Meiotic Studies in Cyanotis Cristata and C. Vaga from Several Localities in North-West India

Meiotic Studies in Cyanotis cristata and C. vaga from Several Localities in North-West India

Arneet Grewal and Poonam Rani*

Department of Botany, Punjabi University, Patiala–147002, Punjab, India

Received June 6, 2018; accepted September 11, 2018

Summary Meiotic studies were carried out on 26 accessions for the evaluation of genetic diversity of Cyanotis cristata and C. vaga from various geographical areas of Una, Hamirpur, Kangra, Palampur, Sirmaur (Himachal Pradesh), Dehradun and Mussoorie (Uttrakhand) in India. All accessions were diploids with n=12. The meiosis was found to be mostly normal in both species. In C. cristata, some meiotic cells showed abnormal ﬁgures such as early disjunction, bridges, laggards, interbivalent connections, vagrant or unoriented bivalents. All accessions of C. vaga showed normal meiotic course with low frequency of interbivalent connections. These species showed high pollen fertility in spite of presence of some meiotic abnormalities.

Key words Cyanotis cristata, Cyanotis vaga, Meiosis, Meiotic abnormality, B chromosome, Pollen fertility.

Cyanotis D. Don. is a cosmopolitan genus belonging Guervin and Le Coq 1966, Heitz 1968, Rao 1970, Jones to family Commelinaceae and includes 56 species. The and Kukkonen 1971, Chimphamba 1973). species are distributed throughout tropical and subtropi- The present study was conducted to investigate cyto- cal Africa, Asia and Australia. The genus is represented logical status of two species of Cyanotis, namely C. cris- by 16 species in India (Karthikeyan et al. 1989). A large tata and C. vaga from various localities of North-West number of species of this genus have been investigated India. The present investigations are aimed chromosome cytologically from various parts of the world (Islam number, meiotic chromosome behavior, and pollen fertil- and Baten 1952, Sharma 1955, Kammathy and Rao ity to provide basic cytogenetic and cytogeographical 1961, Guervin and Le Coq 1966, Morton 1967, Heitz information in two Indian Cyanotis species for their ge- 1968, Jones and Jopling 1972, Bhattacharya 1975). netic variability and phylogeny. The 16 indian species of Cyanotis have been recorded the chromosome number of n=12 or 2n=24 by several Materials and methods workers (Sharma 1955, Kammathy and Rao 1961, 1964, Raghavan and Rao 1961, 1965, Shetty and Subraman- Plant materials were collected from 26 localities yam 1961, 1962, Mitra and Datta 1967, Rao et al. 1968, growing in different regions of North-West India. Col- 1970, Bhattacharya 1975, Mehra and Sachdeva 1976, lected flower buds were fixed in a Carnoy’s fixative Sarkar et al. 1980, Renugadevi and Sampathkumar (ethanol : chloroform : glacial acetic acid=6 : 3 : 1) for 24 h 1983, 1986, Alam and Sharma 1984, Bai et al. 1984, at room temperature, and then transferred in 70% etha- Lalithambika and Kuriachan 1996, 1997). In C. vaga, nol and stored at 4°C until use. Voucher specimens were 2n=24 and 34 were reported (Bhattacharya 1975, Zheng deposited at the Herbarium, Department of Botany, et al.1989). Other chromosome number of 2n=48 in C. Punjabi University, Patiala and accession numbers were adscendens (Raghavan and Rao1961, Lalithambika and obtained (PUN) in Table 1. Meiotic chromosomes were Kuriachan 1997), n=16 in C. papilionacea (Lalitham- obtained from anthers of fixed floral buds by squashing bika and Kuriachan 1996), 2n=20 in C. axillaris and C. in 2% acetocarmine. Microphotographs of chromosome cucullata (Shetty and Subramanyam 1962, Lalithambika counts were taken from freshly prepared slides using a and Kuriachan1996, 1997, Rao et al. 1970, Renugadevi Nikon Eclipse 80i microscope. Pollen fertility was in- and Sampathkumar 1983, 1986, Bai et al. 1984) and vestigated using glycerol–acetocarmine (1 : 1) technique 2n=26 in C. villosa (Faden and Suda 1980) were record- (Marks 1954). The well-stained pollens were considered ed. The Cyanotis is polybasic with x=8, 10, 11, 12, 13, as fertile, while the unstained and shrunken pollens were 14, 15 and 17, and common basic chromosome number considered as sterile. To study pollen morphology, ma- of x=12 was proposed previously (Shetty and Subra- ture flower buds were fixed in 70% ethanol and pollens manyam 1962, Lewis 1964, Raghavan and Rao 1965, were processed using an acetolysis technique (Erdtman 1954). The morphology of pollens was described as per * Corresponding author, e-mail: [email protected] the terminology is given by Nair (1964). The average DOI: 10.1508/cytologia.84.9 size of pollens was estimated on the basis of measure- 10 A. Grewal and P. Rani Cytologia 84(1) ments along the polar diameter (P) and equatorial diam- Morton (1967) and Bhattacharya (1975). Chromosome eter (E) using a micrometer. The microphotographs of number of 2n=20 and 34 (Morton 1967, 1993, Thulin pollen were taken by using a Magnus MLX Plus micro- 1970, Zheng et al.1989) were not observed in this study. scope. Cytological studies revealed that C. cristata and C. vaga exhibit a common meiotic count of n=12 (Figs. 1, 10) Results and discussion thus both species are diploid based on x=12. Abnormal meiotic behavior was observed at vari- Meiotic analysis of 21 accessions of species of C. ous stages of meiosis in almost all the accessions of C. cristata showed chromosome number of n=12 of pol- cristata and C. vaga (Table 2). In C. cristata, in spite len mother cells (PMCs) (Figs. 1, 2). This number is in of normal bivalent formation and equal distribution at accordance with previous reports of 2n=24 (Sharma anaphase I (A I) (Fig. 2), some PMCs showed meiotic 1955, Kammathy and Rao 1961, Raghavan and Rao abnormalities which included unoriented bivalents, an 1961, Shetty and Subramanyam 1962, Fotedar and Roy early disjunction of the chromosome, chromatin bridges, 1969, Bhattacharya 1975, Mehra and Sachdeva 1976, laggards and interchromosomal connections (Figs. 4–8). Sarkar et al. 1980, Alam and Sharma 1984, Renugadevi In C. vaga all the accessions under present study showed and Sampathkumar 1986, Lalithambika and Kuriachan the presence of interbivalent connections (Fig. 12). 1997). Previous reports of 2n=30 (Guervin and Le Spindle apparatus plays an important role in the ori- Coq 1966) and 2n=26 (Jones and Jopling 1972) were entation of bivalents at metaphase I (M I). Bivalents not detected. Three accessions from Una and Kangra positioned away from the equatorial plate are said to be (HP) showed a B chromosome along with 12 bivalents un-oriented bivalents. The accessions of C. cristata col- at diakinesis (Fig. 3) which is a coincidence with the lected from 11 localities (Table 1) showed the presence previous report of 2n=24+0-1B from Bangladesh (Is- of un-oriented bivalents at M I (Fig. 4). Lack of coordi- lam and Baten 1952). Meiotic course studied in five nation between chromosomes and spindle leads to non- accessions of C. vaga collected from two localities of synchronous disjunction of bivalents (Sharma 1976). Kangra and Mussoorie illustrated chromosome number Early disjunction of bivalents has been observed in of n=12 (Figs. 10, 11). The present chromosome number 13 accessions of C. cristata, out of which accession in C. vaga is in agreement with the previous reports of collected from Kufalu (932 m) showed the highest fre-

