Morphometric and Cytological Analysis of Different Cytotypes of Dioscorea Deltoidea Wall., 1850 (Dioscoreaceae) from North-Western Indian Himalayas

Morphometric and Cytological Analysis of Different Cytotypes of Dioscorea deltoidea Wall., 1850 (Dioscoreaceae) from North-Western Indian Himalayas

Savita Rani, Tilak Raj Sharma and Rakesh Kumar Chahota*

Department of Agricultural Biotechnology, CSK Himachal Pradesh Agricultural University, Palampur-176 062, India

Received September 17, 2015; accepted June 14, 2016

Summary Dioscorea deltoidea Wall. (Dioscoreaceae), commonly called “Nepal Yam,” is distributed in the Himalayas, from Kashmir to Assam at altitudes of 450–3100 m. It is an important medicinal plant commer- cially exploited for the extraction of diosgenin, a pioneer for steroid drugs. This valuable species of the Indian Himalayas faces a serious threat of extinction due to over-exploitation of its tubers and gradual shrinkage of its natural habitat. The species shows considerable intraspeciﬁc morphological and cytological variations involving polyploidy and hybridization. In the present study, we examined the meiotic course, microsporogenesis, pollen fertility, and morphological characters of 12 populations of diploid (2x) and four populations of tetraploid (4x) cytotypes of Dioscorea deltoidea. The majority of populations exhibit normal course of meiosis with 100% pollen fertility. However, in six populations (four diploid cytotypes and two tetraploid cytotypes), the individuals show inter-PMC transfer of chromatin material at various stages of meiosis and associated meiotic irregularities such as chromosome stickiness, unoriented bivalents, laggards, micronuclei, and chromatin bridges at different stages of meiosis. Consequently, these populations exhibited varying degree of pollen sterility and heterogenous- sized pollen grains. Analysis of various morphological characteristics of diploid and tetraploid cytotypes revealed that increase in ploidy level in the species is correlated with gigantism of some vegetative characteristics.

Key words Cytotype, Dioscorea deltoidea, Morophotype, Meiotic abnormality, North-Western Himalayas.

The family Dioscoreaceae is popularly known as 450–3100 m above sea level. It is a hairless vine, twining the ‘Yam family’ which comprises of five genera and clockwise; tubers ligneous and irregular; leaves simple 750 species (Murti 2001). According to Caddick et al. alternate, triangular-ovate, heart shaped, the basal lobes (2002), the family includes only four genera, Dioscorea, rounded, long pointed, hairless above; male flowers Trichopus, Taca, and Stenomeris. On the basis of mo- spikes occur solitary in leaf axils; flowers in small clus- lecular phylogenetic studies, Merckx et al. (2006) placed ters, six stamens; female spikes solitary, few flowers. the genus Taca in the family Burmanniaceae. The mem- The flowering and fruiting is seen during the months bers are mainly distributed in the tropical and warm of May–July. It is an endangered medicinal plant of the temperate regions of Asia, South America, and West Western Himalayas. The tubers contain saponin, acrid Africa (Coursey 1967). resin, diosgenin, starch, and calcium oxalate, which are The genus Dioscorea L. has been divided in various used as contraceptives and in the treatment of various subgroups (Burkill 1960) and thus, has been the subject disorders of the genitals. The roots of this species con- of systematic studies by many workers in the recent tain an average of 4.8% diosgenin. Diosgenin is said to years (Perret et al. 2003, Wilkin et al. 2005). The genus be a basic material for hormone preparation. The roots can be easily identified by its twining habit, prominent and leaves are also used for washing shawls and wool- basal nerves, and reticulate venation of leaves, a rare len cloth as well as a vermifuge and an anthelmintic for characteristic in the monocots but a common feature purging intestinal worms. In the Western Himalayas, the of dicots. In India, the genus Dioscorea has 50 species juice of the root tuber is taken in the evening in the treat- (Murti 2001). ment of roundworm. Dioscorea deltoidea is commonly called the ‘Napal As in the literature, the cytological investigations of Yam’ and ‘Grishti’ in Hindi. It is mainly found in moist members of Dioscoreaceae have been made earlier by and damp forests and distributed in the Himalayas several researchers (Chin et al. 1985, Abraham and Nair from Kashmir to Arunachal Pradesh and in Assam at 1991, Egesi et al. 2002, Obidiegwu et al. 2009, etc.) mainly from outside India. So, keeping the medicinal * Corresponding author, e-mail: [email protected] value and existence of polyploid cytotypes in view, the DOI: 10.1508/cytologia.81.301 present cytomorphological study was undertaken to ex- 302 S. Rani et al. Cytologia 81(3) plore the genetic diversity in Dioscorea deltoidea from until used. Smears of appropriate-sized flower buds were different parts of North-West India. From a medicinal made, using standard acetocarmine technique (Marks point of view, the genetic diversity can be evaluated for 1954). About 20–50 fresh slides in each case were pre- screening better cytotypes for future exploitation. pared from different anthers/flowers for different individuals of a particular population and were analyzed Materials and methods in each case. To confirm the chromosome number in case of normal meiosis, around 50 pollen mother cells Plant material (PMCs) were observed at different stages of meiosis, Materials for cytological studies in the form of buds preferably at diakinesis/metaphase-I/anaphase-I or -II. were collected from different districts of Himachal In case of abnormal meiosis, however, more than 300 Pradesh, India in the months of May–July in 2014 and PMCs were considered to ascertain the type and fre- 2015 (Table 1). The propagating materials of these plants quency of various abnormalities per plant. Pollen fertil- were also collected and planted in the experimental ity was estimated by mounting mature pollen grains in fields of Chaudhary Sarwan Kumar Himachal Pradesh glycero–acetocarmine (1 : 1) mixture (Belling 1925). Agricultural University, Palampur, India in a Random- Nearly 400–500 pollen grains were analyzed in each ized Complete Block design with two replications. The case for ascertaining pollen fertility and pollen size. plant to plant distance was kept at 5 cm while the row to Well-filled pollen grains with stained nuclei were taken row distance was kept at 50 cm. as apparently fertile, while shrivelled and unstained pollen grains were counted as sterile. The size of stained Cytological studies pollen grains was measured with an occulomicrometer. For meiotic studies, flower buds were collected from Photomicrographs–chromosome counts and other male plants growing under natural conditions from meiotic abnormalities were analyzed using the Olym- selected areas of the Western Himalayas. These flower pus light microscope and the best plates of chromosome buds were collected from 15 randomly selected plants counts, meiotic abnormalities, and pollen grains were of each population and fixed in Carnoy’s fixative (6 : 3 : 1 photographed from the temporary mounts with the Op- ethanol/chloroform/acetic acid v/v/v) for 24 h. Flower tika Digital Imaging System. buds were washed and preserved in 70% ethanol at 4°C

