoxylin squash technique of Marimuthu and Subramaniam (1960) for somatic counts. So far this technique has not

* Present Address; Senior Botanical Assistan% Sunn Hemp Research Station, Pratapsarh (U.P.),

232

Cytological Studies in Caesulia axillaris Roxb. 233

been employed for meiotic counts in plants probably because the customary aceto-carmine technique affords easy results.

The buds were fixed in 1:3 Acetic Alcohol, hydrolysed in N HCI for 5 minutes at 60 ° C., washed thoroughly, and mordanted in Iron Alum (47o prepared from violet crystals) for one hour. After washing in distilled water for 5 minutes it is put in 0-57o Haematoxylin for two hours. The deeply stained buds were placed on a drop o f 4570 acetic acid taken on the slide. From the softened tissue the anthers alone can be easily separated and the rest of the floral parts discarded. The cover-slip is placed and a gentle pressure gives adequate spreading without tapping. Slides are made permanent by the Acetic Acid: Butanol series.

III. OBSERVATIONS

(a) Somatic Mitosis

The diploid chromosome number is found to be 14 (P1. XII, Figs. 1, 2). Chromosomes within the complement do not show prominent variation in size and as such an idiogram analysis has not been attempted. The centro- meres are mostly median or sub-median.

(b) Meiosis

In general, meiosis can be called regular though evidence exists for structural changes having taken place. It shows seven bivalents having complete pairing (P1. XII, Fig. 3 and Text-Fig. 1).

Asynapsis.--Weak pairing or total asynapsis frequently involving one or two bivalents is a common feature. But this may also be due to a precocious separation of the bivalents (P1. XII, Fig. 4).

Extra-chromosomes.--At least a few among the total PMCs observed had an extra univalent e~ther free or associated with other bivalents (PI. XII, Fig. 5).

Secondary assoeiations.--Secondary association of bivalents is a feature of common occurrence in this material. The range of grouping is from unassociated 7 bivalents to a maximum .of 4 groups, with intermediate groups of 6 and 5 (P1. XII, Fig. 6; Text-Fig. 1). By taking an association of three as two and an association of two as one the maximum number of associations is found to be three, i.e., made up of one group of three bivalents, one group of two bivalents and the other two bivalents unassociated (Table I).

234 K. V. JITENDRA MOHAN

TABLE I

No. of associations No. of bivalents in association

O) (2) (3) No. of cases

0 7 0 0 16

1 5 1 0 9

2 4 0 I 5 3 2 0 6

3 2 1 1 8

TOTAL 44

Reciprocal translocation.--There is indication of reciprocal translocation as could be inferred from P1. XII, Fig. 7, where there is disjunction of 3/3 from a chain of 6.

Inversions.mOccurrence of acentric fragments and bridges such as are indicative of paracentric in~,ersions are noticed in some of the first anaphases. Dicentric chromosomes due to bridge formation follows equal disjunction through a break of the bridge in the middle (P1. XII, Fig. 10). Unequal chromatids in anaphaslc chromosomes (P1. XII, Fig. 9) indicate a possibility of pericentric inversion also. Unequal disjunction of 7 normal and one small chromosome on one pole and six normal and one small on the other pole is noticed as a rare case (P1. XII, Fig. 11). It may be due to a division in advance of second metaphase. Secondary associations are found persisting even in the anaphase stages (P1. XII, Figs. 9, 10 and 11).

Extrusion.---The lagging of the chromatin material in the equator due to extrusion is noticed as a very rare case in the first telophase (Text-Fig. 2).

End-to-end association of second anaphase chromosomes.mSecond anaphase is normal. But the polar separation into the definite four groups is delayed in some cases (P1. XII, Fig 12, Text-Fig. 3). Another feature noticed at this stage is the end-to-end assoclat;.on of certain number of chromosomes. Due to this phenomenon even after the second anaphase separation, chromosomes show reluctance for orientating to their respective poles. However this does not lead to the formation of giant pollen grains.


The tetrad formation is generally regular in spite of the few abnormalities shown in the preceding stages. But ultimately an average of 2 ~ of the pollen

TExT-FI6s. I-4. Fig. 1. Metaphase I showing maximum number of secondary association, × 2,000. Fig. 2. Extrusion of the chromatin in the telophase I, × 2,000. Fig. 3. Same as Plate XII, Fig. 12, indicating chromosomes included in each pole to form the future nucleus, × 2,000. Fig. 4. A normal telophase H, × 2,000.

are found to be sterile as evidenced from the aceto-carmine test (Table II). Pollen is shed in the three-celled stage.

