Cytogenetical Studies in Narenga porphyrocoma I. Study of karyotype and abnormalities in meiosis1

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1 Cytogenetical Studies in Narenga porphyrocoma I. Study of karyotype and abnormalities in meiosis1 D. Jagathesan and T. V. Sreenivasan Sugarcane Breeding Institute, Coimbatore-7, India Received February 16, 1966 The genus Navenga has two species 1) N. fallex and 2) N. porphyrocoma, of which latter is found in Northern India. This genus was formerly included in Saccharum as a species, namely S. navenga. As early as 1913 (Barber 1916) this species was crossed with S. officinavum variety Vellai for introducing characters like erect habit, good tillering and wide adaptability to various conditions of climate and soil. Parthasarathy and Venkatraman (1942) felt that satisfactory habit and marked tillering capacity of Narenga entitled its use as a parent in sugarcane breeding. Panje (1953) has reported that Navenga occurs in the Indian and Nepal terrains and lower Assam valley. He feels that the spread of the species is "probably influenced by the extent of shading and the liability of the patch to either a superficial stagnation of water or the accumula tion of free water in the sub-soil". Bhat (1964) has reported that Navenga and Sclerostachya simultaneously possess a number of economic characters highly desired in sugarcane, viz. resistance to red-rot, smut and mosaic diseases, resistance to waterlogging, very early flowering, high tillering and erect habit of growth. Srinivasan and Batcha (1962) studying the clones of S. spontaneum and allied genera Narenga, Sclerostachya and Erianthus under artificially created waterlogged conditions found that the performance of Narenga was the best. Parthasarathy (1948) and Raghavan (1951) found 5 chromosomes of Narenga and Sclerostachya are homologous to 5 chromosomes in S, officinarum thereby indicating a probable relationship with this species. Attempts are on way at this Institute to employ Navenga porphyrocoma in a systematic way in the breeding programme especially with regard to disease resistance by transferring the desirable segments or genes to S. officinarum as has been done in wheat by Sears (1956). As a first step in this direction, the karyotype and meiotic abnorma lities observed in 20 plants of N. porphyrocoma are reported in this paper. Materials and methods About 20 clones of this species are maintained in the live herbarium of the germplasm bank at this Institute. This includes plants collected by Spontaneum Expedition Scheme from Northern India (Panje 1953). Karyotype analysis was carried out in the metaphase of pollen mitosis according to a schedule developed at this Institute (Jagathesan and Sreenivasan 1966). Ten clear and well spread metaphase plates were drawn with a camera lucida and the chromo some lengths were calculated from the drawings. For a study of meiosis, arrows (a term used for spikes) were fixed in acetic-alcholol (1:3), the acetic acid part of which was saturated with ferric acetate, and the anthers were smeared in aceto-carmine. Pollen fertility was determined on the basis of stainability in aceto-carmine. Those which had regular shape and staining were taken as fertile and empty grains without any staining were counted as sterile ones. For a classification of karyotype asymmetry, the scheme of Stebbins (1958) was used and the nomen clature recommended by Levan et al. (1964) for centromeric position has been adopted. Observations Karyotpye analysis was carried out in all the 20 plants and no critical differences could be detected among them. The haploid complement consists of1 Approved for publication by Director, Sugarcane Breeding Institute, Coimbatore-7, India.