Table 1. List of population number, meiotic chromosome number, and places of collection with district, state, altitude, latitude and longitude, and habitat of different populations of the diploid and tetraploid cytotypes of Dioscorea deltoidea.

Population Meiotic chromosome Place of collection with district, state, altitude in meters (Alt. m), Cytotype number number (2n)/Meiotic course* latitude and longitude, and habitat

Diploid (2x) PP-1 20/A Nurpur, Kangra, Himachal Pradesh, 643 m/ 32°30′N, 75°90′E; slopes in Dalbergia forest PP-2 20/N Jassor, Kangra, Himachal Pradesh, 836 m/ 32°23′N, 76°00′E; growing under Murraya koenigii trees in dry conditions PP-3 20/N Rehlu, Kangra, Himachal Pradesh, 900 m/32°13′N 76°10′E; open and grassy slopes among scattered trees of Dalbergia PP-4 20/A Kotla, Kangra, Himachal Pradesh, 400 m/32°15′N, 76°02′E; on the open slopes PP-5 20/N Nagni, Kangra, Himachal Pradesh, 510 m/32°29′N, 76°08′E; on moist and sunny places PP-6 20/N Gangtha, Kangra, Himachal Pradesh, 620 m/32°14′N, 75°49′E; open and moist grassy slopes among scattered shrubs of Murraya koenigii and Vitex negundo PP-7 20/A Chowari, Chamba, Himachal Pradesh, 1138 m/32°43′N, 76°01′E; on the rocky slopes PP-8 20/A Gola, Chamba, Himachal Pradesh, 510 m/32°30′N, 76°08′E; in the Dalbergia forest PP-9 20/A Samote, Chamba, Himachal Pradesh, 510 m/32°30′N, 76°08′E; in the tropical forest PP-10 20/N Sujanpur Tira, Hamirpur, Himachal Pradesh, 1200 m/31°83′N, 76°51′E; on the open slopes of tropical forests PP-11 20/N Amb, Una, Himachal Pradesh, 478 m/31°56′N, 76°19′E; open slopes among scattered shrubs of Murraya koenigii and Vitex negundo PP-12 20/N Una, Una, Himachal Pradesh, 500 m/31.48°N, 76.27°E; in the Dalbergia forest Tetraploid (4x) PP-13 40/A Salooni-I, Chamba, Himachal Pradesh, 1900 m/32.72°N, 76.05°E; along water course PP-14 40/A Salooni-II, Chamba, Himachal Pradesh, 1900 m/32.73°N, 76.06°E; on moist and shady places PP-15 40/N Boh, Kangra, Himachal Pradesh, 1900 m/32.22°N, 76.17°E; on the rocky slopes PP-16 40/N Tal Mata, Himachal Pradesh, 1365 m/32.28°N, 76.20°E; in moist places

Meiotic Course* N=Normal; A=Abnormal. 2016 Morphometric and Cytological Analysis of Different Cytotypes of Dioscorea deltoidea Wall. 303

Morphological study were much larger in the tetraploid cytotype compared Different qualitative morphometric characters were with the diploid. The values for stomatal size, density, studied for each cytotype to have proper insight on and index were found to be more in the tetraploid com- morphological variation present in these cytotypes. For pared to the diploid cytotype (Table 2). Pollen grains in stomatal studies, mature leaves were treated with 10% the diploid cytotype were almost uniform sized whereas aqueous solution of potassium hydroxide (KOH) at room in the tetraploid cytotype pollen grains were of variable temperature for 10–15 min after which we obtained epi- sizes (Table 3). dermal peels, which were stained with 10% saffanin in 90% ethanol. In order to reveal the significant difference Cytological studies in the stomata and pollen grain size of diploid and tetra- We have cytologically examined the detailed ploid cytotypes, the t-test was performed. male meiosis in the 16 wild accessions of Dioscorea deltoidea collected from the four districts (Kangra, Results Chamba, Hamirpur, and Una) of Himachal Pradesh. The diploid (2n=20) cytotype Morphological studies The diploid cytotype has been found to be more com- The morphological study involved both macro- and mon as confirmed from the presence of meiotic chromo- microscopic characters (Table 2). Macroscopic charac- some number 2n=20. These diploid individuals of 12 ters including plant height, number of leaves per plant, accessions show the presence of 10 small sized bivalents and leaf length were studied from all the populations (Fig. 1) at metaphase (M-I) and 10 : 10 chromosome dis- of the diploid (2n=20) and tetraploid cytotype (2n=40). tribution (Fig. 2) at anaphase (A-I). The meiotic course The tetraploid plants measured in height were much is normal in seven accessions which resulted in 96.5% taller than the diploid plants. Furthermore, the leaves pollen fertility. However, the other five accessions show

Table 2. Comparison of micro- and macroscopic characters of the diploid and tetraploid cytotypes of Dioscorea deltoidea.