Table 1. Information about locality with altitude, accession number, pollen fertility and pollen size of two species of Cyanotis D. Don. of family Commelinaceae.

Accession number Chromosome number Pollen fertility Pollen size (µm) Taxa Locality with altitude (PUN) (n) (%) P E

C. cristata Dehradun, UK (637 m) 62139 12 94.44 63.05±2.13 41.71±0.91 Mussoorie, UK (2006 m) 62140 12 91.78 24.25±0.01 17.46±0.57 Palampur, HP (1472 m) 62141 12 88.88 59.17±17 42.22±0.74 Nagrota, HP (733 m) 62142 12+(0-1)B 94.89 62.71±1.63 38.46±1.03 Bhompur, HP (738 m) 62143 12+(0-1)B 93.67 67.56±1.76 44.28±1.55 Chintpurni, HP (940 m) 62144 12 92.00 59.17±1.50 39.77±1.12 Banganga, HP (733 m) 62145 12 94.49 58.49±1.00 41.03±1.03 Nagarkot, HP (733 m) 62146 12 94.73 46.56±1.04 35.40±0.89 Gupt Ganga, HP (733 m) 62147 12 93.82 52.38±1.27 36.52±0.72 Tirsoo, HP (746 m) 62148 12 91.22 63.05±2.63 37.83±1.33 Nadaun, HP (478 m) 62149 12 84.84 45.59±1.93 35.89±0.91 Kufalu, HP (932 m) 62150 12 92.07 69.16±2.68 43.94±1.70 Ponta Sahib, HP (389 m) 62151 12 93.42 73.04±2.94 43.31±1.83 Rajban, HP (1552 m) 62152 12 91.52 60.14±1.40 37.83±1.52 Shillai, HP (1900 m) 62153 12 95.31 68.19±1.67 48.79±1.23 Chamunda, HP (1662 m) 62154 12 90.56 25.70±0.57 19.40±0.01 Rajban road, HP (1552 m) 62155 12 91.66 25.70±0.57 14.55±0.01 Nahan, HP (932 m) 62156 12 92.06 56.74±1.12 36.86±0.63 Basoli, HP (393 m) 62157 12 92.40 63.68±1.92 44.28±1.12 Jawalamukhi, HP (610 m) 62158 12 97.64 57.23±0.67 40.40±0.99 Gagret, HP (439 m) 62159 12+(0-1)B 90.16 56.55±1.06 38.12±1.24

