Chromosome Botany (2016) 11(1): 14-20 ©Copyright 2016 by the International Society of Chromosome Botany

Meiotic abnormalities in some members of the collected in District Kinnaur, Himachal Pradesh, India

Kamlesh Kumari1 and Manjit Inder Singh Saggoo

Department of Botany, Punjabi University, Patiala-147002, India

1Author for Correspondence: [email protected] Received November 13, 2014; accepted May 5, 2015

ABSTRACT: Male meiotic studies in ten of grasses (Poaceae) were carried out from District Kinnaur, Himachal Pradesh, India, between the altitudinal ranges of 2000 to 3080 m. All the species were worked out cytologically for the first time from the study area. The hexaploid cytotype in Thysanolaena agrostis (n=33) made new addition for the species on a worldwide basis, supplementing the earlier report of n=11 and 12. The meiotic course in all the species studied was found to be abnormal. Cytomixis in two species, Apluda mutica and Elymus dahuricus was reported here for the first time. Besides abnormal meiotic course the microsporogenesis was found to be normal in all the species but these abnormalities results in the reduction of pollen fertility in the .

KEYWORDS: Chromosome number, cytomixis, meiotic abnormalities, pollen fertility, Kinnaur, Himachal Pradesh

Kinnaur is a region of high mountain ranges with narrow and at Botanical Survey of India (BSI) herbarium, valleys of the River Satluj and its tributaries. Most of Dehradun (Rau 1975, Chowdhery and Wadhwa 1984, Kinnaur enjoys a temperate climate due to its high Aswal and Mehrotra 1994). The young unopened spikes elevation with long winters from October to May and were fixed in carnoy’s fixative for 24 h and then stored in Short summers from June to September. Portions of 70% alcohol at 4oC until use. For meiotic studies anthers Kinnaur are situated high in the Himalayas, where were squashed in 2% acetocarmine. Pollen fertility was vegetation is sparse and consists primarily of hardy estimated using glycerol-acetocarmine technique. grasses. Alpine species such as juniper, pine, fir, cypress Photomicrographs were made from freshly prepared slides and rhododendron can be found at elevations between using Nikon 80i Eclipse Microscope. 3,500 and 5,000m, primarily in middle Kinnaur. At lower altitudes temperate- climate trees are found, including oak, RESULTS AND DISCUSSION chestnut, maple, birch, alder, apple and apricot. Based on the classification given by Champion and Seth (1968), the Tribe Thysanolaeneae vegetation of this region can be classified in to 29 types 1. Thysanolaena agrostis Nees. which belong to Himalayan moist and dry temperate Commonly known as Brooms grass is a tall, tufted, reed forests, sub-alpine forests, dry and moist alpine scrub and like perennial grass, its is made in to broom meadows. In the Western Himalayan region, the which is locally known as Phool jhadu. Young leaves and prominent angiosperm families are Asteraceae, Rosaceae, stem tips are used to feed cattle and buffaloes. Its large Poaceae, Ranunculaceae and Brassicaceae (Rau 1975). As are used in making brooms. The grass is the cytomorphological diversity in the grasses of the occasionally planted for ornamental purposes and as a region is unexplored, In the present study, detailed meiotic hedge. Presently, the species has been studied course in ten species of grasses has been carried out and chromosomally on the basis of plants collected from Baba all the species had abnormal meiotic courses with the Nagar (2000m). The hexaploid chromosome count of presence of late disjunction, out of plate bivalents, n=33 (based on X=11, 12), represents a new cytotype, cytomixis, laggards and bridges (Figs. 1-3). The supplementing the earlier report of diploid (n=12) phenomenon of cytomixis has been reported in five cytotype as reported by Kalia (1978) from North Eastern species such as Apluda mutica (n=10), Elymus dahuricus India and Tetraploid (n=22) cytotype as reported by (n=21), Dactylis glomerata (n=7), Eleusine coracana Mehra and Sunder (1969) from North India. Meiotic (n=18) and monspliensis (n=21). course included the phenomenon of out of plate bivalents (33.33%) and late disjunction (30%), laggards (37.5%) MATERIALS AND METHODS and bridges (40%) and consequently reduced pollen For meiotic studies, young spikes of appropriate size were fertility (89.51%) and pollen grains of variable sizes. collected from wild plants during June-August in different localities of District Kinnaur, Himachal Pradesh. Tribe Systematic surveys were conducted to cover different 2. Apluda mutica Linn. altitudinal zones between 2000-3080 m. The Apluda mutica is s perennial grass usually occurs along specimens were identified with the help of regional floras the edges of fields, in moist places along streams and 15 KUMARI AND SAGGOO