Cytotype

S. No. Characters Diploid Tetraploid 2n=20 2n=40

1. Plant height (cm) 2. Number of leaves/plant 30–50 25–45 3. Leaf length (cm) 5.05–11.58×4.76–10.59 (9.76±0.76) (7.87±1.09) 8.00.76–12.87×5.32–11.21 (10.08±1.01) (9.20±0.87) 4. Stomatal size (µm) 1.65–1.87×1.20–1.25 (1.67±0.38) (1.22±0.18) 1.89–2.00×1.32–1.41 (1.93±0.89) (1.38±0.76) 5. Stomatal density (mm-2) 27.80±4.02 (15–40) 34.55(25–60) 6. Stomatal index 38 53 7. Pollen grain size (µm) 28.45–28.94×26.54–26.89 (28.62±0.26) (26.69±0.54) 28.89–29.17×27.34–27.98 (29.00±0.14) (27.60±0.65)

Figures in parentheses represent the mean±standard deviation.

Table 3. Pollen grain size, relative frequency of variable sized pollen grains and pollen sterility in diploid and tetraploid cytotypes of Dioscorea deltoidea.

Pollen grain size (µm) Rf Pollen sterility S. No. Population Diploid Tetraploid (%) (%)

1. PP-1 28.05–28.14×26.04–26.19 (28.10±0.26) (26.10±0.54) 29 2. PP-2 28.76×26.76 00 3. PP-3 28.12×26.87 00 4. PP-4 28.00–28.65×26.10–26.98 (28.28±1.22) (26.54±1.23) 34 5. PP-5 28.08×26.42 00 6. PP-6 28.19×26.53 00 7. PP-7 28.65–29.21×26.89–27.87 (29.00±1.09) (27.32±1.08) 45 8. PP-8 28.87–29.31×26.87–27.43 (29.11±0.80) (27.13±1.04) 19 9. PP-9 29.01–29.54×27.19–27.87 (29.23±1.15) (27.11±1.98) 29 10. PP-10 28.76×26.21 00 11. PP-11 28.89×26.32 00 12. PP-12 28.23×27.87 00 13. PP-13 28.92–29.27×27.14–27.88 (29.10±1.14) (27.60±0.65) 49 14. PP-14 29.34–30.43×28.32–28.90 (30.18±1.32) (28.78) 39 15. PP-15 30.43×28.43 10 16. PP-16 30.49×28.19 14

Figures in parentheses represent the mean±standard deviation. Rf=relative frequency of variable sized pollen grains. 304 S. Rani et al. Cytologia 81(3)

Figs. 1–17. 1) PMC at metaphase-I showing 10 bivalents (Diploid cytotype). 2) PMC at anaphase-I showing 10 : 10 distribution of chromosomes. 3) PMC at metaphase-I showing 20 bivalents (Tetraploid cytotype). 4) PMCs showing transfer of chromatin material (arrow). 5) Group of PMCs showing hypo- and hyperploids cells. 6) Different groups of PMCs involved in cytomixis. 7) Two PMCs showing cytomixis at anaphase-I. 8) PMC showing chromosomal stickiness at metaphase-I. 9) PMC showing bridge at anaphase-I. 10) PMC showing laggards at anaphase-I. 11) PMC at metaphase-I showing unoriented bivalent (arrow). 12) PMC at telophase-II showing multipolaity (ﬁve poles). 13) Diad. 14) Triad. 15) Diad with micronuclei. 16) Tetrad with micronuclei. 17) Heterogenously sized fertile pollen grains. Scale 10 µm. inter-PMC transfer of chromatin material resulting in form sized pollen grains. However, the other two acces- various meiotic abnormalities and heterogenous-sized sion showed inter-PMC transfer of chromatin material pollen grains. The data on cytomixis, meiotic course, resulting in various meiotic irregularities and heteroge- and pollen fertility and pollen size in each accession is nous-sized pollen grains. The data on cytomixis, meiotic provided in Table 4. course, pollen fertility, and pollen size in each accession The tetraploid (2n=40) cytotype is provided in Table 4. Only four accessions growing on the moist, rocky Meiotic irregularities and shady places existed at tetraploid level (based on The phenomenon of cytomixis involving inter-PMC x=10) as conﬁrmed from the presence of 20 small sized transfer of chromatin material exists in the populations bivalents in the PMCs at M-I (Fig. 3). These bivalents of PP-1, PP-7, PP-8, PP-13, and PP-14 (Table 4). The showed regular separation during A-I. Further meiotic transfer of chromatin material takes place during for- course was regular in two accessions resulting in normal mation of single or multiple chromatin channels (Fig. tetrad formation, nearly 100% pollen fertility, and uni- 4). The inter-PMC migration of chromatin is observed 2016 Morphometric and Cytological Analysis of Different Cytotypes of Dioscorea deltoidea Wall. 305

Table 4. Data regarding cytomixis, meiotic course and microsporogenesis in the different populations of Dioscorea deltoidea.

