Meiotic and Ethnobotanical Studies on Rheum Species from Kashmir Himalaya

Shagoon Tabin1, Kuljit Kaur1, Vijay Singh1, Azra Nahaid Kamili2 and Raghbir Chand Gupta1*

1 Department of Botany, Punjabi University, Patiala 147002, Punjab, India 2 Centre of Research for Development University of Kashmir, Srinagar 190006, J&K, India

Received October 23, 2015; accepted June 11, 2016

Summary The Rheum species (R. emodi, R. spiciforme, and R. webbianum) are endangered species and have vanished from lower altitudes. Meiotic studies of the three species show that the polyploidy is reported only in R. webbianum, as it depicts two cytotypes, i.e., diploid (2x) in population from Panzila Top and tetraploid (4x) from Parkhachik and Tangole, of which the diploid cytotype is reported for the ﬁrst time in this species. Moreover, a diploid cytotype (2x) in R. spiciforme is reported for the ﬁrst time in India. Further, meiotic course is abnormal in all the studied populations, as all show meiotic abnormalities in the form of cytomixis, chromatin stickiness, unoriented bivalents, chromatin bridges and laggards, leading to abnormal microsporogensis, ultimately affecting pollen viability. Ethnobotanical information depicts the use of different parts of all the Rheum species by local people to control many diseases like piles, constipation, skin eruptions, intestinal infection, wounds, joint pains and also used as food.

Key words Cytotype, Endangered species, Ethnobotany, Meiosis, Meiotic abnormality, Rheum.

The genus Rheum L. (commonly called Rhubarb) nology may lead to the development of new pharmaceu- belongs to the family Polygonaceae of group Monochla- tical drugs. mydae and is represented by 60 species spread through- out the world (Anjen et al. 2003), of which only seven Materials and methods species have been reported from the Indian subcontinent (Ganie et al. 2014). According to Stewart (1972), all the Meiotic study seven species (including R. emodi, R. spiciforme, and Young flower buds were collected during day time R. webbianum) reported from the Indian subcontinent and fixed in standard Carnoy’s fixative (6 : 3 : 1=abso- are also present in the Kashmir Himalaya. A total of lute alcohol : chloroform : glacial acetic acid v/v/v) for 560 plant species of India are included in the Red List 24 h and preserved in 70% alcohol at 4°C. The collected of Threatened Species by IUCN including Rheum spe- buds were subjected to detailed meiotic analysis for cies (Phartyal et al. 2002). Rheum emodi is included in chromosome number determination, meiotic course, critically endangered species. Rheum, commonly known microsporogenesis, pollen fertility percentage, and pol- as Rhubarb, is a strong vigorous plant and can live long len grain size. In each case more than 20 fresh slides even in tough conditions. Many different species of were prepared from different anthers/flowers of more Rheum are distributed in the temperate and sub-tropical than one plant, using a standard 1% acetocarmine regions of Himalaya from Kashmir to Sikkim (Nautiyal technique. Chromosome numbers were determined at et al. 2003) and growing wild in the mountains of the diakinesis, metaphase-I (M-I), and anaphase I/II (A-I/II) Western and North-Western provinces of China and from freshly prepared slides. For the sake of confirma- adjoining Tibetan territory. In taxonomical focus, inflo- tion of any observation, about 100 pollen mother cells rescence is pyramidal or spherical in which flowers are (PMCs) were analyzed at various stages of meiosis from arranged in panicles and pedicel articulates at or below prophase-I to telophase-II. Pollen fertility was estimated the middle. by mounting pollen grains in 50% glycero–acetocar- The ethnobotanical information provided by tribal mine (1 : 1) as proposed by Marks (1954). Well-filled and local people may provide clues for specific medici- and stained pollen grains were considered as fertile and nal properties and the presence of specific biochemicals, shrivelled or unstained pollen grains as sterile. Measure- which when explored with modern phytochemical tech- ments of pollen grain size were taken using micrometry and a Nikon Eclipse 80i Imaging System. Microphoto- * Corresponding author, e-mail: [email protected] graphs of PMCs depicting various meiotic stages and DOI: 10.1508/cytologia.81.295 pollen grains were taken from freshly prepared tempo- 296 S. Tabin et al. Cytologia 81(3)

