Urginea Indica (Roxb) Kunth

Karyotypic Variations in Different Accessions of Urginea indica (Roxb) Kunth. and Urginea wightii Lakshmin. Hyacinthaceae

H. L. Geetha, M. N. Shivakameshwari* and R. Nijagunaiah

Department of Botany, Jnana Bharathi Campus, Bangalore University, Bangalore 560056, India

Received December 2, 2015; accepted June 22, 2016

Summary Urginea indica Kunth. Hyacinthaceae, commonly called Indian Squill, represents a species complex. Intraspecies variation is common. Karyological studies were made in six accessions of Urginea collected from six different places in India, of which four accessions of Urginea indica were collected from Kanakapura, Trichendur, Kashmir valley, and Kerala. Two accessions of Urginea wightii were from Gubbi and Bellary. Varia- tions in ploidy were noted. Among the Urginea indica accessions, diploids (2n=20) were found in Kanakapura, Trichendur, and Kashmir Valley; a pentaploid (2n=50) was noticed in the Kerala accession, whereas two different ploidies were noticed in the Urginea wightii accessions of Gubbi and Bellary (2n=20, 2n=40) within the same plant. There is close homology between Kanakapura and Tiruchendur with subtelocentric being a major component.

Key words Urginea, Karyotype, Accession, Diploid, Tetraploid, Pentaploid.

Urginea genus belongs to the family Hyacinthaceae. placed Drimia in the Tribe Urgineae, Order Scillodeae, Urginea indica is also called Indian squill. Squill bulbs and Family Asparagaceae. Urginea is known for pheno- have long been used as a source of medicine with phar- typic and genotypic complexities, and variations in ac- maceutical and biocidal applications. There is mention cessions within species lead to the appearance of a wide of Indian squill in British and European pharmacopias. range of intermediate forms, which led to confusion Urginea indica accessions are unexplored from a cyto- taxonomically. taxonomic point of view. The species has been studied Several cytological studies have been conducted by by Shiva Kameshwari and Muniyamma (1999, 2001). Dixit and Yadav (1989), Deb and Dasgupta (1974), Jha Karyotype analysis of different accessions is very use- and Sen (1980, 1983a, b), and Shiva Kameshwari et al. ful in detecting the probable relationship, origin, and na- (2014) to solve these complexities. However, even to ture of chromosome variations. The present report deals this day, none of the studies have completely elucidated with karyomorphology of Urginea indica and Urginea the complexities. Thus, Urginea is a taxonomically, wightii. The karyotype differentiation between acces- genetically, and phenotypically complex genus. Further sions was elucidated. Urginea indica is an aggregate of detailed studies of Urginea are required, with a large diploid, tetraploid, aneuploid, pentaploid, and hexaploid collection of specimens from various localities to sup- cytotypes growing in different localities in India. Mor- port the research. In the present study, karyomorpho- phological and cytological differences have been detect- logical studies were made in U. indica (four accessions) ed within the accessions of U. indica (Shiva Kameshwari and U. wightii (two accessions) aiming to characterize et al. 2010). Urginea wightii is morphologically and these species cytologically and explore the pattern of cytologically distinct from Urginea indica. choromosomal differentiation and karyological varia- The plant is known for its cardiac glycosides (Mulhol- tions within and between the species. The basic number land et al. 2013). It is gaining global importance due to of Urginea according to Goldblatt et al. (2012) is X=10. its multiple properties as a medicine (Nath et al. 2014). According to Manning et al. 2004, U. indica and their Materials and methods accessions are controversial and need further resolution. The medicinal plant Urginea is separated from Bulbs of Urginea indica of four different accessions Urginea and included in Drimia (Jessop 1977, Stedje were collected from Kashmir, Kanakapura, Thiruchen- 1987). Molecular studies by Pfosser and Speta (1999) dur, and Kerala, and two accessions of Urginea wightii were collected from Gubbi and Bellary and grown in * Corresponding author, e-mail: [email protected] the department of Botany, Bangalore University, Banga- DOI: 10.1508/cytologia.81.329 lore, under uniform environmental conditions. Root tips 330 H. L. Geetha et al. Cytologia 81(3)

Fig. 1. a–d. Somatic metaphase chromosomes and idiograms of accessions of U. wightii. a: Somatic metaphase chromosomes of Bellary accession showing 2n=40. b: Somatic metaphase chromosomes of Bellary accession showing 2n=20. c: Idiogram of Bellary accession 2n=40. d: Idiogram of Bellary accession 2n=20. Scale bar=10 µm.