Cytomixis Meiotic course Microsporogenesis

PMCs with PMCs with PMCs with No. of PMCs with Sporads (tetrads and Population Meiotic % age of PMCs chromosome bridges at laggards at PMCs unoriented polyads with and stage/s involved stickiness at A-I/T-I, A-I/T-I, involved bivalents (%) without micronuclei) M-I (%) A-II/T-II (%) A-II/T-II (%)

PP-1 P-I, M-I 5.60 (7/125) 2–4 4.87 (4/82) 5.21 (6/115) 4.30 (4/93) ̶ 16.40 (21/128) PP-4 M-I ̶ ̶ 2.38 (2/84) 2.86 (2/70) ̶ 4.03 (5/124) 5.26 (6/114) PP-7 P-I, M-I 5.06 (4/79) 2–4 ̶ ̶ 2.63 (2/76) 4.23 (5/118) 3.34 (4/120) PP-8 M-I, A-I 7.24 (10/138) 2–4 4.47 (6/134) 4.16 (5/120) 2.91 (3/103) 1.81 (2/110) 16.03 (17/106) PP-9 M-I ̶ ̶ ̶ 1.38 (6/132) ̶ 1.76 (2/113) 10.76 (7/65) PP-13 M-I 4.16 (3/72) 2–3 2.77 (2/72) ̶ 1.89 (2/106) ̶ 5.21 (6/115) PP-14 P-I, A-I 5.26 (4/76) 2–4 2.22 (2/90) 5.69 (7/123) ̶ 4.95 (5/101) 8.23 (7/85)

