_??_1987 by Cytologia, Tokyo Cytologia 52: 151 -155 , 1987

Colchiploidyin (L.) F. Muell.

A. R. P. Shinha

Botany Department, Government College, Port-Blair-744 104, India

Accepted December 7, 1985

Lindernia crustacea (L.) F. Muell. of the family is an annual medicinal weed commonly growing in the paddy field, garden and on the bank of ponds and rivers. It flowers over a long period but the best period of flowering is from July to March. Morphological variations among the , general cytology and chromosome number of this species has already been reported by Sinha (1984). Chromosome number of the was reported by earlier workers also. Raghavan and Srinivasan (1940), Bhattacharyya (1968) and Biswas (1975). For the present investigation plant with gametic number n=14 was select ed for colchicine treatment. In order to improve the variety of the plant polyploid was induced for the first time.

Material and methods Seedlings were treated with different concentration of aquous solution of colchicine for different duration. Five seedlings of the same age group were left untreated and were allowed to grow in the same environmental condition in which the treated plants were growing. Flower buds of the treated and control plants were fixed separately in Carnoy's solution and anthers were squashed in two percent acetocarmine for cytological studies. Fertility of the pollen grains were studied on the basis of their stainability in acetocarmine. Stained pollen grains were scored as fertile and unstrained as sterile.

Observation Treatment of the seedlings with 0.1 percents aqueous solution of colchicine for 20 hours proved to be ineffective while treatment with 0.3, 0.4 and 0.5 percent colchicine solution for 20 and 14 hours respectively proved to be lethal. Positive result was obtained after treatment with 0.2 percent aqueous solution of colchicine for 20 hours. In case of treatment with 0.2 percent aquous solution of colchicine two seedlings died at the very initial stage whereas the rest eight seedlings initially showed stunted growth therefater grew into plants which were morphologically highly distinguishable from the control. Treated plants were profusely branched, taller with larger leaves than the control plants. Detailed morphological characters of the treated and control plants are listed in Table 1. Cytological examination revealed that these morphologically vigorous plants were tetra ploid with a gametic chromosome number of n=28 while the control plant possessed the gametic number n=14. Meiosis in the colchiploids has been found to be irregular. At the first place, it was greatly non-synchronized and stages from diakinesis to anaphase II could be seen in one anther squash. Clumping and precocious separation of chromosomes was observed to be another pronounced anomaly. Both at diakinesis and metaphase I there were cells in which 28 bivalents (Fig. 1) could clearly be counted. However, the occurrence of multivalents and univalents (Figs. 2, 3) was 152 A. R. P. Sinha Cytologia52 observed in a majority of the pollen mother cells. The nature and frequency of chromosome associations analysed in 25 pollen mother cells at metaphase I is given in Table 2. Multivalents of higher order were not seen.

Table 1. Comparativestatement of morophologicalcharacters of colchicine treatedand controlplants of Linderniacrustacea

Table 2. Nature and frequency of chromosome associations analysed in 25 PMCs at metaphase I in Lindernia crustacea

At anaphase I, some of the pollen mother cells showed equal separation of chromosomes (28:28) to the two poles (Fig. 4). However, in most of them unequal separation of chromo somes and laggards were observed. In a few cases, complete failure of anaphasic separation was noticed. These irregularities have been found in later stages too. Consequently monads to hexads and micronuclei (Figs. 5, 6) were recorded at the quartet stage. Pollen grains were of variable size. Percentage of micropollen was about 6.58 percent and that of giant pollen was about 3.69 percent. Pollen sterility was estimated to be about 89.73 percent (Table 3).

Table 3. Analysis of pollen grains in treated and control plants of Lindernia crustacea

Discussion It is evident from the data mentioned above that the plants originating from colchicine treatment shows gigantic morphological characters in respect of plant length, leaf size, number of stomata, size of stomata etc. over a controlled diploid plant. This is due to differential dose of genome present in colchiploid plant. Such changes in morphological features of colchi ploids (autotetraploids) were reported in different plants by Bose and Panigrahi (1969), Biswas 1987 Colchiploidyin Linderniacrustacea (L.) F. Muell 153 and Bhattacharyya (1971), Raghuvanshi and Singh (1977), Murty and Satyavathi (1978) and many others. Cytology of colchiploid Lindernia crustacea was irregular and greatly non-synchronized. At metaphase I univalents and multivalents were present but the frequency of such PMC con taining univalents and multivalents was only 44 percent. Range of multivalents per PMC was 0 to 7 and univalents was 0 to 5 which is much lower than theoretically possible for the PMC having n=28 in autotetraploid plant. The low frequency of multivalent present in the present