2 12 D. Jagathesan and T. V. Sreenivasan Cytologia chromosomes. They are broadly divided into 5 categories on the basis of their length and position of constrictions. The r value (ratio of long arm/short arm) for all the 15 chromosomes lies between 1.0 and 1.7 and all possess centromere in the median region. Type A: Very long chromosomes (>4ƒÊ in length) with one constriction in the median region. Type B: Comparatively long chromosomes (3 to 4ƒÊ) with constriction in the median region. Type C: Long chromosomes (about 3ƒÊ) with two constrictions, one at the median region and the other in the long arm at the terminal region (or a satellite). Chromosome 7 belongs to this class. Type D: Similar to type C (2 to 3ƒÊ) but without a secondary constriction. Type E: Short chromosomes (1 to 2ƒÊ) with median centromere. Fig. 1. Idiogram of haploid chromosomes of Narertga porphyrocoma. This karyotype belongs to type 2 b (i.e. ratio between the largest and smallest chromosome in the com plement varying between 2:1 and 4:1 and with 1 to 50 percent of chromo somes with an arm ratio <2:1) of Stebbin's classification, which indi cates greater asymmetry (Fig. 1). Meiosis was studied in all the plants and the data are presented in Table 1. In plant No. 16 only meiosis and pollen mitosis was regular. fifteen plants PMC's from the same anther had both 14 and 15 bivalents at M 1, though majority of cells had 15 bivalents only (Fig. 2). The percentage of cells with abnormal number at M I varied from 1.81 to (Table 1). Diakinesis was also studied and in majority of cells only one bivalent was associated with the nucleolus, indicating the presence of one pair of satellited chromosomes. This was confirmed by the study of karyotype in pollen mitosis. Anaphase I was studied in all the plants and the data are presented in Table 2. The abnormalities included laggards, dicentric bridges with fragments and unequal seperation of chromosomes (Figs. 4-5). In plant No. 2 two cells with two dicentric bridges were observed. The percentage of cells showing abnormal anaphases varied from 1.90 to in different plants. Pollen mitosis was studied in all the plants and the data are included in Table 2. Plant Nos. 7 and 16 had 15 chromosomes in all the pollen grains studied. In others the chromosome number varied from 13 to 16 (Figs. 6-8). Percentage of pollen grains with abnormal chromosome numbers varied from 4.0 to Idiograms of chromosomes from cells containing different numbers were drawn and critically studied. It was observed that in general in 14 chromosome pollen In

3 1967 Cytogenetical Studies in Narenga I 13 Table 1 1 One cell had 13 bivalents in this plant. grains, the missing chromosome was one of the long median constricted type D probably either chromosome 7 or 8. In pollen grains with 13 chromosomes, the satellited one was also found missing. In plant 17, one pollen grain with 9 and another with 7 chromosomes were observed. In plant Nos. 18 and 19 large pollen grains with 30 chromosomes were also observed along with normal ones (Fig. 9). In addition, one pollen grain with 11 chromosomes was also noted in plant No. 19. Pollen grains with 30 chromosomes were always larger in size. All these observa tions were recorded from pollen grains of the same anther. Pollen fertility was studied in all the plants and included in Table 2. Pollen fertility ranged from 34 to 91%. Pollen fertility was significantly low in Plant Nos. 17 to 20 where abnormalities in meiosis and pollen mitoses are high. Seed fertility could not be studied since this genus is self-incompatible and practically no seed setting takes place. It is vegetatively propagated. A detailed study of habit, foliar and floral characters revealed no differences of importance among them. Discussion Twenty different plants of Narenga porphyrocoma were studied cytologically. Out of these, plant Nos. 15 to 20 were collected by Spontaneum Expedition

4 14 D. jagathesan and T. V. Sreenivasan Cytologia 32 Table 2 1 One cell with 9 and another with 7 chromosomes were observed. 2 Ten cells with 30 chromosomes were observed.3 Three cells had 30 chromosomes and one 11 chromosomes. Scheme, identified and maintained at this Institute. Chromosome number of Narenga porphyrocoma was first reported by Bremer (1924) as 2n=30 and later confirmed by Janaki Ammal (1941). The present investigation has shown that pollen mother cells and pollen grains with different chromosome numbers occur from cells of the same anther in all the plants. In all plants except plant No. 16, there were irregularities either in meiosis or pollen mitosis. In several plants it has been reported that cells with different chromosome numbers co-exist in the same tissue along with full comp lement and such cells have been termed "chromosome mosaics" (Frankhauser 1945). Plants with different chromosome numbers have been recorded in polyploids and experimental amphidiploids (Sachs 1952). Bhaduri and Sharma (1946) in Datura, Datta (1952) and Panda (1961) in Corchorus, recorded the occurrence of PMC's in the same plant having different chromosome numbers. Chopra (1960) reported that in one monosomic line of Tyiticum aestivum different PMC's had numbers ranging from 16 to 21. Shahare and Shastry (1963) also recorded such mosaics in roses. Spindle abnormalities accompanied by unequal separation of chromosomes or chromosome elimination could be responsible for the origin of plants with different chromosome numbers in the present study as indicated by Darlington and Thomas (1937) in Festuca-Lolium hybrids or in cotton by Brown (1950).

1967 Cytogenetical Studies in Narenga. I 15 Figs. 2-9. 2. meiosis showing 15 bivalents at diakinesis. 3. meiosis showing 14 bivalents at diakinesis. Note a bivalent attached to nucleolus. 4-5.

