2019 年 84 巻 2 号 p. 183-188
Present study on male meiotic analysis of wild grass species from Uttarkashi district hills, Uttarakhand, India provides chromosome numbers for 10 species belonging to tribe Aveneae, family Poaceae. The study reports chromosome numbers firstly for Helictotrichon uniyalii (2n=4x=28, Rarhi Valley), Trisetum aeneum (2n=2x=14, Assi Ganga Valley) and Tr. clarkei (2n=4x=28, Assi Ganga Valley), and confirmed in the Indian species, Avena fatua (2n=6x=42, Purola Valley), Koeleria macrantha (2n=2x=14, Bhagirathi Valley), Phleum alpinum (2n=4x=28, Har Ki Dun Valley), P. pratense (2n=4x=28, Assi Ganga Valley), Trisetopsis aspera (2n=6x=42,; Assi Ganga Valley), T. virescens (2n=4x=28, Assi Ganga Valley and Bhagirathi Valley) and Tr. spicatum (2n=4x=28, Assi Ganga Valley, Bhagirathi Valley, and Har Ki Dun Valley). The meiotic course was normal in most of the species, although suggested a paracentric inversion in a plant of Tr. spicatum.
Tribe Aveneae also called ‘oat tribe’ is one of the main tribes of the family Poaceae and is distributed in temperate and cold regions of the world. The tribe is represented by more than 57 genera and 1050 species (Clayton and Renvoize 1986) and is characterized by annuals and perennials with well-developed membranous ligules. The present study is in a line of endeavor to explore the phytogeographically distinct and unexplored regions of Northwest Himalaya. The aims of this study were to record the exact chromosome number in the high altitudinal grass species of the tribe Aveneae from Uttarkashi district. Also, the purpose was to analyze the detailed meiotic course at different stages including microsporogenesis and estimation of pollen fertility.
Wild accessions of 10 species in six genera of tribe Aveneae for detailed male meiosis and pollen fertility were collected from Uttarkashi district hills, Uttarakhand state of India (Table 1). Young and unopened panicles were fixed in Carnoy’s fixative (ethanol : chloroform : acetic acid=6 : 3 : 1). After 48 h, the materials were transferred to 70% ethanol and stored in a refrigerator. Meiocyte preparations were made by squashing the developing anthers from the unopened florets using 1% acetocarmine. Chromosome numbers and meiotic course were studied from the freshly prepared slides having pollen mother cells (PMCs) at diakinesis, metaphase I (M I), anaphase I (A I) and telophase I, II (T I, II). Pollen fertility was estimated through a stainability test by squashing the mature anthers in a mixture of glycerol and 1% acetocarmine (1 : 1). Well-filled pollen grains with completely stained nuclei and cytoplasm were scored as fertile, while partially stained and shriveled ones as sterile. Preparations of PMCs with well-spread bivalents/chromosomes, meiotic irregularities, and pollen grains were selected for photomicrographs using a Nikon 80i Eclipse and Leica Qwin microscopes equipped with a digital imaging system. Vouchers of cytological examined specimens were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).
| Name of taxon | Locality of the collection with altitude | Accession number (PUN) | Gametophytic chromosome number | Ploidy level | Pollen fertility (%) | Previous chromosome number reports (2n) |
|---|---|---|---|---|---|---|