Figures in parentheses denote observed number of abnormal PMCs in the numerator and total PMCs observed in denominator. PMC=pollen mother cell; M-I=Metaphase-I; P-I=Prophase-I; A-I/T-I=Anaphase-I/Telophase-I; A-II/T-II=Anaphase-II/Telophase-II. from the early prophase to telophase stage. The migra- differences in morphological characteristics are depen- tion of chromatin material takes place through narrow dent upon the change of environment, genetic recombi- as well as broad cytoplasmic channels. The transfer of nation, and mutations. chromatin among PMCs is observed to be both unidirec- In the presently studied species, different intraspecific tional as well as bidirectional. Hypo-, hyperploid, and cytotypes show variation in macro- and microscopic enucleated PMCs resulted due to partial and complete characters attributed to the variation in chromosome transfer of chromatin material (Figs. 5–7). The migration numbers as reported earlier in Andropogon gerardii of chromatin material during microsporogenesis causes (Keeler and Davis 1999), Dactylis (Amirouche and Mis- various meiotic irregularities. Chromatin stickiness in- set 2007) and Centaurea stoebe (Španiel et al. 2008). volving a few bivalents or the whole complement is one This increase in the ploidy level is correlated with slight of the most common meiotic irregularities, which are gigantism for some of the features such as macro-mor- encountered in the individuals of four populations from phological characters like plant height, number of leaves prophase to metaphase-I (Fig. 8). The most characteristic per plant, and size of leaf or micro-morphological char- abnormality observed is the occurrence of chromatin acters as size, density, and index of stomata. bridges at anaphases and telophases (Fig. 9). Other The morphological comparison of diploid and tetra- prominent meiotic abnormalities include the occurrence ploid cytotype of Dioscorea deltoidea revealed scorable of laggards at anaphases/telophases (Fig. 10, Table 4) increase in plant height, length, numbers of leaves per and disorientation of chromosomes during anaphases plant, size of stomata, stomatal density, stomatal index due to spindle irregularities (Fig. 11, Table 4). These and pollen size of tetraploid cytotype than diploid cy- laggards and unoriented chromosomes failed to get inte- totype. Such genetic and morphological variations are grated at the poles during telophases, and constituted the more common among the individuals of different popu- micronuclei and multipolar PMCs (Fig. 12). As a result lations than among the members of a same population of irregular meiotic behaviour during the different stag- (Španiel et al. 2008). es, microsporogenesis is also observed to be abnormal, evidenced by the presence of dyads (Fig. 13), triads (Fig. Chromosomal status 14), and polyads with micronuclei (Figs. 15–16). Conse- A perusal of cytological literature shows that the quently, reduced pollen fertility and heterogenous-sized primary basic number for the genus Dioscorea is given pollen grains were observed (Fig. 17). as x=10 (Essad and Maunoury 1984). However, recent molecular studies have suggested the doubled number Discussion x=20 to be the basic number (Bousalem et al. 2006). On the basis of chromosomal data compounded at present Morphological variations for the genus, it is clear that the chromosome numbers in According to Stahlin (1929), an increase in chromo- 93 species through 173 cytotypes range from 2n=20 to some number is directly tied to an increase in plant size 2n=40, and most of these are based on x=10. The genus but only up to a certain limit, beyond which the further is polybasic with x=10, 12, 14, 15 and 18 (x=10 being increase of chromosome numbers has no effective role more common). The basic numbers x=17 and 19 are to in increase in plant size but may lead to physiological be taken with caution. Further, polyploidy is noticed in imbalance. The morphological variations are more com- 77 species and with 28 species showing intraspecific mon in the wild taxa due to their widespread distribution euploidy, whereas, 14 species reveal intraspecific an- (Turesson 1930), whereas Stebbins (1950) inferred that euploidy. In India, 23 species are found cytologically 306 S. Rani et al. Cytologia 81(3) with 21 species showing polyploidy, 10 species being an meiosis-I and meiosis-II. Many authors have associated example of intraspecific euploidy, and four species pre- the phenomenon of cytomixis to other meiotic abnor- senting cases of aneuploidy. At present, 16 populations malities such as the increase or decrease in chromosome of Dioscorea deltoidea have been cytologically investi- number, unorganized chromatin, interbivalent connec- gated from different localities of the Western Himalayas. tions, chromosome stickiness, desynapsis, chromosomal The present chromosomal count of 2n=20 conforms to laggards and chromatin bridges (Mary and Suvarnalatha the earlier report (Srivastava and Purnima 1990), where- 1981, Lakshmi et al. 1989), and the same is confined as that of 2n=40 adds a new tetraploid cytotype for the in the present cases also. Although environmental fac- species in the Western Himalayas. Earlier, chromosome tors certainly influence the process, cytomixis seems to number of Dioscorea deltoidea (2n=40) was reported be a natural phenomenon under genetic control in the from southen parts of India by Raghavan (1958) and out- presently investigated species, as has been suggested side India (Martin and Ortiz 1963). by many authors (Bellucci et al. 2003, Malallah and At- tia 2003, Haroun et al. 2004, Kumar and Singhal 2011, Meiotic abnormalities Rani et al. 2014). It leads to the formation of hypo- and Meiosis is a genetically programmed processes, thus, hyperploid PMCs, abnormal tetrad formation and pollen it is controlled by a large array of genes (Pagliarini 2000, sterility (Malallah and Attia 2003, Rani et al. 2014). Villeneuve and Hillers 2001). Mutation in any of these At present, heterogeneous-sized pollen grains have genes causes various meiotic abnormalities which may also been observed with reduced pollen fertility. Previ- lead to adverse effects on pollen fertility and overall re- ously, similar observations related to heterogeneous- productive efficiency of the species (Kumar et al. 2010). sized pollen grains as a consequence of chromatin trans- The different abnormalities at any stage of meiosis are fer have also been reported in other plants (Haroun et primarily expressions of the genotypical composition of al. 2004, Boldrini et al. 2006, Fatemeh et al. 2010, Rani the plant governing degrees of absence as well as pres- et al. 2014). The cytological status of such apparently ence of biochemical characters or substances (Whyte fertile pollen grains could not be ascertained presently, 1975). These chromosome abnormalities play a major but its role in the origin of intraspecific polyploids and role in the genetic system producing isolation barriers thereby in evolution cannot be ruled out. between the species (Haque and Ghoshal 1980). Ac- cording to Darlington (1965), the structural changes in Chromatin stickiness the chromosomes act mainly as the means of holding It is the phenomenon in which chromosomes form ir- together certain favourable genes and therefore promote regular clumps of chromatin masses and lose their own their fitness at the expense of flexibility. shape completely. Chromatin stickiness at M-I results In Dioscorea deltoidea, different meiotic abnormali- in the delayed disjunction of bivalents, thus, the forma- ties have been recorded in the form of cytomixis, chro- tion of chromatin bridges and laggards at anaphases and matin stickiness, unoriented bivalents, chromosomal telophases. Sometimes, chromatin stickiness impairs bridges and laggards at