Fig. 1. Meiotic chromosome number in different species of Rheum from Kashmir Himalaya. a, b: Rheum emodi (2n=22): PMC

at M-I showing 11II. PMC at A-I showing 11 : 11 distribution of chromosomes. c: Rheum spiciforme (2n=22): PMC at M-I showing 11II. d, e, f: Rheum webbianum (2n=22, 44): PMC at M-I showing 11II, PMC at A-I showing 11 : 11 distribution of chromosomes, PMC at A-I showing 22 : 22 distribution of chromosomes. rary slides using Leica Qwin and Nikon Eclipse 80i mi- bridges at anaphases/telophases (Fig. 2f, i), and unori- croscope Digital Imaging Systems. The specimens were ented bivalents at M-I (Fig. 2c), leading to abnormal mi- submitted to the Herbarium, Department of Botany, crosporogenesis with the presence of monads, triads, and Punjabi University, Patiala and University of Kashmir, tetrads with micronuclei (Fig. 2n, o, q), which ultimately Kashmir. The data regarding the cytologically known leads to the decrease in pollen fertility (72.80%). species, number of cytotypes, and frequency of poly- ploids of a particular genus has been compiled world- Rheum spiciforme Royle wide as well as in India from various Chromosomal Cytologically, both populations of the species showed Atlases and Indexes to Plant Chromosome Numbers by a diploid chromosome count 2n=22 (Fig. 1c), which was Darlington and Wylie (1955), Fedorov (1974), Moore elucidated cytologically for the ﬁrst time from India and (1973, 1974, 1977), Goldblatt (1981, 1984, 1985, 1988), is in accordance with the reports by Jaretzky (1928) and Goldblatt and Johnson (1990, 1991, 1994, 1996, 1998, Edman (1929) from outside India (Table 1). The detailed 2000, 2003), Kumar and Subramaniam (1986), and Kha- course of meiosis was abnormal with the presence of toon and Ali (1993), various journals, the Internet, as various irregularities like laggards and chromatin bridg- well as the species in this study. es at A-I/T-I (Fig. 2g, j), chromatin stickiness at M-I (2l), unoriented bivalents at M-I (Fig. 2d), and cytomixis Ethnobotany between different PMCs at M-I (Fig. 2b), which leads to The surveys and collection of Rheum species was abnormal microsporogenesis with the presence of micro- mainly on high altitudes, and information was taken nuclei in tetrads (Fig. 2r) and resulted in decreased pol- from gujjars, bakkerwals, and some local people. For len fertility (78.23%). gaining knowledge and information on medicinal plants, these people were interviewed during the course of the Rheum webbianum Royle present study. During meiotic course, one accession of the species collected from Panzila Top (Zanaskar area) showed the Results presence of diploid cytotype showing 11 bivalents at M-I and 11 : 11 distribution of chromosomes at A-I (Fig. Rheum emodi Wall. 1d, e). Two accessions collected from Parkhachik and The meiotic studies of the present accessions reveal Tangole (Kargil district) are tetraploid showing 22 : 22 the presence of a diploid chromosome count 2n=22 (Fig. distribution of chromosomes at A-I (Fig. 1f). Previously, 1a, b) (Table 1), which is in accordance with the previ- the tetraploid cytotype (2n=44) of the species has also ous reports by various workers from India (Gohil and been reported by Gohil and Rather (1986), and Saggoo Rather 1986, Saggoo and Farooq 2011). Further, the and Farooq (2011) from Kashmir Himalaya (Table 1), meiotic course was abnormal with the presence of chro- whereas, 2n=22 adds a new diploid cytotype for the matin transfer at M-I (Fig. 2a), laggards and chromatin ﬁrst time on a worldwide basis and reports the presence 2016 Meiotic and Ethnobotanical Studies on Rheum Species from Kashmir Himalaya 297

Fig. 2. Meiotic abnormalities in the studied species of Rheum from Kashmir Himalaya. a, b: PMCs showing chromatin transfer. c–e: PMCs at M-I showing unoriented bivalents. f–h: PMCs showing laggards at A-I and T-I. i–k: PMCs showing chromatin bridges at A-I and T-I. l, m: PMCs showing chromatin stickiness at M-I. n: Monad. o, p: Triad. q, r: Tetrads with micronuclei. s: Fertile and sterile pollen grains. of intraspeciﬁc polyploidy. The detailed meiotic course was collected by interacting with local and tribal people. of the diploid cytotype shows various abnormalities as During the surveys, it was observed that tribal people unoriented bivalents at M-I (Fig. 2e), chromatin laggards used to preserve and conserve the Rheum species for and bridges at anaphases/telophases (Fig. 2h, k), and their living. After interviews of local people of high al- chromatin stickiness at M-I (Fig. 2m) (Table 2). These titude, it was observed that Rheum species are used for abnormalities lead to abnormal microsporogenesis with many diseases listed in Table 4. triads and tetrads with micronuclei (Table 3) and resulted in the reduction of pollen fertility (69.15%), whereas Discussion the tetraploid cytotype shows a normal meiotic course with high pollen fertility (97.38%). Chromosome number The genus is comprised of 60 taxonomically known Ethnobotany species, out of which 42 species/51 cytotypes are cyto- Information regarding local uses of Rheum species logically elucidated worldwide. It is a monobasic genus 298 S. Tabin et al. Cytologia 81(3)

Table 1. Data containing accession numbers, locality with altitude, meiotic chromosome number, ploidy level, meiotic course, pollen size, pollen fertility, and previous reports with the remarks of presently studied three species of Rheum from Kashmir Himalaya.