Fig.1. a. Thysanolaena agrostis (n=33) A PMC showing 33 bivalents at M-I, b. A PMC showing 33:33 distribution of chromosome at mixed anaphase-I, c. A PMC showing late disjunction at A-I, d. A PMC showing Unoriented bivalents at M-I, e. A PMC showing laggards at A-I, f. A PMC showing a chromatin bridge at A-I, g. Sterile and fertile pollen grains, h-i. Apluda mutica (n=10) A PMC showing 10 bivalents at Diakinesis and M-I, j. A PMC showing 10:10 distribution of chromosomes at A-I, k. A PMC showing cytomixis at T-I, l. A PMC showing unoriented bivalents at M-I, m. heterogeneous sized pollen grains, n. parviflorum (n=20) A PMC showing 20 bivalents at M-I, o. A PMC showing 20:20 chromosomes at A-I, p. A PMC showing laggards at A-I, q. Sterile and fertile pollen grains, r. Elymus dahuricus (n=21)A PMC showing 21 bivalents at M-I, s. A PMC showing 21:21 chromosomes at A-I, t. A PMC showing cytomixis at M-I.

MEITOTIC ABNORMALITIES IN SOME MEMBERS OF THE POACEAE 16

Fig.2. a. A PMC showing late disjunction at A-1, b. A PMC showing laggard at A-I, c. A PMC showing bridge at A-I, d. Sterile and fertile pollen grains, e-f. Elymus semicostatus (n=14) A PMC showing 14 bivalents at Diakinesis and M-I, g. A PMC showing 14:14 chromosomes at A-I, h. A PMC showing laggards at A-I, i. Sterile and fertile pollen grains, j-k. Dactylis glomerata (n=7) A PMC showing 7 bivalents at Diakinesis and M-I, l. A PMC showing 7:7 chromosomes at A-I, m. A PMC showing cytomixis at Diakinesis, n. A PMC showing laggard at A-I, o. A PMC showing bridge at A-I, p. Sterile and fertile pollen grains, q. Poa jaunsarensis (n=14) A PMC showing 14:14 chromosomes at A-1, r. A PMC showing laggard at A-I, s. A PMC showing Bridge at T-I, t. Sterile and fertile pollen grains. 17 KUMARI AND SAGGOO

00 KUMARI AND SAGGOO

Fig.3. a-b. Brachiaria villosa (n=18) A PMC showing 18 bivalents at Diakinesis and M-I, c. A PMC showing laggard at A-I, d. Sterile and fertile pollen grains, e. Eleusine coracana (n=18) A PMC showing 18 bivalents at Diakinesis, f. A PMC showing cytomixis at Diakinesis, g. A PMC showing laggard at A-I, h. A PMC showing bridge at A-I, i. Sterile and fertile pollen grains, j. Polypogon monspeliensis (n=21) A PMC showing 21 bivalents at Diakinesis, k. A PMC showing 21:21 chromosomes at A-I, l. A PMC showing Unoriented bivalents at M-I, m. A PMC showing cytomixis at M-I, n. A PMC showing laggard at A-I, o. A PMC showing bridge at A-I, p. Sterile and fertile pollen grains. meadows on slopes below 2000m. The young plants can as irregular distribution of chromosomes at anaphase. be used as forage grass. Meiotic chromosome count of B-chromosomes had been reported by Saxena and Gupta n=10 is confirmed on the basis of plant collected from 1970 and Kalia 1978. Subsequent course of meiosis is Baba Nagar (2,000 m) which is in conformity with the abnormal with the presence of cytomixis at Telophase-I previous reports from Central India by Gill et al. (1980). (33.33%), out of plate bivalents at metaphase-I (30%) and Cytomixis was reported for the first time in this species, reduced pollen fertility (81.39%). earlier Sahni and Bir (1985) recorded abnormalities such MEITOTIC ABNORMALITIES IN SOME MEMBERS OF THE POACEAE 18

orts and remarks.

with 2n=42 confirmed. is with report Previous cytotype 4x of Remarks ever First chromosome report 6x cytotype of 2n=66with for species.the Previous report of 2x cytotype 2n=20with confirmed. is Previous report of 4x cytotype 2n=40with confirmed. is Previous report of 6x cytotype Previous report of 2n=28 is confirmed. Previous report of 2x cytotype 2n=14with confirmed. is Previous report of 2n=28 is confirmed. 2n=36with confirmed. is Previous report of 4x cytotype 2n=36with confirmed. is Previous report of 6x cytotype 2n=42with confirmed is