different stages of meiosis. As a chromosome segregation and leads to the formation of result of these meiotic abnormalities, anomalous micro- pycnotic nuclei and chromatin degeneration (de Souza sporogenesis in the form of production of monads, diads, and Pagliarini 1997). The phenomenon of stickiness triads or polyads with or without micronuclei, have been and clumping of chromosomes has been attributed to recorded. Consequently, variable sized pollen grains are environmental factors (Sudhakaran 1972), genetic fac- formed with low pollen fertility. These meiotic abnor- tors (Beadle 1932), low temperature (Eriksson 1968) or malities are discussed below. partial dissociation of nucleoprotein (Kaufman 1956). Various authors associated the chromatin stickiness Cytomixis with the phenomenon of cytomixis and other meiotic ir- It is a phenomenon of inter-pollen mother cell transfer regularities (Rani et al. 2014). However, Gaulden (1987) of chromatin material. Cytomixis may occur at any stage suggested that chromatin stickiness may be the result of meiosis, during Prophase-I (Chauhan 1981) as well of some changes by mutation in structural genes in the as from Prophase-I to tetrad stage (De 1981). In recent specific non-histone proteins that are the essential com- years, various cytologists reported this phenomenon in a ponents of chromosomes. Chromosome stickiness may large number of plants (Lattoo et al. 2006, Gupta 2009, lead to meiotic abnormalities and reduced pollen fertil- Kumar et al. 2010, Fatemeh et al. 2010, Rani et al. 2011, ity. Jeelani et al. 2011). However, few workers (de Souza and Pagliarini 1997, Unoriented bivalents Malallah and Attia 2003) earlier reported that the diploid At metaphase-I, all the bivalents orient themselves on species is the most common occurrence of cytomixis. the equator of the spindle with the help of chromosomal In most of these species, the occurrence of cytomixis fibres. The bivalents which fail to adjust on the spindle has been recorded in the PMCs during all the stages of are known as unoriented bivalents. During the present 2016 Morphometric and Cytological Analysis of Different Cytotypes of Dioscorea deltoidea Wall. 307 studies, mostly one or two bivalents fail to orient on the observations have also been made earlier in many an- equator of the spindle. According to Nirmala and Rao giospermic plants by various workers (Sheidai et al. (1996), any malformation or breakage in the spindle fi- 2008, Gupta 2009). The formation of heterogenous-sized bres might be the possible cause of random sub-grouping pollen grains may lead to the formation of unreduced 2n of chromosomes. Due to these extra groupings of biva- pollen grains (Sheidai et al. 2008, Fatemeh et al. 2010, lents, laggards are formed during anaphases and telo- Guan et al. 2012). phases and ultimately pollen malformation. Conclusion Chromosomal bridges and laggards The phenomenon of chromatin bridges has been com- On the basis of morphological study, both the dip- monly observed in plants showing abnormal meiotic loid and tetraploid cytotypes are distinguishable in the behaviour. At present the phenomenon is reported in the field from each other. The diploid cytotype has a wider form of single or multiple bridges at either of the meiotic distribution in the Western Himalayas compared to the stages as A-I/T-I/A-II/T-II. However, chromosomal lag- tetraploid cytotype. Additionally, the occurrence of vari- gards are the bivalents or chromosomes which lag be- ous meiotic abnormalities in the diploid and tetraploid hind at anaphases and telophases. cytotypes may be attributed to the genetic imbalance in Mostly, late disjuncting bivalent/multiple bridges are the species. there in all the species, and these seem to be the result of chromatin stickiness and late disjunction of bivalents. Acknowledgements Consequently, pollen malformation occurs as seen earlier also by Rani et al. (2014) and Kumar et al. (2010). The authors are grateful to the Science and Engi- Other types of chromatin bridges may originate from neering Research Board (SERB) under Young Scien- chiasma formation in heterozygous inversions (Rothfels tist Fellowship Scheme [Registration No. SERB/LS- and Siminovitch 1958). 527/2013] to Dr. Savita Rani. Thanks are also due to the The laggards are of various nature, and mostly chro- Head, Department of Agricultural Biotechnology, CSK, mosomal laggards are observed to be common in the Himachal Pradesh Agricultural University, Palampur, present investigated species. These might have been for necessary laboratory facilities. formed due to lack of synapsis at early Prophase stages or precocious separation of bivalents/chromosomes at References anaphases. Pagliarini (1990) and Souza et al. (2006) reported that laggards may result from late chiasma termi- Abraham, K. and Nair, P. G. 1991. Polyploidy and sterility in rela- nalization. According to Gupta and Priyadarshan (1982), tion to sex in Dioscorea alata L. (Dioscoreaceae). Genetica 83: 93–97. asynapsis, desynapsis, failure of chiasma formation and Amirouche, N. and Misset, M. T. 2007. Morphological variation and premature disjunction of bivalents might be the possible distribution of cytotypes in the diploid-tetraploid complex of the reasons of laggards formation. Presently, the laggards in genus Dactylis L. (Poaceae) from Algeria. Plant Syst. Evol. 264: most of the PMCs failed to reach at the poles during ana- 157–174. phases and telophases and thus, micronuclei have been Beadle, G. W. 1932. A gene for sticky chromosomes in Zea mays. Z. Indukt. Abstamm. Vererbungsl. 63: 195–217. formed during microsporogenesis which confirms to the Belling, J. 1925. Chromosomes of Canna and of Hemerocallis. J. recent observations of Jiang et al. (2011). Hered. 16: 465–466. The formation of chromatin bridges and laggards may Bellucci, M., Roscini, C. and Mariani, A. 2003. Cytomixis in pollen be attributed to various other reasons, such as due to in- mother cells of Medicago sativa L. J. Hered. 94: 512–516. terlocking of bivalents (Bhattacharjee 1953), paracentric Bhattacharjee, S. K. 1953. Cytogenetics of Lens esculenta Monesch. inversions (Sinha and Godward 1972) or abnormal spin- Caryologia G. Citol. Citosistematica Citogenet. 5: 159–166. Boldrini, K. R., Pagliarini, M. S. and do Valle, C. B. 2006. Abnor- dle formation (Tarar and Dyansagar 1980). In addition to mal timing of cytokinesis in microsporogenesis of Brachiaria these, a number of authors (Lakshmi et al. 1989, Sheidai humidicola (Poaceae: Paniceae). J. Genet. 85: 225–228. et al. 2009) associated the formation chromosomal lag- Bousalem, M., Arnau, G., Hochu, I., Arnolin, R., Viader, V., Santoni, gards and chromatin bridges to the phenomenon of cy- S. and David, J. 2006. Microsatellite segregation analysis and tomixis. cytogenetic evidence for tetrasomic inheritance in the American yam Dioscorea trifida and a new basic chromosome number in the Dioscoreae. Theor. Appl. Genet. 113: 439–451. Abnormal microsporogenesis and pollen sterility Burkill, I. H. 1960. The organography and the evolution of the All populations/species with an abnormal course Dioscoreaceae, the family of the yams. Bot. J. Linn. Soc. 56: of meiosis end up with anomalous microsporogenesis 319–412. in the form of production of monads, diads, triads or Caddick, L. R., Wilkin, P., Rudall, P. J., Hedderson, T. A. J. and Chase, M. W. 2002. Yams reclassified: A recircumscription of polyads with or without micronuclei. Ultimately, these Dioscoreaceae and Dioscoreales. Taxon 51: 103–114. taxa reveal the formation of heterogeneous-sized pol- Chauhan, A. K. S. 1981. Cytomixis in Papaver rhoeas. In: Manna, G. len grains accompanied by low pollen fertility. Similar K. and Sinha, U. (eds.). Perspectives in Cytology and Genetics. 308 S. Rani et al. Cytologia 81(3)