| Avena fatua L. =A. fatua var. fatua =A. fatua var. glabrata =A. pilosa Scop. | Purola, Uttarkashi, 1330 m | 62416 | 21 | 6x | 82 | 2n=14 Russia (Rodionov et al. 2006). 2n=14, 28 India (Mehra and Remanandan 1973). 2n=28 India (Singhal et al. 2014). 2n=42 Armenia, Bulgaria (Kožharov and Petrova 1991), Canada (Huskins 1926), China (Yu et al 199 L), Czech Republic (Javůrková-Jarolimová 1992), Egypt (Philp 1938), England (Dempsey et al. 1994), Greece (Rajhathy and Morrison 1959), India (Mehra et al. 1968), Italy (Martinoli 1955), Japan (Kihara 1919), Libya (Faruqi and Quraish 1979), Nepal (Nakao and Mori 1956), Poland (Pogan et al. 1982), Russia (Nikolaeva 1929), Slovakia (Váchová and Májovský 1988), South Africa (Spies et al. 1996), Sweden (Lövkvist and Hultgård 1999), Texas (Gould 1968), United Kingdom (Bullen and Rees 1972). 2n=42+0-1B India (Kaur 2011). 2n=42+0-2B India (Parkash 1979). |
| Helictotrichon uniyalii Kandwal and B.K. Gupta | Rarhi, Uttarkashi, 2200 m | 62407 | 14 | 4x | 100 | |
| Koeleria macrantha (Ledeb.) Schult. =K. albescens=K. gracilis Pers. =K. latifrons (Domin) Rydb.=K. pontarlieri Domin =K. tokiensis Domin | Dunda, Uttarkashi, 1100 m | 62424 | 7 | 2x | 88 | 2n=14 Bulgaria (Kozuharov et al. 1988), Canada (Löve and Löve 1981), Czech Republic (Pecinka 2006), Hungary (Ujhelyi 1961), India (Kaur et al. 2011), Netherlands (Gadella et al. 1968), Poland (Frey 1993), Russia (Sokolovskaya and Probatova 1975), Slovakia (Májovský et al. 1978), Switzerland (Bajon 1985), Colorado (Reeder 1977). 2n=14, 15 Russia (Fedorov 1969). 2n=14, 28 France (Bajon 1985). 2n=28 England (Hedberg and Hedberg 1961), France (Ladadie 1976), Slovakia (Holub et al. 1972), United Kingdom (Dixon 2001). 2n=28+0-6B Austria (Larsen 1982). 2n=28, 30 England (Maude 1939). |
| Kuteti Devi, Uttarkashi, 1100 m | 62425 | 7 | 2x | 95 | ||
| Phleum alpinum L. =P. commutatum Gaudin =P. rhaeticum (Humphries) Rauschert | Kalkati Dhar, Uttarkashi, 3100 m | 60511 | 14 | 4x | 98 | 2n=14 Austria (Joachimiak and Grabowska-Joachimiak 2000), Germany (Cai and Bullen 1991), Bulgaria (Kožharov and Petrova 1991), Czech Republic (Měsiček 1992), France (Litardiere 1948), Georgia (Daviliazidze and Mosulischvili 1984), Greece (Strid and Franzen 1981), India (Mehra and Sunder 1969), Italy (Contadriopoulos and Gamisans 1974), Poland (Michalski 1955,), Romania (Kula et al 2006), Russia (Magulaev 1984), Scotland (Joachimiak and Grabowska-Joachimiak 2000), Slovakia (Májovský et al. 1970), Sweden (Gregor and Sansome 1930), Switzerland (Müntzing 1935), Ukraine (Pashuk 1987). 2n=14+0-2B Austria (Joachimiak and Kula 1996), Bulgaria (Petrova and Stoyanova 1998). 2n=14, 28 Germany (Roessler 1983), Poland (Joachimiak and Kula 2006), Sweden (Joachimiak and Grabowska-Joachimiak 2000). 2n=14, 28+0-2B Sweden (Joachimiak and Kula 1996). 2n=24 Scotland (Joachimiak and Kula 1996). 2n=28 Canada (Bowden 1960), France (Heitz 1967), Greece (Strid and Franzen 1981), Georgia (Bennett et al. 1982), Greenland (Böcher and Larsen 1950), India (Mehra and Remanandan 1973), Japan (Tateoka 1964), Norway (Laane 1965), Russia (Sokolovskaya 1968), Scotland (Gregor and Sansome 1930), South America (Rahn 1960), Sweden (Müntzing 1935), Switzerland (Scholte 1977), Turkey (Doğan 1983), USA (Clausen et al. 1940). 2n=28, 56 Norway (Knaben and Engelskjön 1967). |
| Kalkati Dhar, Uttarkashi, 3200 m | 62411 | 14 | 4x | 95 | ||
| Kalkati Dhar, Uttarkashi, 3250 m | 61011 | 14 | 4x | 95 | ||
| Har Ki Dun, Uttarkashi, 3450 m | 61012 | 14 | 4x | 92 | ||
| Har Ki Dun, Uttarkashi, 3500 m | 62410 | 14 | 4x | 92 | ||
| Morinda Lake, Uttarkashi, 3800 m | 62408 | 14 | 4x | 95 | ||
| Ratadoi, Uttarkashi, 3900 m | 62409 | 14 | 4x | 95 | ||