-

3B,

-

1B,42,35

-

4B,40,22 4B,28,21,27,

- -

2n=36,36+1B Previous reports*** 2n=22,24 2n=20,20+2B,40, 60,70 2n=20, 20+1B, 20+2 2n=28,42 2n=28 2n=14, 14+1 14+1 42 2n=28 2n=18,27,36,36+1 2B,37, 38, 39,43 2n=14, 28,28+0

85.38 75.96 67.82

Pollen fertility (%) 89.51 81.39 80.29 79.87 83.23 88.94 64.19

4x 4x 6x

Ploidy Ploidy level (x) 6x 2x 4x 6x 4x 2x 4x

18 18 21

Meiotic chromosome number (n) 33 10 20 21 14 7 14

Peo

3080 Chora Locality Locality altitudewith (m) Nigulsari 2050 Baba Nagar 2000 Baba Nagar 2000 Hango Chitkul 3450 Pooh 2662 Chuling 3000 2050 Baba Nagar 2000 Reckong 2670

.

(R. Stapf Br.)

(Linn.) Desf

Nees.

(Nees ex Steud.) Meld.

Linn.

(Lam.) Camus(Lam.) A.

(Linn.) Gaertn.

Turcz.Grieseb. ex

Bor.

Linn.

mutica mutica

Andropogoneae Eragrostideae Aveneae

jaunsarensis

Elymus dahuricus Elymus Tribe:Paniceae Taxa/Accession number(PUN) Tribe:Thysanolaeneae Thysanolaena agrostis (58696) Tribe: Apluda (58706) Capillipedium parviflorum (58702) Tribe:Triticeae (58698) Elymus semicostatus (58699) Tribe: Poeae Dactylisglomerata (58511) Poa (58711) Brachiaria villosa (58688) Tribe: Eleusine coracana (58709) Tribe: Polypogon monspeliensis (58509)

Data showing DataTaxa showing Accession with number (PUN),Locality altitude, with chromosome meiotic number, Ploidy number, previous rep