Hindasia Publishers, New Delhi. pp. 309–312. Lakshmi, N., Prakash, N. S., Harini, I. and Rama, Y. R. 1989. A case Chin, H. C., Chang, M. C., Ling, P. P., Ting, C. T. and Dou, F. P. 1985. of spontaneous cytomixis coupled with desynapsis in Capsicum A cytotaxonomic study on Chinese Dioscorea L. The chromo- annum L. Cytologia 54: 287–291. some numbers and their relation to the origin and evolution of the Lattoo, S. K., Khan, S., Bamotra, S. and Dhar, A. K. 2006. Cyto- genus. Acta Phytotaxon. Sin. 23: 11–18. mixis impairs meiosis and influences reproductive success in Coursey, D. G. 1967. Yams: An Account of the Nature, Origins, Chlorophytum comosum (Thunb) Jacq.̶An additional strategy Cultivation and Utilisation of the Useful Members of the Diosco- and possible implications. J. Biosci. 31: 629–637. reaceae. Longmans, London. Malallah, G. A. and Attia, T. A. 2003. Cytomixis and its possible Darlington, C. D. 1965. Cytology. Churchill Ltd., London. evolutionary role in a Kuwait population of Diplotaxis harra De, M. 1981. Cytomixis in pollen mother cells of an apomict orna- (Boraginaceae). Bot. J. Linn. Soc. 143: 169–175. mental Tabemaemontana coronaria willd. Proc. Ind. Sci. Cong. Marks, G. E. 1954. An acetocarmine glycerol jelly for use in pollen Assoc. 68: 90. fertility counts. Stain Technol. 29: 277. de Souza, A. M. and Pagliarini, M. S. 1997. Cytomixis in Brassica Martin, F. W. and Ortiz, S. 1963. Chromosome numbers and behav- napus var. oleifera and Brassica campestris var. oleifera (Bras- iour in some species of Dioscorea. Cytologia 28: 96–101. sicaceae). Cytologia 62: 25–29. Mary, T. N. and Suvarnalatha, B. 1981. Cytomixis and deviation of Egesi, C. N., Pillay, M., Asiedu, R. and Egunjobi, J. K. 2002. Ploidy chromosome numbers in pollen mother cells of Gossypium spe- analysis in water yam, Dioscorea alata L. germplasm. Euphytica cies. J. Indian Bot. Soc. 60: 74. 128: 225–230. Merckx, V., Schols, P., Kamer, H. M., Maas, P., Huysmans, S. and Eriksson, G. 1968. Temperature response of pollen mother cells in Smets, E. 2006. Phylogeny and evolution of Burmanniaceae (Di- Larix and its importance for pollen formation. Studia Forestalia oscoreales) based on nuclear and mitochondrial data. Am. J. Bot. Suecica 63: 1–131. 93: 1684–1698. Essad, S. and Maunoury, C. 1984. Variation geographique des Murti, S. K. 2001. Flora of Cold Deserts of Western Himalaya. Vol. 1. nombres chromosomiques de base et polyploıdie dans le genre (Monocotyledons). Botanical Survey of India, Calcutta. Dioscorea a propos du denombrement des especes transversa Nirmala, A. and Rao, P. N. 1996. Genesis of chromosomal numerical Brown, pilosiuscula Bert et trifida L. Agron. 4: 611–617. mosaicism in higher plants. Nucleus 39: 151–175. Fatemeh, F., Sheidai, M. and Asadi, M. 2010. Cytological study the Obidiegwu, J., Loureiro, J., Ene-Obong, E., Rodriguez, E., Koles- genus Arenaria L. (Caryophyllaceae). Caryologia G. Citol. Cito- nikova-Allen, M., Santos, C., Muoneke, C. and Asiedu, R. 2009. sistematica Citogenet. 63: 149–156. Ploidy level studies on the Dioscorea cayenensis/Dioscorea Gaulden, M. E. 1987. Hypothesis: some mutagens directly alter spe- rotundata complex core set. Euphytica 169: 319–326. cific chromosomal proteins (DNA topoisomerase II and periph- Pagliarini, M. S. 1990. Meiotic behavior and pollen fertility in eral proteins) to produce chromosome stickiness, which causes Aptenia cordifolia (Aizoaceae). Caryologia G. Citol. Citosiste- chromosome aberrations. Mutagenesis 2: 357–365. matica Citogenet. 43: 157–162. Guan, J. Z., Wang, J. J., Cheng, Z. H., Liu, Y. and Li, Z. Y. 2012. Pagliarini, M. S. 2000. Meiotic behavior of economically important Cytomixis and meiotic abnormalities during microsporogenesis plant species: The relationship between fertility and male steril- are responsible for male sterility and chromosome variations in ity. Genet. Mol. Biol. 23: 997–1002. Houttuynia cordata. Genet. Mol. Res. 11: 121–130. Perret, M., Chautems, A., Spichiger, R., Kite, G. and Savolainen, V. Gupta, A. 2009. Exploration of cytomorphological diversity in grasses 2003. Systematics and evolution of tribe Sinningieae (Gesneria- from Haryana and adjoining Shiwalik hills. Ph.D. Thesis, Pun- ceae): Evidence from phylogenetic analyses of six plastid DNA jabi University, Patiala. regions and nuclear ncpGS. Am. J. Bot. 90: 445–460. Gupta, P. K. and Priyadarshan, P. M. 1982. Triticale: Present status Raghavan, S. 1958. A chromosome survey of Indian Dioscorea. Proc. and future prospects. Adv. Genet. 21: 255–345. Ind. Acad. Sci. 48: 59–63. Haque, M. S. and Ghoshal, K. K. 1980. Behaviour of meiotic chromo- Rani, S., Kumar, S., Jeelani, S. M., Kumari, S. and Gupta, R. C. 2011. somes with special reference to their relationship in pollen steril- Cytological studies in some members of Ranunculaceae from ity of Salvia L. Cytologia 45: 743–752. Western Himalaya (India). Caryologia G. Citol. Citosistematica Haroun, S. A., Al Shehri, A. M. and Al Wadie, H. M. 2004. Cytomixis Citogenet. 64: 405–418. in the microsporogenesis of Vicia faba L. (Fabaceae). Cytologia Rani, S., Kumari, S., Gupta, R. C. and Chahota, R. K. 2014. Cytologi- 69: 7–11. cal studies of Angiosperms (174 species) from District Kangra, Jeelani, S. M., Rani, S., Kumar, S., Kumari, S. and Gupta, R. C. 2011. Himachal Pradesh (India). Plant Syst. Evol. 300: 851–862. Meiotic studies in some members of Caryophyllaceae Juss. from Rothfels, K. H. and Siminovitch, L. 1958. The chromosome comple- the Western Himalayas. Acta Biol. Craco. Bot. 53: 86–95. ment of the rhesus monkey (Macaca mulatta) determined in kid- Jiang, Y., Ding, C., Yue, H. and Yang, R. 2011. Meiotic behavior and ney cells cultivated in vitro. Chromosoma 9: 163–175. pollen fertility of five species in the genus Epimedium. Afr. J. Sheidai, M., Bahmani, F., Enayatkhani, M. and Gholipour, A. 2009. Biotechnol. 10: 16189–16192. Contribution to cytotaxonomy of Silene: Chromosome pairing Kaufman, B. P. 1956. Cytological studies of changes induced in cel- and unreduced pollen grain formation in sec. Sclerocalycinae. lular materials by ionizing radiations. Ann. N. Y. Acad. Sci. 59: Acta Biol. Szeged. 53: 87–92. 1–53. Sheidai, M., Nikoo, M. and Gholipour, A. 2008. Cytogenetic vari- Keeler, K. H. and Davis, G. A. 1999. Comparison of common cyto- ability and new chromosome number reports in Silene L. species types of Andropogon gerardii (Andropogoneae, Poaceae). Am. J. (Sect. Lasiostemones, Caryophyllaceae). Acta Biol. Szeged. 52: Bot. 86: 974–979. 313–319. Kumar, P. and Singhal, V. K. 2011. Chromosome number, male meio- Sinha, S. S. N. and Godward, M. B. E. 1972. Radiation studies in sis and pollen fertility in the plants of Polypetalae from Indian Lens culinaris. Meiosis: Abnormalities due to gamma radiation cold deserts of Lahaul-Spiti and adjoining areas in Himachal and its consequences. Cytologia 37: 685–695. Pradesh (India). Plant Syst. Evol. 297: 271–297. Souza, M. M., Pereira, T. N. S., Dias, A. J. B., Ribeiro, B. F. R. and Kumar, P., Singhal, V. K., Kaur, D. and Kaur, S. 2010. Cytomixis Viana, A. P. 2006. Structural, hystochemical and cytochemical and associated meiotic abnormalities affecting pollen fertility in characteristics of the stigma and style in Passiflora edulis f. fla- Clematis orientalis. Biol. Plant. 54: 181–184. vicarpa (Passifloraceae). Braz. Archiv. Biol. Technol. 49: 93–98. 2016 Morphometric and Cytological Analysis of Different Cytotypes of Dioscorea deltoidea Wall. 309