| P. pratense L. =P. brachystachyum (Salis) Gamisans, A.T. Romero and C. Morales =P. nodosum L. | Darwa, Uttarkashi, 3400 m | 62413 | 14 | 4x | 95 | 2n=14 Canada, Frence, Germany (Cai and Bullen 1991), England (Gregor and Sansome 1930), India (Koul and Gohil 1987), Poland (Pogan et al. 1980), Portugal (Fernandes and Queirs 1969), Russia (Magulaev 1984), Sweden (Müntzing 1935). 2n=14+0-4B Sweden (Bosemark 1957). 2n=14, 28 Hungary (Soo 1947). 2n=14, 28, 42 Poland (Joachimiak and Kula 1993). 2n=14, 35, 36, 42 Sweden (Müntzing 1935). 2n=14, 42 France (Litardiere 1948), Germany (Rohweder 1937), Sweden (Nordenskiold 1937), Russia (Guzik 1984), USA (Wilton and Klebesadel 1973). 2n=28 Bulgaria (Kozuharov and Petrova 1991), India (Koul and Gohil 1991), Russia (Magulaev 1976), Slovakia (Májovský et al. 2000). 2n=28, 42 Italy (Cenci 1978), Poland (Joachimiak and Kula 1993). 2n=42 Armenia (Ghukasyan 2004), Belgium (Cai and Bullen 1991), Bulgaria (Petrova and Stoyanova 1997), Canada (Bowden 1960), Denmark (Simonsen 1972), England (Montgomery et al. 1997), Finland (Arohonka 1982), Greece (Strid and Franzen 1981), Georgia (Gvinianidze and Avazneli 1982), Germany (Kattermann 1930), Hungary (Polya 1950), India (Mehra et al. 1968), Italy (Cenci 1979), Japan (Tateoka 1953), Norway, Russia (Simonsen 1972), Poland (Skalińska et al. 1978, Slovakia (Májovský et al. 1974), Sweden (Nordenskiold 1937), USA (Gregor and Sansome 1930). |
| Darwa, Uttarkashi, 3500 m | 62414 | 14 | 4x | 95 | ||
| Darwa, Uttarkashi, 3700 m | 62415 | 14 | 4x | 90 | ||
| Darwa Top, Uttarkashi, 3800 m | 62412 | 14 | 4x | 88 | ||
| Trisetopsis aspera (Munro ex Thwaites) Röser & A. Wölk =Helictotrichon asperum (Munro) Bor | Dharkot, Uttarkashi, 2500 m | 62426 | 21 | 6x | 100 | 2n=28 India (Christopher and Abraham 1971). 2n=28, 42 India (Parkash 1979). 2n=42 India (Mehra and Sood 1974). |
| T. virescens (Nees ex Steud.) Röser & A. Wölk =Helictotrichon virescens (Nees ex Steud.) Henrard | Dharkot, Uttarkashi, 2500 m | 61010 | 14 | 4x | 92 | 2n=28 India (Mehra and Sharma 1977). 2n=42 India (Sharma and Sharma 1978). 2n=56 India (Sharma and Kumar 1980). |
| Kacheru, Uttarkashi, 2700 m | 62429 | 14 | 4x | 95 | ||
| Bhojwasa, Uttarkashi, 3600 m | 62430 | 14 | 4x | 95 | ||
| Chirwasa, Uttarkashi, 3600 m | 62427 | 14 | 4x | 95 | ||
| Devgaad, Uttarkashi, 3600 m | 62428 | 14 | 4x | 95 | ||
| Trisetum aeneum (Hook.f.) R.R. Stewart | Darwa, Uttarkashi, 3700 m | 62418 | 7 | 2x | 100 | |
| Darwa Top, Uttarkashi, 3800 m | 62417 | 7 | 2x | 100 | ||
| Tr. clarkei (Hook.f.) R.R. Stewart | Dodital, Uttarkashi, 3200 m | 62420 | 14 | 4x | 100 | |
| Darwa, Uttarkashi, 3400 m | 62419 | 14 | 4x | 100 | ||
| Tr. spicatum (L.) K.Richt.=Tr. seravschanicum Roshev.=Tr. mongolicum (Hultén) Peschkova | Dodital, Uttarkashi, 3000 m | 60485 | 14 | 4x | 98 | 2n=14, 28, 42 Russia (Krogulevich 1976). 2n=28 Alaska (Johnson and Packer 1968), Austria (Jonsell et al. 1975), Canada (Mosquin and Hayley 1966), China (Chen and Hsu 1962), France (Kupfer 1974), Greenland (Holmen 1952), Iceland (Jonsell et al. 1975), India (Mehra and Sunder 1969), Japan (Tateoka 1953), Kazakhstan (Probatova et al. 2012), Mexico (Beaman 1962), New Zealand (Murray et al. 2005), Norway (Flovik 1938), Russia (Zhukova 1965), Sweden (Jonsell et al. 1975), USA (Myers 1947). 2n=28, 42 Canada (Bowden 1960), Greenland (Böcher and Lasen 1950), Iceland (Jonsell et al. 1975), Sweden (Lövkvist and Hultgård 1999), USA (Löve and Löve 1965). 2n=42 Canada (Moore et al. 1976), Dominican Republic (Davidse and Pohl 1972). |
| Gangotri, Uttarkashi, 3100 m | 62422 | 14 | 4x | 98 | ||
| Kankhu, Uttarkashi, 3200 m | 62421 | 14 | 4x | 98 | ||
| Bhojwasa, Uttarkashi, 3700 m | 60467 | 14 | 4x | 92 | ||
| Morinda Lake, Uttarkashi, 3700 m | 62423 | 14 | 4x | 95 |
Presently 10 grass species of six genera belonging to tribe Aveneae (Poaceae) were analyzed for gametophytic chromosome number, male meiosis, and pollen fertility. Information covering the name of the taxon, sites of the collection with altitude, accession numbers, gametic chromosome number, ploidy level, pollen fertility, and previous chromosome number reports with the country are given in Table 1.
