Proc. Indian Acad. Sci., Vol. 86 B, No. 5, November 1977, pp 295-302, ~) Printed in .

Cytological study of cookei (Stapf) Schenck ex Henx. (Tribe Maydeae)

J VENKATESWARLU, PANUGANTI N RAO and D S NARAYANA Department of Botany, Andhra University, Visakhapatnam 530 003

MS received 14 June 1977; revised 17 September 1977

Abstract. Meiotic studies were reported in Trilobachne cookei (Stapf) Schenck ex Henr. for the first time. Chromosome number was found to be n=lO. This is not in agreement with one of the earlier reports of 2n=10 but confirms the more recent report of 2n=20. Several hundred cells were examined at various stages of meiosis in many . Meiosis was regular, and pollen fertility and seed set were high. But irregularities like univalents, aneuploid numbers, quadrivalents, secondary asso- ciation of bivalents into groups, interlocking of bivalents, pseudocytomixis, bridges, laggards and micronuclei were found in a low proportion of the cells. Two pairs of satellited chromosomes were present in the somatic metaphase plates. These facts taken together with the previous report of 2n=10 indicate the probable tetraploid aqature of the present material. Intergeneric crosses attempted between Trilobachne and maize, teosinte, and job's tears (some of the other members of the tribe Maydeae) did not succeed.

Keywords. Trilobachne cookei; asiatic Maydeae; meiotic irregularities; tetraploid; intergeneric crosses.

1. Introduction

The monoecious tribe Maydeae of the grass family comprising seven genera is recognised into two geographical groups, the american (occidental) Maydeae consti- tuting maize and its two american relatives, teosinte and tripsacum, and the asiatic (oriental) Maydeae with five genera, , , , Sclerachne and Trilobachne. Some information is known only on Coix and Chionachne among the asiatic Maydeae. For a clear understanding of the phylogeny of maize, when even taxa, outside Maydeae, belonging to the neighbouring tribe Andropogoueae are being sought (Chandravadana and Ga]inat 1976), it is considered essential that adequate information be obtained on the less known members of the asiatic Maydeae. While maize, teosinte, Chionachne and Sclerachne have a chromosome number of 2n=20, tripsacum has 2n=36 and 72, Coix 2n=10, 20 and 40, and Polytoea has 2n=40. For Trilobachne a chromosome number of 2n=10 was reported (Anonymous 1960-61), but more recently 2n=20 was recorded from somatic cells (Sapre 1977). Information on meiotic chromosome behaviour is completely lacking. In the present investiga- tion detailed cytological studies were made on several plants and these together with the results on attempted intergeneric crosses with a few other Maydeae are reported here.

295 296 J Venkateswarlu, Panuganti N Rao and D $ Narayana

2. Materials and methods

Trilobachne Schenck with only one species, 7". cookei (Stapf) Schenck ex Henr., is characterised by the presence of a persistent trilobed glume that adheres to the caryopsis (Bhagwat and Deodikar 1961). Its distribution is restricted to Western Ghats, Maharashtra (Bor 1960). It is erect, annual and grows up to 2 meters high, occurring abundantly on the hill slopes. Seeds for the present investigation which were collected in 1969 from Relegoan hill, Junnar, Poona District, were provided by the Botanical Survey of India. The populations were being grown since 1970 in the Experimental Field Station, Andhra University. Acetocarmine squash technique was employed for the study of meiosis in pollen mother cells and somatic chromosomes in root tip cells. Pretreatment of root tips with a saturated solution of para-dichlorobenzene for 3 to 3½ hr gave a satisfactory contraction and spread of the chromosomes. The maize used in intergeneric crosses was' Ganga Hybrid' (a commercial Double Cross Hybrid). Seeds oftripsacum were collected from Mexico, and those ofteosinte from Central America. Job's tears were raised from seeds collected from plants which ran wild in the University campus.