5 1967 Cytogenetical Studies in Narenga. I 15 Figs meiosis showing 15 bivalents at diakinesis. 3. meiosis showing 14 bivalents at diakinesis. Note a bivalent attached to nucleolus anaphase I and telophase show ing laggards pollen mitosis showing 16, 14 and 15 chromosomes. In Fig. 8, arrow indicates a small micro-satellite. 9. small and big pollen grains with 15 and 30 chromo somes at pollen mitosis. In all PMC's with abnormal chromosome numbers, 14 bivalents were invariably found, one bivalent being missed (see Figs. 2 and 3 for clear 14 and 15 bivalents during diakinesis). It is very interesting that a pair of homologous chromosomes is always eliminated in the mitosis. The occurrence of different numbers in the early stages of meiosis indicates that mosaics must have been produced by irregularities at pre-meiotic cell division.

6 16 D. Jagathesan and T. V. Sreenivasan Cytologia 32 Abnormalities in the later stages of meiosis (i.e.) anaphase I and telophase I included laggards and bridges. One pair of chromosomes were always found to divide later than others and lag behind during anaphase separation. This particular pair of homologous chromosomes probably possesses a weak centromere, lags behind and gets eliminated during cell divisions resulting in the formation of pollen grains containing deficient and extra chromosomes. Hence pollen grains with both 14 and 16 chromosomes are produced. These were probably responsible for the occurrence of different chromosome numbers in pollen grains. Pollen grains with different chromosome numbers have been reported in colchicine induced tetraploid Ribes nigrum (Vaarama 1949) and in polyploid Lilium longiflorum (Emsweller 1949). Button and Hull (1956) observed different chromosome numbers in the root tip tissues of blackberry seedlings. They also reported giant pollen grains in the flowers of colchicine treated blackberries. In the present study giant pollen grains with 30 chromosomes were observed in Plant No. 18. We have not come across any pollen mother cell with 30 bivalents in this or any other plant in this study. This indicates that giant polyploid pollen grains could have arisen due to post-meoitic abnormalities. The occurrence of different chromosome numbers in pollen mother cells and pollen grains shows that cells with deviation from full chromosome complement are still able to divide along with normal ones. Sachs (1952) has reported that in experimental amphidiploids of Triticinae polyploid tapetal cells secrete some substances which are connected with the initiation of meiosis. Montalenti et al. (1950) found a similar mechanism in the secretory cells of the testis in Ascellus. This secretion is responsible for the initiation of meiosis in aneuploid cells and thus account for the ability of aneuploid cells to undergo meiosis in Triticinae. A similar mechanism probably operates in Narenga since in plants the tapetum which surrounds the cells undergoing meiosis also contains polyploid cells (Brown 1949). Sharma (1956) reported variation in chromosome number in somatic cells of vegetatively propagated plants. The probability of Narenga plants being chimeras can be ruled out since the majority of cells have normal chromosome complement and the root tip cells had 30 chromosomes in the aberrant plants studied. A few interesting points emerge from the present study on chromosome mosaics. Firstly the occurrence of PMC's with 14 and 15 bivalents and pollen grains with 14, 15 and 16 chromosomes indicates that one pair of homologous chromosomes possess a weak centromere. Secondly the occurrence of different numbers indicates the polyploid nature of Nayenga Porphyrocoma and confirms the speculations of earlier investigators (see Li et al. 1959) since mosaicism occurs only in polyploid plants. Thirdly the chances of obtaining viable aneuploid plants from these "mosaics" are bright.