Table 1. Karyometric data in different accessions of Urginea indica Kunth.

Total chromosome Sl. no. Place of collection Chromosome number Ploidy Karyotype formula Karyotype length in µm

1. Kanakapura 2n=20 Diploid St10 81.64 Asymmetrical 2. Trichendur 2n=20 Diploid St10 99.72 Asymmetrical 3. Kashmir 2n=20 Diploid Sm2+St7+T1 115.5 Asymmetrical 4. Kerala 2n=50 Pentaploid Sm2+St20+t3 301.51 Asymmetrical 5. Gubbi (U. wightii) 2n=20 Diploid St10 94.64 Asymmetrical 2n=40 Tetraploid Sm5+St15 222.45 6. Bellary (U. wightii) 2n=20 Diploid Sm5+St5 82.22 Asymmetrical 2n=40 Tetraploid Sm6+St14 189.08 were excised from potted plants between 9 : 30 a.m. to mosome numbers of all the accessions were recorded 11 : 30 a.m. and pretreated with 0.1% 8-hydroxy quino- (Table 1). Karyotypes were made for each accession line and kept at 4°C for 6 h and ﬁxed in acetic alcohol following the methods employed by Levan et al. (1964). (3 : 1) for 24 h and stored in 70% alcohol. The roots were Symmetry and asymmetry have been determined ac- hydrolysed in 1 N HCl for 15 min and kept in feulgen cording to the system proposed by Stebbins (1958). stain for 1 h and squashed in 45% acetic acid. Temporary preparations for feulgen staining were made. The chro- 2016 Karyotypic Variations in Different Accessions of Urginea indica (Roxb) Kunth. and Urginea wightii Lakshmin. Hyacinthaceae 331

Fig. 2. a–d. Somatic metaphase chromosomes and idiograms of accessions of U. wightii. a: Somatic metaphase chromosomes of Gubbi accession showing 2n=40. b: Somatic metaphase chromosomes of Gubbi accession showing 2n=20. c: Idiogram of Gubbi accession 2n=40. d: Idiogram of Gubbi accession 2n=20. Scale bar=10 µm.

Results and discussion Such inconsistency has been reported in over 240 species belonging to angiosperms by Nirmala and Rao The cytological variations obtained in the present (1996). Similar cases of polysomaty have been reported study provided a basis for recognizing them as distinct in growing shoot apex by Sharma (1956). cytotypes (Figs. 1–4). The four cytotypes of Urginea Karyotype asymmetry in different accessions of U. indica investigated revealed three diploids of 2n=20 and indica varies greatly due to different conditions of the one pentaploid of 2n=50. The karyotypes were asym- locality. Similar studies have been made by Wang et metrical in all the accessions, with subtelocentric be- al. (2013) in the ophiopogoneae tribe from Southwest ing the major component of the karyotype followed by China. submetacentric, terminal point, and ﬁnally telocentric Currently, we do not know how many more cytotypes chromosomes. These accessions are phenotypically dis- from the U. indica complex are present in South India. tinct types. The karyotype complementary shows that it Cytotaxonomic studies in the genus Urginea and karyo- should be diploid with the basic chromosome number of type variation in U. indica revealed by Oyewole (1987) X=10. Diploid and pentaploid accessions were detected have aided our understanding of karyotypic variations. in Urginea indica. The high frequency of diploid cyto- He reported that the difference in the chromatin material types is most common. is correlated with both morphological differences and In the Gubbi and Bellary accessions of Urginea ecological preferences. wightii, chromosomal variations were observed in the The main objective of this study is to identify the ploi- cells of the same root tip. Of the cells studied, 50% dy level of the Urginea accessions and to compare their showed 2n=20 chromosomes, while the remaining 50% morphological characteristics with plants collected from showed 2n=40 chromosomes. Such variations within the different localities of India with the same chromosome same tissue are termed chromosome mosaics. number or different cytotypes. The results obtained sug- 332 H. L. Geetha et al. Cytologia 81(3)