3. Results and discussion

3.1. Cytological studies

First cytological examination of pollen mother cells in a clearly revealed ten bivalents at diakinesis and metaphase I. In view of the deviation in chromosome number from the previous report of 2n=10, male spikelets from 15 plants in 1970 and 26 plants in 1971 were examined cytologically for locating the chromosomal races and abnormalities. AII of them showed n=10 which is in agreement with the number reported recently (Sapre 1977). In a low proportion of cells, however, they showed the occurrence of univalents up to two per cell and two or more nucleoli at diakinesis. Otherwise meiosis was regular with high pollen fertility and seed set. Subsequently, in 1976, twelve more plants were studied in detail at different stages of meiosis. Pachytene chromosomes were more or less tangled and were not amenable for study. However, the observations showed that the chromosomes are not differen- tiated into e# and heteropycnotic regions (differentiated chromosomes are found in Coix and tripsacum) and knobs are absent (knobs are present in american Maydeae and in Coix lacryma-jobi (Venkateswarlu et al 1976). Among the asiatic Maydeae, inso far as is known, undifferentiated and knobless chromosomes are found in Polytoca macrophylla (Cameron 1943), Chionachne koenigii and Scleraehne punctata (unpublished). The pachytene chromosomes in TnTobachne, however, present a typical chromomeric pattern. In all the 12 plants, though the chromosome number was n=10 with regular meiosis in a majority 0fthe cells (figures l, 9, 16, 19 and 22), some abnormalities were found in a few cells. Abnormal chromosome numbers (2n=18, 19, 21 and 22; figures 4, 5-7 and 10) occurring within the same anthers, were found in 0.90~ of the cells (table 1). When an excess or deficient number was found, the chromosome(s) concerned was not always the same, since sometimes a large chromosome (figures 5. Cytology of Trilobachne cookei 297

Figures 1-16. Meiosis ( × 1500) in Trilobachne cookei, see text for explanations. 298 J Venkateswarlu, Panuganti N Rao and D S Narayana

++ ~i+~+i ,+,+ u" +~ ~~"~ ?~i++;++. + ~ ~r • + j .i,

...... +~i .... ] • " + +++++ " +J+ e + ++++ "~- • b o +>-!

~+ .++ .... ++ ,,+ ~.(~ :~+ ?++::.+ ++++ i!++f~+ ...... ~l+'+ -+ + ~,+~+ +++ "

++++'+ 2 ++++++ +

Figt~res I?-2~. 17-24. Meiosis (× 1500) and 25. Somatic chromosomes (× 3000) in Trilobachne cookei, see text for explanations. Cytology of Trilobachne cookei 299

Table 1. Chromosome bohaviour at diplotene, diakinesis and metaphasc I in Trllo- bachne cookei.

Number of cells Chromosome bohaviour Diplotene Diakinesis Mctaphase I Total Per cent

11 II -- -- 2 2 0"23 10 II+ 1 I (short chromosome) -- 2 -- 2 0.23 10 II+ 1 I (large chromosome) -- 1 ~ 1 0.11 10 II 316 146* 47** 509 58.24

9 II+2 1 (small chromosomes) m 3 -- 3 0.35 9 II m 1 -- 1 0.11 9 II+ I I (small chromosome) -- 1 ~ 1 0.11 9 II+ 1 I (large chromosome) w 1 ~ 1 0.11 1-7 fro¢ II+rest in one group 67 51 56 174 19.91 1-3 free II+rest in two groups 3 18 12 33 3.78 1-3 fr¢~ II+rest in thre~ groups -- 6 ~ 6 0.69 All II in one group 65 28 18 111 12.70 2 groups with variable number of II in each 7 -- 2 9 1-03 3 groups with variable number of II in each 10 -- 2 12 1.37 4 groups with variable number of II in each w 2 -- 2 0.23 Irregular orientation of chromosomes -- -- 7 7 0.80