7 1967 Cytogenetical Studies in Narenga I 17 Summay Meiosis and pollen mitosis were studied in twenty plants of Narenga porphyrocoma. Karyotype was analysed and found to be asymmetrical and belong to 2b type according to Stebbin's (1958) system of classification. Meiotic abnorm alities such as bridges, laggards and cells with different chromosome numbers were observed. It is suggested that these mosaics have probably arisen due to one pair of homologous chromosomes possessing a weak centromere and abnormalities in postmeiotic cell divisions. Acknowledgment The authors wish to express their thanks to Dr. J. Thuljaram Rao, Director, Sugarcane Breeding Institute, for facilities provided and encouragement and Dr. Ichizo Nishivama, presently, Visiting Professor at Agronomy Department, University of Wisconsin, Madison, U. S. A., for his valuable suggestions and comments on the manuscript. References Barber, C. A Studies in Indian sugarcanes. No. 2. Memoirs Dept. of Agri. India. 8: 3. Bhaduri, P. N. and Sharma, A. N Cytogenetics of Datura fastuosa L. Bull. Torr. Bot. Club 73: Bhat, N. R Potentialities of Sclerostachya fusca and Yarenga porphyrocoma in sugarcane breeding. Ind. J. Sug. Res. Dev. 8: 121. Bremer, G De chromosomen bij primitieve vormen van het geslacht Saccharum. Mededeelingen Profestation 16: Britton, D. M. and Hull, J. W Mitotic instability in blackberry seedlings. J. Hered. 47: Brown, M. S Polyploids and aneuploids derived from species hybrids in Gossypium. Proc. 8th Inter. Congr. Genet., Hereditas Suppl.: Cotton from Bikini. J. Hered. 41: Chopra, V. L Pre-meiotic somatic reduction in wheat. Curr. Sci. 29: Darlington, C. D. and Thomas, P. T The breakdown of cell division in Festuca-Lolium derivative. Ann. Bot. N. S. 1: Datta, R. M Irregular meiosis in Corchorus olitorius Linn. Sci. & Cult. 18: 149. Emsweller, S Polyploidy in Lilium longiflorum. Amer. J. Bot. 36: Frankhauser, G The effect of changes in chromosome numbers on amphibian development. Quart. Rev. Biol. 2: Jagathesan, D. and Sreenivasan, T. V A pollen grain squash technique for Saccharum and related genera. Stain Tech. 41: Janaki Ammal, E. R Intergeneric hybrids of Saccharum. J. Genet. 41: Levan, A., Fregda, K. and Avery, A. Sandherg Nomenclature for centromeric position on chromosomes. Hereditas 52: Li, H. W., Shang, K. C., Hsiao, Y. Y. and Yong, P. C Cytological studies of sugarcane and its relatives, XVI. Basic chromosome number of Saccharunn officinarum L. Cytologia 24: Montalenti, G. et al The supply of ribonucleic acid to male germ-cells during meiosis in Ascellus aquaticus. Heredity 4: Mukerjee, S. K Search for wild relatives of sugarcane in India. Internatl. Sugar Jour. 52: Panda, B. S. 1961, Chromosome mosaics and variable microsporocytes in Corchorus aspleni folius Burch. a wild jute species of Africa. Sci. & Cult. 27: Panje, R. R Studies in S. spontaneum. 3. Recent exploration for spontaneum and related grasses in India. Proc. 8th Cong. I. S. S. C. T., Barbados, 1953:

8 18 D. Jagathesan and T. V. Sreenivasan Cytologia 32 Parthasarathy, N Origin of noble sugarcane (S. officinarum L.). Nature 161: and Venkatraman, T. S Yet more parents for sugarcane breeding. Curr. Sci. 11: 150. Raghavan, T. S The sugarcanes of India-Some cytogenetic considerations. J. Hered. 42, Sachs, L Chromosome mosaics in experimental amphidiploids in Triticinae. Heredity 6: Sears, E. R The transfer of leaf-rust resistance from Aegilops umbellulata to wheat. Brook haven Sym. Biol. 9: Shahare, M. L. and Shastry, S. V. S Meiosis in garden roses. Chromosoma 13: Sharma, A. K A new concept of a means of speciation in plants. Caryologia 9: Srinivasan, K. and Batcha, M. B. G. R Performance of clones of Saccharum species and allied genera under conditions of waterlogging. Proc. 11th Cong. I. S. S. C. T., Mauritius 1962: Stebbins, G. L Longevity, habitat and release of genetic variability in higher plants. Cold Spr. Harb. Sym. Quant. Biol. 23: Vaarama, A Spindle abnormalities and variation in chromosome number in Ribes nigrum. Hereditas 35:

AN ASYNAPTIC MUTANT IN RICE. (ORYZA 8ATIvA).

AN ASYNAPTIC MUTANT IN RICE. (ORYZA 8ATIvA). AN ASYNAPTIC MUTANT IN RICE (ORYZA 8ATIvA). BY S. RAMAMUJAM, B.A. (HONS.), AND N. PARTHASARATHV, B.A., B.SC.,.~griczdtural Research Institute, Cobnbatore. Received June 18, 1935. (Communicated by Mr. K.