Španiel, S., Marhold, K., Hodálová, I. and Lihová, J. 2008. Diploid ane sulphonate and radiation induced meiotic abnormalities and tetraploid cytotypes of Centaurea stoebe (Asteraceae) in in Turnera ulmifolia L. var. angustifolia wild. Cytologia 45: central Europe: Morphological differentiation and cytotype dis- 221–231. tribution patterns. Folia Geobot. 43: 131–158. Turesson, G. 1930. Studien Über Festuca Ovina L. II. Hereditas 13: Srivastava, A. K. and Purnima 1990. Agamospermy in some poly- 177–184. doi: 10.1111/j.1601-5223.1930.tb02521.x ploidy grasses. Acta Bot. Indica 18: 240–246. Villeneuve, A. M. and Hillers, K. J. 2001. Whence meiosis? Cell 106: Stahlin, A. 1929. Morphologische und zytologische Untersuchungen 647–650. an Gramineen. Pﬂanzenbau 1: 330–397. Whyte, R. O. 1975. The geography of abnormal meiosis in plants. Stebbins, G. L. 1950. Variation and Evolution in Plants. Columbia Nucleus 18: 183–203. University Press, New York. Wilkin, P., Schols, P., Chase, M. W., Chayamarit, K., Furness, C. A., Sudhakaran, I. V. 1972. Inﬂuence of gamma rays on cell division in Huysmans, S., Rakotonasolo, F., Smets, E. and Thapyai, C. 2005. the seed roots of irradiated dry seeds of Vinca rosea L. Cytologia A plastid gene phylogeny of the yam genus, Dioscorea: Roots, 37: 445–456. fruits and Madagascar. Syst. Bot. 30: 736–749. Tarar, J. L. and Dnyansagar, V. R. 1980. Comparison of ethyl meth-