8. Sr. No. 1. 2. 3. 4. 5. 6. 7. 9. 10.

Table. 19 KUMARI AND SAGGOO

3. Capillipedium parviflorum (R. Br.) Stapf outside India and n=7 by Koul and Gohil (1990) from Capillipedium is a tufted perennial grass with erect, India. Further meiotic course is observed to be abnormal slender culms and Spikelets are present in pairs. due to the presence of cytomixis (22.85%), laggards Inflorescence is purplish in color. This grass grows on (9.52%) and bridges (9.09%) which ultimately leads to hills and mountain slopes. This grass is eaten by cattle and reduced pollen fertility (88.94%) buffaloes. Presently the species for meiotic study has been collected from Baba Nagar (2000m). Meiotic course of the 7. Poa jaunsarensis Bor. species shows n=20 bivalents at metaphase-I which is in Poa jaunsarensis is a Perennial plant with elongated conformity with the previous report by Ahsan et al. (1994) rhizomes, usually found on the bank of rivers or shady from Pakistan and Mehra (1982) from India. Further the places. Leaves are mostly basal. Spikelets comprising 2-3 species is found to have abnormal meiotic course with the fertile florets. Meiotic chromosome count of n=14 on the presence of laggards at anaphase-I which ultimately leads basis of plant collected from Chuling (3000m) is to reduced pollen fertility (80.29%). confirmed by the presence of 14 :14 chromosomal distribution during A-I and is in conformity with the Tribe Triticeae previous report of Mehra and Sunder (1969). Further the 4. Elymus dahuricus Turcz. ex Grieseb. species is found to have abnormal meiotic course with the Commonly known as Dahurian wildrye is said to have presence of laggards (27.05%) and bridges (21.42%) and high saline tolerance and is adapted to all soil textures reduced pollen fertility of (64.19%). (Dobb and Burton 2013). It is a tall plant with leaves growing high up on the stem providing excellent forage Tribe Paniceae for pasture. It is a short lived perennial with quick 8. Brachiaria villosa (Lam.) A. Camus establishment and is used as an early forage component This is an annual weedy species recognized by its short, with other slower growing species. The hexaploid broad, usually soft hairy leaf blades and small spikelets. It chromosome count of n=21 (based on x=7) is confirmed is found on hilly slopes, fields, roadsides and other weedy on the basis of plant collected from Hango (3080m), places. Presently the species has been studied which is in conformity with the previous reports by Von chromosomally on the basis of plant collected from Chora Bothmer et al. (2005) from and Gohil and Koul (2050m). Further meiotic study of the species reveals (1986) from India. Phenomenon of cytomixis (38.46%) n=18 bivalents at Diakinesis and metaphase-I and is in was reported for the first time in the species. Other conformity with the previous report by Basappa and meiotic abnormalities include laggards (16.12%), bridges Muniyamma (1981) from outside India and Mehra (1982) (23.07%) which ultimately leads to reduced Pollen fertility from Eastern India. Meiotic course is abnormal with the (79.87%). presence of laggards at anaphase-I and reduced pollen fertility (85.38%). 5. Elymus semicostatus (Nees ex Steud.) Meld. Elymus is a of tufted perennial grasses which Tribe Eragrostideae contains many of the main cereal grains. Plants of this 9. Eleusine coracana (Linn.) Gaertn. genus are generally known as wheatgrass. A very variable Eleusine coracana is an annual plant commonly known as species ranging from small slender plants to tall robust African finger millet. It is adaptable to higher elevations ones. Presently the species has been collected from and is grown in Himalayas up to 2,300 m elevation. Finger Chitkul (3450m). Meiotic studies of the species shows the millet straw makes good fodder and is used in traditional presence of 14 bivalents at diakinesis and metaphase-I medicine as an internal remedy for leprosy or liver disease which is in conformity with the previous report by (Van Wyk and Gericke 2000). Finger millets are also rich Salomon and Lu (1992). Further meiotic course is in dietary fibre hence help in easy digeston. Finger millet abnormal with the presence of laggards (25%) at anaphase is rich in essential amino acids and contains a higher level I which ultimately leads to reduced pollen fertility of calcium and iron than other cereals (Pore and Magar (83.23%). 1979, Shukla et al. 1985, Babu et al. 1987). Presently the chromosome number in the species is determined on the Tribe Poeae basis of plants collected from Baba Nagar (2000m). 6. Dactylis glomerata Linn. Meiotic studies reveals the presence of n=18 bivalents at Commonly known as orchard grass is widely used as a diakinesis which is in conformity with the previous report hay grass and for pastures because of its high yields and by Devarumath et al. (2005) and Mehra (1982). Further sugar content, which makes it sweeter than most other meiotic course is abnormal with the presence of cytomixis temperate grasses. It is distributed in temperate and (26.92%), laggards (14.81%) and bridges (15%) which subtropical regions throughout the world. The present ultimately leads to reduced pollen fertility (75.96%). meiotic study carried out on plants collected from Pooh (2662m) reveals the presence of n=14 bivalents at Tribe Aveneae diakinesis and metaphase-I. Earlier reports reveals the 10. Polypogon monspeliensis (Linn.) Desf. existence of n=14 by Petrova and Stoyanova (1997) from MEITOTIC ABNORMALITIES IN SOME MEMBERS OF THE POACEAE 20