Total 468 260*** 146 874

*including 2 cells with one quadrivalent each, **including 2 cells involved in pseudocytomixis, ***including 11 cells showing interlocking of bivalents. and 6) and at other times a Smaller chromosome (figure 7) was involved. In a low frequency of cells, two univalents were present (figure 3; table 1). In cells with n----10 invariably there were two bivalents associated with the nucleolus (figures 1, 4, 5, 7 and 8). In several cells interbivalent connections were observed, and in afew cells quadrivalents with a frequency of not more than one per cell were present (figure 8). True interlocking involving two bivalents was found in 4.23 ~o of thecells at diakinesis (figure 8; table 1). At diplotene, diakinesis and metaphase I, among the several hundred cells examined, secondary association of bivalents into variable number of groups was observed in 40.51 Yo of the cells (figures 2, 3, 12-14; table 1). At metaphase I such associations were so complete in some cases, that all the bivalents formed a single sticky mass and individual bivalents in it could never be identified (figure 14). In addition to such sticky masses, irregular orientation of bivalents leading to multi- polar organization of spindle was observed in many cells (figure 11 ; table 1). In a few metaphase I cells, 20 bivalents were observed due to pseudocytomixis resulting from dissolution of the cell walls between adjacent pollen mother cells (figure 15; table 1). The number of chiasmata determined from 141 cells at diakinesis and metaphase I varied between 15 and 20 per cell with an average frequency of 17.84+0.1937. There were generally 5 to 10 ring bivalents with an average of 7.87 per cell, and none to 5 rod bivalents with a mean of 2.11 per cell. More than 90 Yo of the chiasmata were terminalized at diakinesis and metaphase L 300 J Venkateswarlu, Panuganti N Rao and D S Narayana

At anaphase I among 238 cells observed, 10 (4.20 %) cells with one bridge (figure 17), 2 (0.84 %) cells with two bridges resulting from delayed disjunction of bivalents were found, 4 (1-68 ~o) cells had laggards varying from 1 to 5 per cell (figure 18). The rest of the cells showed I0 : I0 separation (figure 15). At telophase I a persistent bridge, and a persistent laggard were found in 1 (0"79 %) and 2 (1-59 ~o) cells respectively out of 126 cells studied. Out of the 65 metaphase II daughter cells observed, 55 (84.62 ~o) showed 10 chromo- somes (figure 19), 1 (1.54 %) had 11 chromosomes (figure 20) and 9 (13.84 %) showed only 9 chromosomes (figure 21). At anaphase II, 118 (95.16~o) ceils showed 10 : 10 separation (figure 22) but in 3 (2.42 %) cells each, a bridge (figure 23), and a laggard were found out of 124 cells examined. Among 388 pollen quartets, 12 (3-09%) showed one micronucleus, and 2 (0.52 %) had two micronuclei (figure 24). Judging from the previous report of 2n=10 (Anonymous 1960-61), it is apparent that the present material with 2n=20 is tetrapoloid. The occurrence of two nucleolar bivalents, occasional formation of quadrivalents, secondary association of bivalents, interbivalent connections and meiotic irregularities seem to agree with this contention. To determine the various morphological categories of chromosomes in the comple- ment, somatic metaphases from root tip cells were examined. Apart from cells with 2n-----20, some cells with 21 chromosomes also were found (figure 25). The chromo- some size variation from longest to shortest is gradual, there are 1 pair with median, 4 pairs with submedian and 5 pairs with subterminal centromeres. One of the pairs with submedian and one of those with subtcrminal centromere bear satellites in the short arm (indicated by arrows in figure 25). This fact also supports the tetraploid nature of the material, but the different chromosomes of the complement are not in multiples of 4. Thus both diploid and tetraploid races appear to exist in this species. It is probable that the tetraploid after originating as an autopolyploid has undergone differentiation in its genomes and become stabilised more or less as a functional diploid. Nevertheless, residual homologies between the different sets still seem to persist.