TABLE I I

F lower s Plant No. on the Total grains Fertile Sterile

same head ~o Sterility

i

2

3

a 124 121 b 115 114 c 103 100 a 132 128 b 115 112 c 102 99 a 120 116 b 104 102 c 99 97

3 2"45 1 0.88 3 3.00 4 3.00 3 2.60 3 3-00 4 3"30 2 1.98 2 2.01

Average 2.44~0 Maximum 3 .3~ Minimum 0.88~

236 K . V . JITENDRA MOHAN

IV. DISCUSSION

1. Extra Chromosomes

Among the PMCs examined in the M I nearly 2~o showed one ch~'omosome in excess of the original complement of 14. Fhis extra chromosome occurred as a separate univalent or associated with other bivalents. It is doubtful whether tlaese extra chromosomes can be called' supernumerary chromosomes ' since this feature was not common in the somatic complement nor was this a regular and frequent feature in meiotic cells.

However Swanson (1957) considered that 'supernumerary chromosomes' may arise as a result of structural changes of chromosomes. The occurrence of supernumerary chromosomes in the related Haplopappus spinulosus (Ning Li and Jackson, R. C.) renders this a possibility in this genus also. Enough material was not available to scrutinise a Iarge number of somatic and meiotic configurations.

They are reported (Swanson, 1957) to arise as a result of structural changes involving unequal distributions in translocation heterozygotes due to imperfect chiasma formation. Swanson also indicates the possibility of overlapping inversion loops and consequent cross-over in the region of overlapping which may lead to the production of supernumerary chromosomes. The phenomena of translocations and inversions with or without the formation of bridges and fragments are prevalent in this material. It is therefore likely that the extra chromosomes that are found in a small percentage of PMCs are in the nature of supernumerary chromosomes.

2. Structural Changes of Chromosomes Structural changes of chromosomes have presumably played a role

in the evolution of this species. Inversions are comparatively more frequent than translocations. In the present study anaphase separation shows bridges and fragments which are the consequence of paracentric inversions. Peri- centric inversion is not so easy to detect in the anaphase stages. It is found that the bivalents of the inverted chromosomes are the last to separate and orientate to the respective poles. Such chromosomes immediately after separation reveal the presence of unequal chromatids.

3. Secondary Associations The secondary association of bivalents is believed to be an expression

of ancestral homology of chromosomes. An analysis of secondary associations is found to throw some light upon the primary constitution of secondary polyploids along with other genetical evidences. Secondary association


may itself be gone-controlled. Hence lack of secondary pairing does not mean lack of homology.

It was Kuwada (1910) who first observed secondary associations in Oryza sativa. Thc basic chromosome number on the basis of maximum grouping has been inferred in quite a few genera. Darlington and Moffet (1930) and Moffet (1931) in this manner have derived the 17 paired Pomoideae from 7 paired ancestors. Lawrence (1931) in the light of evidence from secondary association assumed the basic number of Dahlia as 8. Nandi (1936) derived the basic number of Oryza as 5, whose diploid number is 24. Ragha- van and Sreenivasan (1940) draw out a probable derivation of the diploid number of 20 in Angelonia grandiflora from a basic number of 5. They interpret the association of three bivalents to be a consequence of structural chai, ges between two homologues and one non-homologue. Raghavan and Krishnamurthy (1947) have suggested that the diploid number of Sesamum orientale (2n = 26) is derived from a basic number of 7, through interspecific hybridization, restoration of fertility by amphidiploidy, followed by the deletion of two homologous chromosomes. Raghavan (1938) thinks that Gynan- dropsis pentaphylla is quadruply hexasomic and that it is a secondary poly- plold having arisen from a primary basic number of 7.

In the present material secondary association is a common feature. By analysis, the maximum grouping is found to be four, made up of I (3), 1 (2), and2 (1). The diploid number of 14 of Caesulia axillaris Roxb. can be derived from this basic number of 4. Even though the meiosis of the plant is generally regular, occurrence of weak pairing or asynapsis, unequal disjunction, extrusion and ultimately a small percentage of infertile pollen indicate hybridity. Combining the two evidences of secondary association and hybridity it can be presumed that it is secondarily derived polyploid.

V. SUMMARY

I. The chromosome number of Caesulia axillaris Roxb. has been deter- mined for the first time as 2n = 14 and n =7.

2. A haematoxylin squash technique has been adapted for application to meiotic cells.

3. One extra chromosome found in many meiotic metaphases is believed to be a supernumerary.

4. Structural changes of chromosomes such as reciprocal translocations and inversions have been recorded.

238 K . V . JITENDRA MOHAN

5. The basic chromosome number is suggested to be 4 and it is suggested to be a secondarily balanced polyploid.

6. Pollen grains show a maximum of 3 .3~ sterility.

V[ . ACKNOWLEDGEMENTS

This work was done under the supervision and guidance of Prof. T. S. Raghavan, t~.~,., t,h.v. (Lond.), F.~,.Sc. and I am highly indebted to him for the same. I am grateful to the Ministry of Scientific Research and Cultural affairs for the award of schelarship, during the tenure of which the present study has been carried out. I express my gratitude to Very Rev. Msgr. Francis Kalacherry, Principal, St. Berchmans' College, Changanacherry, for giving the facilities for research. Thanks are due to Dr. C. A. Ninan, Reader in Botany, University of Kerala, for allowing me to avail of the photographic facilities of the University Botany Laboratory, Trivandrum.