Polypogon monspliensis commonly known as annual Dobb, A. S. and Burton. 2013. Rangeland Seeding Manual for rabbits foot grass is an annual grass growing to heights British Columbia, B. C. Min. Agri., Sust. Agri. Mgmt. Br., between 5 centimeters to one meter. The soft, fluffy Abbotsford, B. C. inflorescence is greenish in color, sometimes divided in to Ghukasyan. 2004. Extent of karyological study of Armenian lobes. The spikelets have long, thin, whitish awns, which grasses (Poaceae). Fl. Rastitel’n. Resursy Armenii 15:85-89. give the inflorescence its texture. This grass is usually Gill, B. S., Bir. S. S., Singhal, V. K. and Bedi, Y. S. 1980. found in moist places and streamsides below 3000m. For Cytological studies on some grasses from Pachmarhi forests the present studies the material was collected from (central India). J. Cytol. Genet. 15:51-57. Reckong Peo (2670m) and meiotic studies revealed n=21 Gohil, R. N. and Koul, K. K. 1986. SOCGI Plant chromosome bivalents at diakinesis and 20:20 distribution of number reports-IV. J. Cytol. Genet. 21:155. chromosomes at anaphase-I. Earlier reports reveals the Kalia, V. 1978. Cytological investigations in some grasses of existence of n=14 by Ghukasyan (2004) from outside North Eastern India. Tribes: Andropogoneae, Arundineae, India and n=21 by Koul and Gohil (1991) from India. oryzeae, Arundinelleae, Chlodeae, Eragrosteae and Further meiotic course is abnormal with formation of out Sporoboleae. Ph.D Thesis, Punjab University 1978, 17 spp. of plate bivalent (13.63%), cytomixis (24%), laggards Koul, K. K. and Gohil, R. N. 1990. Cytological studies on some (12.90%), bridges (11.11%) which ultimately leads to Kashmir grasses…V. Dactylis glomerata Linn. J. Cytol. reduced pollen fertility (67.82%). Genet. 25: 74-77. Koul, K. K. and Gohil, R. N. 1991. Cytogenetic studies on some ACKNOWLEDGMENTS Kashmir grasses. VIII Tribe Agrostideae, Festuceae and The authors are thankful to UGC, New Delhi for financial assistance Paniceae. Cytologia 56: 437-452. under DRS-SAP II, ASIST programme and to DST for grant under FIST Mehra, P.N.1982. Cytology of East Indian grasses.Y. programme. Thanks are also due to the Director Botanical Survey of Mehra, P. N. and Sunder, S. 1969. Cytological studies in the India (Dehradun) for identification of the species and Head, Department North Indian Grasses. Part II. Res. Bull. Punjab. Univ.,Sci. of Botany, Punjabi University, Patiala for providing the necessary 20: 503-539. laboratory and library facilities. Petrova, A. and Stoyanova, K. 1997. Mediterranean chromosome number reports 7 (783-802). Fl. Medt. 7:204-213. LITERATURE CITED Pore, M. S. and Magar, N. G. 1979. Nutrient composition of hybrid varieties of finger millet. Indian J. Agric. Sci. Ahsan, S. M. N., Vahidy, A. A. and Ali, S. I. 1994. Chromosome 49:526-531. numbers and incidence of polyploidy in Rau, M. A. 1975. High altitude flowering plants of Western (Poaceae) from Pakistan. Ann. Missouri Bot. Gard. Himalaya. Calcutta: Botanical Survey of India. 81(4):775-783. Sahni, M. and Bir, S. S. 1985. SOCGI Plant chromosome Aswal, B. S. and Mehrotra, B. N. 1994. Flora of Lahaul-Spiti. number reports-III. Journal of Cytology and Genetics. 20: Dehradun: Bishen Singh Mahendra Pal Singh. 205-206. Babu, B.V., Ramana, T. and Radhakrishanan, T. 1987. Chemical Salomon, B. and Lu. B. R. 1992. Genomic groups, morphology composition and protein content in hybrid varieties of finger and sectional delimitation in Eurasian Elymus (Poaceae, millet. Indian. J. Agric. Sci. 57:520-522. Triticeae). Pl. Syst. Evol. 180: 1-13. Basappa, G. P. and Muniyamma, M. 1981. In chromosome Saxena, B. K. and Gupta, B. K. 1970. Chromosome numbers of number reports LXXII. Taxon 30:703. some grasses of Dehra Dun. Bulletin of Botanical Survey of Champion, H. G. and Seth, S. K. 1968. A revised survey of the India. 11(3/4): 433-444. forest types of India. Delhi, India: Manager of Publications, Shukla, S. S., Gupta, O. P., Sawarkar, N. J., Sharma, Y. K. 1985. Govt. of India Press. Studies on macro and micro mineral nutrient contents of Chowdhery, H. J. and Wadhwa, B. M. 1984. Flora of Himachal some cultivars of ragi (Eleusine coracana Gaertn). Ind. J. Pradesh analysis. 3 vols. Culcutta: Botanical Survey of India. Nutr. Dietet. 22: 249-252. Devarumath, R. M., Hiremath, S. C., Rao, S. R., Kumar, A. and Van Wyk, B. E. and Gericke, N. 2000. Peoples plants : a guide Bewal. 2005. Genome analysis of finger millet E. coracana to useful plants of Southern . Briza, Arcadia. by interspecific hybridization among diploid wild species of Von Bothmer, R., Salomon, B., Enomoto, T. and Watanabe, O. Eleusine (Poaceae). Cytologia 70: 427-434. 2005. Distribution, Habitat and status for perennial triticeae species in Japan. Bot. Jahrb. Syst.126 (3): 317-346.