Avena fatua L. of Purola locality showed the gametic chromosome number of n=21 as confirmed from the presence of 21 bivalents in PMCs at diakinesis (Fig. 1A) and M I (Fig. 1B). Majority of the PMCs at A I showed equal segregation of chromosomes. However, 10.6% of the PMCs depicted some meiotic irregularities in the form of late disjunction and laggards (Fig. 1C, D). The lagging chromosomes seem to be organized into micronuclei (Fig. 1E). In a plant accession, 18% of pollen grains were observed as sterile (Fig. 1F). Based on the base number of x=7 the analyzed accession exists at 6x ploidy level. A perusal of earlier chromosome reports reveals that the species exhibits intraspecific euploidy with three ploidy levels (2x, 4x, 6x). Out of these, 4x individuals with (2n=28) exist only in Indian taxa (Mehra and Remanandan 1973, Singhal et al. 2014) while the 2x (2n=14) and 6x (2n=42) are found in India as well as out of India. It has also been inferred that individuals of 6x cytotype depicted much wider distribution as in Africa (Egypt, Libya, South Africa), Asia (Armenia, China, India, Japan, Nepal, Russia), Europe (Bulgaria, Czech Republic, England, Greece, Italy, Poland, Slovakia, Sweden) and North America (Canada, USA) (Table 1).
Helictotrichon uniyalii Kandwal & B.K. Gupta collected from the Rhododendron forest region of the Rarhi valley, exists at 4x ploidy level with a gametic chromosome number of n=14 ascertained from the presence of 14 bivalents in PMCs at diakinesis (Fig. 1G). The meiotic course was normal with fertile pollen grains.
Two accessions of Koeleria macrantha (Ledeb.) Schult. were collected from the sub-tropical Chir pine forests and their PMCs revealed a cytotype with a chromosome number of n=7, confirmed from the presence of seven bivalents at diakinesis (Fig. 1H) and equally separated 7 : 7 chromosomes at A I (Fig. 1I). This chromosome number (2n=14) has been recorded by Kaur et al. (2011) from Kangra district, Himachal Pradesh, India and intraspecific 2x (2n=14) and 4x (2n=28) cytotypes has been reported in France (Bajon 1985).
Seven accessions of Phleum alpinum L. were collected in the subalpine and alpine meadows of Har Ki Dun Valley. In PMCs 14 bivalents were observed at diakinesis (Fig. 1J) and M I (Fig. 1K), and 14 : 14 chromosomes at poles at A I (Fig. 1L). This species had the tetraploid chromosome number of n=14. Meiotic course in all the accessions was noticed to be perfectly normal remarked with normal sporads and high pollen fertility. Presently recorded 4x chromosome number 2n=28 is in line with the previous records of Mehra and Remanandan (1973) and Mehra and Sharma (1977) from Tangmarg and Gulmarg localities of Kashmir Himalaya, respectively. Available chromosome number reports reveal that the species exhibits intraspecific euploidy with 2x, 4x and 8x ploidy levels, along with 2x and 4x cytotype reports from the Himalayan regions of India (Table 1).
P. pratense L. collected from the inception zone localities in between 3400–3900 m in Assi Ganga valley have the gametic chromosome number of n=14 ascertained from the occurrence of 14 bivalents at diakinesis (Fig. 2A) and 14 : 14 chromosomes at A I (Fig. 2B). All, the analyzed individuals (PUN 62412-15) showed regular meiosis and high pollen fertility. An account of previous chromosome number reports of the species from Himalayan regions suggests that it is a complex species with polyploids of 2x (Koul and Gohil 1987), 4x (Koul and Gohil 1991) and 6x (Mehra et al. 1968) cytotypes.