Figs. 1-6. 1, PMC at metaphase I showing 28 bivalents. •~750. 2, PMC at metaphase I showing 20

bivalents, 2 quadrivalents and 8 univalents. •~1500. 3, PMC at metaphase I showing univalents alongwith bivalents and quadrivalents. •~750. 4, PMC at anaphase I showing equal separation of

chromosomes (28: 28). •~750. 5, PMC showing pentad. •~750. 6, PMC showing hexad. •~750. 154 A. R. P. Sinha Cytologia52 investigation indicates that the presence of large number of multivalent is not the main feature of an autotetraploid rather it indicates that the pairing of homologous chromosomes even in autotetraploid plant is controlled by gene directly or indirectly. The genotypically controlled tendency to form only bivalents was also found by Miintzing and Prakken (1940). Giles and Randolph (1951) has reported reduction in multivalents frequency in the colchicine induced tetraploid of Zea mays. Riley and Law (1965) has reported that the pairing of chromosomes related genetically or evolutionarily can be induced or narrowed by gene action. A gene con trolled suppression of homologous pairing in the tetraploid race has also been discussed by Sisodia (1971) and Kaul and Bhan (1977). In addition to quadrivalents trivalents were also observed in few pollen mother cells. Pre sence of trivalent configuration in tetraploids have also been reported by Raghuvanshi and Singh (1977) and Indira and Abraham (1977). Clumping and precocious separation of chromosomes was observed quite frequently. Unequal separation of chromosomes at the poles at anaphase I and II, laggards and lagging bivalents and simple bridges were observed quite frequently. All these anomalies at various phases of division ultimately lead towards pollen variability as well as lead towards the vari ation at quartet stage with the result that monad to hexad were observed most frequently at quartet stage. These further lead towards pollen sterility. Sterility due to these anomalies has also been observed in induced polyploids of Amaranthus hypochondriacus L. and Amaranthus dubius Mart. ex Thell. by Behera and Patnaik (1975), in Mulber by Das et al. (1970) and in Job's tears by Venkateshwarlu et al. (1976). Fertile pollen grains were very few in number and it was only 10.27 percent. Gene born physiological inbalance may also be one of the possible cause of this high degree of sterility.

Summary Autotetraploid has been raised in Lindernia crustacea, a medicinal weed, by treating the seedlings with 0.2 percent of aqueous solution of colchicine for 20 hours. Morphological and cytological characters of colchiploid and diploid were studied in detail. Cytology of colchiploid was highly non-synchronized and one interesting phenomenon was observed that the frequency of multivalents were very low whereas the sterility of pollen grains was very high. Other anom alies like clumping of chromosome at various stages, precocious separation of chromosomes, formation of univalents and unequal distribution of chromosomes at anaphase I and II were also observed and discussed. Pollen grains were variable in size.

Acknowledgement

I wish to express my deep sense of gratitute and reverences to late Prof. V. Thakur, the then University Professor and Head of the Department of Botany, Magadh University, Bodh Gaya for his consistance guidance throughout the course of the present investigation. I am thankful to U. G. C. for financial assistance through FIP.

References

Behera, Bharati and Patnaik, S. N. 1975. Induced polyploid in Anaranthus dubius Mart. ex Thell. Cytologia 40: 157-168. Bhattacharyya, N. K. 1968. Chromosomal diversities in Lindernia. Nucleus, International Seminar on Chro mosomes-its structure and function: 102-114. Biswas, A. K. and Bhattacharyya, N. K. 1971. Induced polyploidy in Legumes I. Cyamposis psoraloides DC. Cytologia 36: 469-479. 1987 Colchiploidy in Lindernia crustacea (L.) F. Muell 155

Biswas, M. 1975. Cytogenetics and evolution of some common weeds of Bihar. Ph. D. Thesis , Magadh Uni versity, Bodh-Gaya (unpublished). Bose, S. and Panigrahi, U. C. 1969. Studies on induced polyploidy in Zinnia linearis Benth. Cytologia 34: 103-111. Das, B. C., Prasad, D. N. and Sikdar, A. K. 1970. Colchicine induced tetraploids of mulberry. Caryologia 23: 283-293. Giles, A. and Randolph, L. F. 1951. Reduction of quadrivalent frequency in an autotetraploid maize during period of 10 yrs. Amer. J. Bot. 38: 12-17. Indira, C. and Susan, Abraham 1977. Morphological and cytological studies on a radiation induced polyploid in Capsicum annum L. Cytologia 42: 371-375. Kaul, M. L. H. and Bhan, A. K. 1977. Cytogenetics of polyploids VI. Cytology of tetraploid and hexaploid Cuscuta reflexa Roxb. Cytologia 42: 125-136. Muntzing, A. and Prakken, R. 1940. The mode of chromosome pairing in Phleum twins with 63 chromosomes and its cytogenetics consequences. Hereditas 26: 463-501. Murty, U. R. and Satyavathi, D. 1978. Colchicine induced polyploids in Apluda mutica L. Nucleus 21: 34 -38. Raghavan, T. S. and Srinivasan, V. K. 1940. Studies in the Scrophulariaceae: The cytology of Angelonia grandiflora C. Merr. and some related genera. Cytologia 11: 37-54. Raghuvanshi, S. S. and Singh, A. K. 1977. Polyploid breeding in Trigonella foenum-graecum L. Cytologia 42: 5-12. Riley, R. and Law, C. N. 1965. Genetic variation in chromosome pairing. Adv. Genet. 13: 57-114. Sinha, A. R. P. 1984. New basic chromosome number and cytology for some Scrophulariacious weeds of Bihar (India). Cytologia 49: 781-787. Sisodia, K. P. S. 1971. Studies on cytogenetics of some speices in Urochloa L. Cytologia 36: 383-391. Venkateshwarlu, J., Rao, P. N. and Rao, M. K. 1976. Cytomorphological study of induced atutotetraploid Job's tears. Nucleus 19: 4-7.