3.2. Intergeneric crosses

To understand the crossability relationships of 1". cookei, intergeneric crosses were attempted between T. cookei and maize (Zea maya, 2n=20), teosinte (Zea mexicana, 2n=20), tripsacum (, 2n=72) and Job's tears (Coix lacryma- jobi var. typica, 2n=20). In crosses where maize and Job's tears were involved they were used as pistillate parents and in the rest T. cookei was the female parent. In maize, the styles were clipped to 1-2 cm length and pollinated. In the rest of the crosses, the female parent was isolated, enclosed in a large muslin bag and male spikelets were removed periodically and pollinated. There was no seed set excepting in cross T. cookei× teosinte where two apparently normal seeds were formed which, however, failed to germinate. Thus once again hybridization attempts between american and asiatic Maydeae did not succeed as in the previous attempts (Mangels- doff and Reeves 1939; Harada et a11954, 1955; Venkateswarlu 1962, 1963; Venkates- warlu and Chaganti 1965; Koul and Paliwal 1965; Rao 1975), suggesting that May- deae is an artificial assemblage of monoecious grasses and that genetic relationships between american and asiatic Maydeae, and among the members of the latter do not exist. However, the apparent experimental hybridization between maize and job's Cytology of Trilobachne cooket 301

tears utilizing embryo culture technique recently (Nowacki et al 1972), though the seedlings died of chlorosis, indicates that such special techniques are to be employed before the phylogenetic relationships between the taxa concerned axe entirely ruled out.

Acknowledgements

We are grateful to R S Rao, K Hemadri, Raju S K Chaganti and H G Wilkes for supplying the seod materials for the investigation. The help rendered by R N Rao in photomicrography is greatly appreciated. One of us (DSN) is thankful to the University Grants Commission for awarding a Junior Research Fellowship.

References

Anonymous 1960-61 Annual Report, Maharastra Association for the Cultivation of Science, Poona Bhagwat S D and Deodikar G B 1961 Trilobachne, an imperfectly known . In Professor S P Agharkar Commemoration Volume ed T S Mahabale (Poona: University of Poona) 139-144 Bor N L 1960 Grasses of Burma, Ceylon, India and Pakistan (blew York: Pergamon Press) Cameron J W 1943 Chromosomes of a maize relative, Polytoca macrophylla Bonth; Am. J. Bot. 30 776-778 Chandravadana P and Galinat W C 1976 Comparative cytology of certain Maydoae and Andro- pogoneae genera in reference to their evolution; ,I. Hered. 67 161-166 Harada K, Murakami M, Fukushima A and Nakazima M 1954 Breeding study on the forage crops: Studies on the intergeneric hybridization between the genus Zea and Coix (Maydoae). I (In Japanese with English summary); Sci. Rep. Saiko Univ. Agric. No. 6 139-145 Harada K, Umekage O and Nakazima M 1955 Studies on the intorgenefic hybridization between the genus Zea and Coix (Maydeae), (In Japanese); Jpn. J. Breed. 4 288 Koul A K and Paliwal R L 1965 Cytogenetic basis for taxonomic rearrangement of the tribe May- deae (family Gramineae); Kashmir Sci. 2 114-121 Mangelsdoff P C and Reeves R G 1939 The origin of Indian corn and its relatives; Texas dgric. Exp. Sta. Bull. No. 574 Nowacki E, Aniol A and Biebcr D 1972 An attempted cross of Zea mays and Coix lacryma-job. and the serological relationships of these species: Bull. l'Acad. Polanaise Sci. 20 695-697 Rao P N 1975 Male sterility in Chionachne koeniqii; Maydica 20 197-202 Sapre A B 1977 Karyotype of Trilobachne cookei (Stapf) Schenck Ex Henrarcl; Curt. ScL 46 357 Venkateswarlu J 1962 Origin of maize in proccedings of Summer School in Botany (¢ds P Mahesh- waft, B M Johri and I K Vasil) 494-504 Venkateswarlu J 1963 Cytogenetic evolution in Angiospcrms--Maydeae; Mere. Indian hot. Soc. 4 65-73 Venkate~warlu J and Chaganti R S K 1965 Apomixis in Coix; Maize News Lett. 39 183 Venkate~warlu J, Chaganti R S K and Ran P N 1976 Pachytene chromosome morphology and its bearing on interspecific and intergeneric relationships of Coix; Bot. Mus. Leafl. Harvard Univ. 24 205-224