Meiotic analysis of Trisetopsis aspera (Munro ex Thwaites) Röser & A. Wölk collected from the open slope of Dharkot locality in Assi Ganga Valley, depicts the gametophytic chromosome number of n=21 confirmed from the presence of 21 bivalents at diakinesis (Fig. 2C) and M I (Fig. 2D). The bivalents were heterogeneous in size with large 14II and small 7II, which indicate the allopolyploid nature of taxon. Cytological analysis of Indian accessions of the species depicts the existence of intraspecific polyploidy with 4x (Christopher and Abraham 1971, Mehra and Sharma 1972, 1975, Parkash 1979) and 6x (Mehra and Sood 1974, Parkash 1979, Mehra and Sharma 1975) ploidy levels.
T. virescens (Nees ex Steud.) Röser & A. Wölk scored from the localities of Assi Ganga Valley and Bhagirathi Valley in the district has the chromosome number of n=14 confirmed from the presence of 14 bivalents in PMCs (Fig. 2E, F). All the accessions showed a perfectly regular meiotic course. The tetraploid chromosome number of 2n=28 was reported from Kashmir Himalaya and Parvati Valley in Kullu district, Himachal Pradesh by Mehra and Sharma (1977), Singhal et al. (2014). This reports new localities of tetraploid cytotype. The 6x cytotype with 2n=42 has been reported in the plants from Kasauli, Himachal Pradesh (Sharma and Sharma 1979) and octoploid, 2n=56 (Sharma and Kumar 1980) from Shimla Hills, Himachal Pradesh. It is thus apparent that T. virescens in the north-west Himalayas have a polyploid complex of three cytotypes with a chromosome number of 2n=28, 42 and 56.
Trisetum aeneum (Hook.f.) R.R. Stewart has been collected from high hills in Assi Ganga Valley in the district. Both the accessions possess gametic chromosome number of n=7 confirmed from the presence of seven bivalents at M I (Fig. 2G). Meiotic course in the studied accessions was regular resulting in complete fertile pollen grains. The species has been worked out for chromosome number for the first time.
Two accessions of Tr. clarkei (Hook.f.) R.R. Stewart collected from Assi Ganga Valley has the tetraploid chromosome number of n=14 confirmed from the presence of 14 bivalents in PMCs at diakinesis (Fig. 2H) and M I (Fig. 2I). Meiotic course in the PMCs was noticed to be regular and high pollen fertility.
Male meiotic analysis of five accessions of Tr. spicatum (L.) K. Richt. scored from the different localities of Assi Ganga Valley, Bhagirathi Valley and Har Ki Dun Valley depicted the existence of species as a tetraploid cytotype with a chromosome number of n=14 confirmed from the presence of 14 bivalents in PMCs at M I (Fig. 2J). Majority of the PMCs were observed with the regular meiotic course, normal sporads, and high pollen fertility. But, the few PMCs of one accession (PUN 60467) were observed with an occurrence of an event of paracentric inversion ascertained from the presence of a chromatin bridge along with the fragment in the PMCs at A I (Fig. 2K) and later as micronuclei in dyad sporads (Fig. 2L). Consequent to the presence of chromosomal inversions, the analyzed individual showed 8% sterile pollen grains. All the Indian taxa of the species so far investigated chromosomally exist at 4x level (Mehra and Sunder 1969, Mehra and Sharma 1975, 1977, Mehra and Sood 1975, Parkash 1979). But outside of India, the species depicts the presence of intraspecific diploid (2n=14), tetraploid (2n=28) and hexaploid (2n=42) cytotypes (Table 1).
The results of this study from a cytologically unexplored region of western Himalayas adds the chromosome number for three species (H. uniyalii, Tr. aeneum, and Tr. clarkei). Paracentric inversion was detected in one plant of Tr. spicatum.
Authors wish to thank the University Grants Commission (UGC), New Delhi for financial support under DSA-I scheme; Department of Biotechnology (DBT), New Delhi under DBT-IPLS project (BT/PR4548/INF/22/146/2012) and for the award of UGC-BSR-Fellowship to Jaswant Singh (Award letter no. 15610/Research/03/06/2015). Thanks are also due to the Head, Department of Botany, Punjabi University, Patiala, for providing necessary laboratory, Herbarium (PUN) and Internet facilities, and Co-ordinator, University Sophisticated Instrumentation Centre for photography. Thanks are also due to Scientist In-charge, Botanical Survey of India, Dehra Dun for providing the herbarium facility, for identification of voucher specimens.