Babcock, E. B. and Navashin, M.

Darlington, C. D. and Janaki Ammal, E. K.

and Moffet, A. A.

Deshpande, P. K.

Hooker, J. D.

Jitendra Mohan, K. V., Girija, P. and Panikkar, A. O. N.

*Kuwada, Y.

Lawrence, W. J. C.

Madmuthu, K. M. and Subra- maniam, M. K.

*Moffet, A. A.

Nandi, H. K.

VII. R~FE~NCES

"The genus Crepis Biblog," Genet., 1930, 6, I.

"Basic chromosome number in plants with special reference to Compositae," New Phytologist, 1934, 33.

Chromosome Atlas, George Allen and Unwin Ltd,, 1945.

. . "Primary and secondary chromosome balances in Pyrus," Journ. of Gen., 1930, 22, 129.

. . "Morphology of the endosperm in Caesulia axillaris Roxb.," Curr. ScL, 1960, 29, 56.

.. Flora of British India, London, 1894, 3.

"Chromosome numbers in some Compositae," Curr. Sci., 1962, 31, 207.

. . " A cytological study of Oryza sativa," Bot. Mag. Tokyo, 1910, 24.

. . "The genetics and cytology of Dahlia variabilis," Journ. Genet., 1929, 24, 257.

" A haematox~,lin squash for root-tips of Dolichos lab lab Linn.," Curr. ScL, 1960, 29, 482.

. . "The chromosome constitution of Pomoideae,'" Proc. Roy. Soc., 1931, 1008.

.. "The chromosome morphology, secondary association and origin of cultivated rice," Journ. Gen., 1936, 23, 315.

K. V. Jiten~ra Mohan Proc. Ind. Acacl. Sci., B, I7ol. LX, PI. XII

. • • .

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• , 7

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5 • ~):.,.,~,;.-!,7 27". {?:~- , ~ 9

FI~S, 1-12

Cytological Studies in Caesulia axillaris Roxb.

Ning Li. and Jackson, R.C. ..

Raghavan, T.S. ..

~ - - and Srinivasan, V. K.

and Venkatasubban, K . R .

and Krishnamurthy, .. K.V.

Ramanujam, S. and Srinivasa- .. char, D.

Rarnayya, N. ..

*Shimotomai, N. ..

* Not seen in original.

239

"Cytology of supernumerary chromosomes in Haplopappu$ spinulosus var. cotula," Am. Journ. Bot., 1961, 48, 5.

"Morphological and cytological studies in Capparidaceae," Ann. Bot., New Series, 1938, 2 (5).

"The cytology of Angelonia grandiflora and some related genera," Cytologia, 1940, 11, 37-54.

"Contribution to the cytology of Tridaxprocumbens Linn.," Proc. Ind. Acad. Sci., 1941, 13, 85.

"Cytological studies in Sesamum," Ibid., 1941, 26, 236--75.

"Cytogenetic investigation in the genus Brassica and artificial synthesis of Brassica juneea," Ind. Journ. of Genet. and PL Breeding, 1943, 3, 73-88.

"Capitulumof Caesulia axillaris, Roxb.," Curr. Sci., 1962) 31, 24.

"Chrysanthemum," Journ. So. Hiroshima Univ. , 1933, Set. B., Div. 2, 7, I.

Fro. 1. FxG. 2. Ftos. 3.

FIo. 4.

Flo. 5. FIG. 7. Flo. 8. FIG, 9.

F~o. 10.

FIo. 11.

Fio. 12.

EXPLANATION OF PLATE XlI

Somatic metaphase (2n = 14), ×2,000. )

Somatic anaphase, × 2,000. and 6. Metaphase I showing secondary associations, ×2,000.

Metaphase I showing hasty separation (or asynapsis ?), × 2,000.

Anaphase I showing an extra chromosome, × 2,000. Anaphase I showing breakage of a ring of six chromosomes, × 2,000. A normal anaphase showing 7 chromosomes at each pole, × 2,000. Anaphase I showing unequal chromatids in one chromosome in one pole each. Also

shows persistence of secondary association, × 2,000. Anaphase I showing an acentric fragment in the centre. × 2,000. Anaphase I showing 6 and 1 small chromosome in one poie and 7 and 1 in the other

pole. Also shows persistence of secondary association, × 2,000.

Anaphase II showing hesitation of polar separation and end-to-end association of chromosomes, x 2,000.

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CYTOLOGICAL STUDIES IN CAESULIA AXILLARIS ROXB