Meiotic Ploidy level S. Taxa Locality with Pollen size Pollen chromosome (x)/Meiotic Previous reports Remarks No. (Accession no.) altitude (m) (μm) fertility (%) number (2n) course (N/A)

Family: Polygonaceae 1. Rheum emodi Wall. 2n=22: Previous P-I Dhara, Srinagar 22 2x/A 28.53×28.06 72.80 Gohil and Rather reports (PUP 59513) (3751) (1986); conﬁrmed P-II Sonamarg, 22 2x/A 27.81×27.00 76.28 Saggoo and (KU 2010 KASH) Ganderbal Farooq (2011) (3462) P-III Gulmarg, 22 2x/N 29.54×28.92 92.12 Baramula (4476) P-IV TarsarMarsar 22 2x/A 28.00×27.28 73.00 (4657) 2. Rheum spiciforme Royle 2n=22: First report P-I (PUP 59516) Chakwali, 22 2x/A 25.63×23.89 78.23 Jaretzky (1928); of diploid Gurez (4684) Edman (1929) cytotype P-II (KU 2012 Satni Mountain 22 2x/A 26.12×25.92 76.14 from India KASH) (3962) 3. Rheum webbianum Royle 2n=44: First report of P-I (PUP 59515) Panzila top, 22 2x/A 23.92×23.16 69.15 Gohil and Rather diploid cy- Zanskar (4593) (1986); totype from P-II (PUP 59514) Parkachik, 44 4x/N 25.68×25.12 97.38 Saggoo and world. Kargil (3938) Farooq (2011) P-III (KU 2011 Tangole, Kargil 44 4x/N 26.00×25.62 99.00 KASH) (4266)

Table 2. Data on cytomixis and detailed meiotic course with different abnormalities in the studied accessions of three different Rheum species.

Cytomixis Meiotic course

PMCs with PMCs with PMCs with PMCs with Taxa PMCs involved No. of PMCs chromatin unoriented chromatin laggards (%) involved stickiness at bivalents at bridges (at A-I/II, (at A-I/II, M-I (%) M-I (%) T-I/II) (%) T-I/II) (%) R. emodi Wall. 6.6 (7/105) 2–3 5.1 (5/98) 2.8 (3/105) 7.8 (9/114) 11.21 (12/107) R. webbianum Royle 4.6 (5/107) 1–2 3.5 (3/84) 6.12 (6/98) 3.92 (4/102) 10.18 (11/108) R. spiciforme Royle 2x (P-I) 2.4 (3/125) 2–4 5.8 (6/102) 11.11 (14/126) 7.62 (9/118) 12.19 (15/123) 4x (P-II) ̶/̶ ̶/̶ ̶/̶ ̶/̶ ̶/̶ ̶/̶

Figures in parentheses represent number of PMCs with abnormalities to total number of PMCs observed.

Table 3. Data on abnormal microsporogenesis in three different studied species of Rheum.

Dyads Triads Tetrads Taxa WMN (%) WM (%) WMN (%) WM (%) WMN (%) WM (%)

Rheum emodi Wall. 1.9 (2/102) ̶/̶ 3.9 (4/102) ̶/̶ 96.07 (98/102) 2.9 (3/102) Rheum spiciforme Royle ̶/̶ ̶/̶ 4.2 (5/119) 2.5 (3/119) 95.79 (114/119) 4.2 (5/119) Rheum webbianum Royle 2x (P-I) 2.7 (3/109) ̶/̶ 6.4 (7/109) 4.5 (5/109) 98.16 (107/109) 1.8 (2/109) 4x (P-II) ̶/̶ ̶/̶ ̶/̶ ̶/̶ 100 (98/98) ̶/̶

WMN=Without micronuclei; WM=With micronuclei. Figures in parentheses represent number of PMCs with abnormalities to total number of PMCs observed. based on x=11 represented by 30 diploid and 21 tetra- Meiotic abnormalities ploid cytotypes. Intraspeciﬁc polyploidy is reported in In normal meiosis, four haploid (n) gametes are nine species. From India, three species are cytologically formed, but recombination and independent assortment elucidated. are the major sources to create change (structural/nu- merical) in gametes, and serve as an important source 2016 Meiotic and Ethnobotanical Studies on Rheum Species from Kashmir Himalaya 299

Table 4. Ethnobotany of Rheum species.