C. vaga Nagrota, HP (733 m) 62137 12 83.87 50.44±1.52 40.25±1.24 Nagarkot, HP (733 m) 62136 12 85.93 50.92±1.29 40.74±0.73 Palampur, HP (1472 m) 62138 12 91.02 50.44±1.44 40.74±0.63 Mussorie, UK (2006 m) 62134 12 90.81 46.56±0.50 36.86±1.11 Mussorie, UK (2006 m) 62135 12 91.96 44.62±0.70 32.98±1.06 2019 Meiotic Studies in Cyanotis from North-West India 11

Figs. 1–13. 1–8. Cyanotis cristata: 1) PMC showing 12II at M I, 2) PMC showing 12 : 12 equal distribution of chromosome at A I, 3) PMC showing 12II at diakinesis with B chromosome, 4) PMC showing chromatin bridge at A I, 5) PMC showing vagrant at M I, 6) Laggards at A I, 7) PMC showing interbivalent connections, 8) Heterogenous sized pollen

grains. 9–13. Cyanotis vaga: 9) PMC showing 12II at diakinesis, 10) PMC showing 12II at M I, 11) PMC showing interbivalent connections, 12) Fertile pollen grain, 13) Sterile pollen grain. Scale bars=10 µm. quency (16.66%) of early disjunction at M I (Fig. 8) between the two poles (Saylor and Smith 1966). followed by accessions from Dehradun (637 m) and Cha- Six accessions of C. cristata showed the presence munda (1662 m) with frequency of 15.38% and 14.28% of laggards at A I (Fig. 6). This phenomenon has been respectively. Accessions scored from Banganga (733 m) observed with the highest frequency (20.00%) in Ra- showed the lowest frequency (2.85%) of this phenom- jban road (1552 m) accession and the lowest frequency enon. (2.06%) in Palampur (1472 m) accession. The failure In the present study, chromatin bridges have been of chromosome movement or delayed terminalization, observed in three plants of C. cristata collected from asynapsis and premature disjunction of bivalents have Palampur (1472 m), Banganga (733 m) and Rajban road been suggested as possible causes of laggard formation (1552 m) (Fig. 5). Out of which maximum percentage (Gupta and Priyadarshan 1982, Jayabalan and Rao1987, (10.00%) of chromatin bridges have been observed in Soheir et al. 1989). accession from Rajban road and minimum (2.06%) in Interchromosomal connections were observed in all Palampur. Anaphasic bridges may arise either due to accessions of C. cristata (Fig. 7) and two accessions interlocking of chromosomes or failure of chiasmata in of C. vaga (Table 2, Fig. 12). Interbivalent connections a bivalent to terminalize and chromosomes get stretched may be attributed to the formation of chromatic knots, 12 A. Grewal and P. Rani Cytologia 84(1)

Table 2. Data on abnormal meiotic course in different accessions of Cyanotis from different localities of North-West India.

No. of PMCs Abnormal PMCs (%) Species PUN number observed Early disjunction Bridges Laggards Interbivalent connections Unoriented bivalents

C. cristata 62139 145 15.38 ̶ 2.56 35.89 2.56 62140 90 9.45 ̶ 5.40 74.32 8.10 62141 99 6.20 2.06 2.06 16.55 2.42 62142 120 7.69 ̶ ̶ 42.30 ̶ 62143 103 ̶ ̶ ̶ 71.42 ̶ 62144 140 7.69 ̶ ̶ 69.23 ̶ 62145 160 2.85 ̶ 5.71 45.71 5.71 62146 100 ̶ ̶ ̶ 8.10 ̶ 62147 96 8.10 ̶ ̶ 32.43 5.40 62148 79 8.10 8.10 ̶ 35.13 2.70 62149 125 ̶ ̶ ̶ 61.53 ̶ 62150 136 16.66 ̶ ̶ 33.33 ̶ 62151 100 ̶ ̶ ̶ 75.00 ̶ 62152 98 ̶ ̶ ̶ 41.66 8.33 62153 142 8.00 ̶ 4.00 34.00 2.00 62154 89 14.28 ̶ ̶ 71.42 ̶ 62155 123 6.66 10.00 20.00 26.66 ̶ 62156 156 ̶ ̶ ̶ 40.00 ̶ 62157 98 ̶ ̶ ̶ 20.00 5.00 62158 112 ̶ ̶ ̶ 17.07 9.7 62159 130 7.69 ̶ ̶ 38.46 11.53

C. vaga 62137 80 ̶ ̶ ̶ 29.41 ̶ 62136 100 ̶ ̶ ̶ 13.33 ̶ 62138 110 ̶ ̶ ̶ ̶ ̶ 62134 120 ̶ ̶ ̶ 29.03 ̶ 62135 99 ̶ ̶ ̶ 34.21 ̶

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