Fig. 3. a–d. Somatic metaphase chromosomes and idiograms of accessions of U. indica. a: Somatic metaphase chromosomes of Kerala accession showing 2n=50. b: Somatic metaphase chromosomes of Kanakapura accession showing 2n=20. c: Idio- gram of Kerala accession 2n=50. d: Idiogram of Kanakapura accession 2n=20. Scale bar=10 µm. gest that the speciﬁc categories assigned to the bulbs col- terning might be involved in rearranging small gene lected from different localities should be autopolyploidy segments. Hence, U. indica seems to be a stable poly- and mixoploidy, which is reported in Gubbi and Bellary morphism in which the different forms have attained with 2n=20 and 2n=40 at equal frequency. reproductive isolation and genetic variability with each Each accession varies in their morphology and karyo- form retaining its morphological identity. According to type. The total length of chromatin material at meta- Oyewole (1987), the species is aggressive in its exhibi- phase of each karyotype differs from the others. All the tion of various ecological niches and habitat diversiﬁca- accessions were brought from their original habitat and tion. Reproduction through vegetative bulbs as compen- grown under uniform environmental conditions; still the sation for reduced fertility brings about and stabilizes morphological differences were retained. The recogni- chromosomal aberrations (Stebbins 1950, Zhu and Xu tion of different karyotypes that correspond to different 1999). In situ hybridization and banding are needed to morphological forms is noteworthy. The morphological study the cause of diversity in the U. indica. differentiation followed by karyological differentiation Detailed karyotype analysis in different accessions of in these accessions suggests that chromosome repat- U. indica and U. wigtii in the present study were helpful 2016 Karyotypic Variations in Different Accessions of Urginea indica (Roxb) Kunth. and Urginea wightii Lakshmin. Hyacinthaceae 333

Fig. 4. a–d. Somatic metaphase chromosomes and idiograms of accessions of U. indica. a: Somatic metaphase chromosomes of Tiruchendur accession showing 2n=20. b: Somatic metaphase chromosomes of Kashmir accession showing 2n=20. c: Idiogram of Tiruchendur accession 2n=20. d: Idiogram of Kashmir accession 2n=20. Scale bar=10 µm.

to ascertain the differences within the species and assign separate ideogram has been drawn. them as different cytotypes both at species and intraspe- In Gubbi, 2n=20, with subtelocentric being the ma- cific levels. jor component, and an absolute chromosomal length of Differences in absolute chromosome size reflect dif- 94.64 µm were observed. We also found 2n=40, with ferent amounts of gene duplication either in a tandem subtelocentric being the major component followed fashion or through polytene multiplication of chromo- by submetacentric and a total chromosomal length of nemata. The accessions with longer chromatins are sup- 222.45 µm. posed to be primitive, whereas accessions with shorter In Bellary, we found 2n=20, with an absolute chro- chromatins were treated as advanced (Stebbins 1971). mosomal length of 82.22 µm. We also found 2n=40, Thus, the present study revealed that amongst the four with subtelocentric being the major component followed accessions of U. indica, Kerala is the most advanced and by submetacentric, and a total chromosomal length of Kanakapura is the most primitive, indicating that karyo- 189.08 µm. type specialization has taken place in Kerala, Tiruchen- A somatic chromosome number of 2n=20 was ob- dur, Kanakapura, and Kashmir, with close homology served in three accessions of U. indica. Thus, they are observed between Kanakapura and Trichendur. homoploids and cannot be distinguished by chromosome a) Kanakapura: 2n=20. Subtelocentric being the ma- number alone. jor component with total chromosomal length of The total chromosomal length and karyotype formula 81.64 µm. did not reveal any significant difference between the dip- b) Tiruchendur: 2n=20. Subtelocentric being the ma- loid accessions, but the diploid accession from Kashmir jor component with total chromosomal length of deviated in karyotype formula and total chromosomal 99.72 µm. length, while U. indica collected from Kerala (Trissur) c) Kashmir: 2n=20. Subtelocentric being the major was pentaploid, showing 2n=50 and a total chromo- component followed by submetacentric and telocen- somal length of 301.50 µm, which is five times longer tric with total chromosomal length of 115.5 µm. than the mean haploid chromatin length of the diploid d) Kerala: 2n=50. Subtelocentric being the ma- accessions. jor component followed by submetacentric and In U. wightii homoploids with 2n=20 and 40, 50% acrocentric with total chromosomal length of of diploid cells and 50% of tetraploids in both acces- 301.50 µm. sions collected from Gubbi and Bellary is significant. 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