Rheum Diseases Part used Preparation species

R. emodi (Pumbhaak, Pumbchaalan) 1 Wounds and boils Roots Root is powdered in the form of paste and applied on wounds directly without massage. 2 Jaundice Leaves Leaves are boiled in water with a pinch of salt and taken a cup orally twice a day. 3 Diarrhea/indigestion Roots Root (50 g) is powdered and boiled in 500 mL water and taken a cup thrice a day. 4 Cough and asthma Leaves Leaves are crushed and boiled in water and then taken half cup orally thrice a day. 5 Headache Leaves Leaves are crushed and boiled and then taken one cup twice a day. 6 Muscular pain/body ache Roots The root is grinded into powdered form and mixed with some oil and then applied externally on affected parts. 7 Freckle/skin eruptions/ Roots Root is crushed, grinded, and mixed with vinegar, and then that paste is applied skin diseases externally on affected parts. Powder of root is applied with fresh milk also. 8 Swelling and inflamma- Leaves/roots Fresh leaves and fresh roots are grinded so that an oil-like substance comes out from tion/rheumatic pain fresh roots and leaves, and that oil is applied on affected parts. 9 Food Leaves/stalks Leaves and its stalks are cooked as a vegetable. 10 Constipation Root Root powder (50 g) is mixed with 10 g of ginger powder, then mixed with 200 mL of water and boiled, and then taken half cup four times a day. 11 Reducing obesity, weight/ Root A grinded paste is made from 10 g root and 3 g ginger. The mixture is boiled in fat 300 mL of water and then taken two spoons morning and evening. R. webbianum (Revandchini, Lachoo) 1 Rheumatic pain/wound Roots The root powder is made into fine paste and used on affected parts. 2 Piles Leaves Leaves are crushed and boiled with water and taken orally thrice a day. 3 Chronic bronchitis Leaves Leaves are boiled and then the boiled water is taken thrice a day. 4 Constipation Roots Roots are grinded into a fine powder and then mixed with water, which is then taken by the teaspoon thrice a day. R. spiciforme (Pumbhaak, Jarochotal) 1 Wound/swelling Roots The powder form of roots is mixed with warm water for making fine paste and then applied on swellings and wounds. 2 Bone fracture/backache/ Roots The powdered form of roots are mixed with water and one spoon of turmeric, mak- joint pain ing a fine paste, and then applied on affected parts and two spoons taken orally our times a day. 3 Food Leaves Leaves are cooked as a vegetable. 4 Rheumatic pain Roots The root is powdered into fine paste and used against rheumatic pain and wounds. The 3–5 g powder once in a week with milk. The paste is applied on affected portion externally. 5 Stomachache/intestinal Roots Tribal people chew roots for any stomach problem. Root powder is mixed with warm infections/dysentery water and taken orally half cup twice a day for irritation in stomach, dysentery and intestinal infections. of genetic diversity (Schwarzacher 2003). During the matin transfer and stickiness might be the results of ge- present investigations, various types of meiotic ir- netic/environmental factors or genomic–environmental regularities, such as cytomixis, chromatin stickiness, interaction (Baptista-Giacomelli et al. 2000, Bellucci et nonsynchronous disjunction of bivalents at metaphase-I, al. 2003, Fatemeh et al. 2010, Kumar and Singhal 2011). chromatin bridges, chromosomal laggards, and unori- The chromosome/chromatin that fails to reach their ented bivalents, have been observed in almost all the respective poles at anaphase I/II or telophase-I/II are studied species. These irregularities during the meiotic regarded as laggards. The reason behind such failure in course usually lead to abnormal microsporogenesis like segregation might be in relation to impaired attachment monads, dyads, triads, and polyads with and/or without of kinetochores to the spindle fibers (Nicklas and Ward micronuclei, which mostly reduces the pollen fertility. 1994), or through late chiasma terminalization, especial- Cytomixis is a phenomenon of transferring chromatin ly in large-sized bivalents (Pagliarini 2000, Souza et al. from one PMC/s to the neighboring PMC/s through cy- 2006). The chromatin stickiness, late or non-disjuncting toplasmic channels by direct fusion, and was recorded bivalents and chromatinl laggards seem to be respon- for the first time by Kornicke (1901) in Crocus sativus. sible for chromosomal bridges (Kumar et al. 2013), but Such chromatin transfer results in a number of hypo/ a more appropriate cause for its formation is heterozy- hyperploid cells or even syncytes and ultimately affects gous paracentric inversions (Sinha and Godward 1972). the reproductive potential by producing non-viable and All these mentioned abnormalities lead to the abnormal unreduced gametes. The diverse accountable sources for microsporogenesis with the formation of dyads, triads, cytomixis are either abiotic (Narain 1976), abiotic/biotic and tetrads with micronuclei, and polyads and ultimately (Ghanima and Talaat 2003), or strictly biotic or genetic result in the heterogenous-sized pollen grains and high factors (Bellucci et al. 2003, Haroun et al. 2004). Chro- pollen sterility. 300 S. Tabin et al. Cytologia 81(3)

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