© 2019 The Mendel Society Cytologia 84(3): 207–210

Report of Intraspecific Polyploidy (2x, 6x) in palustris, from Cold Deserts of Ladakh Division (J & K),

Nissar Ahmad Khan*, Vijay Kumar Singhal, Younas Rasheed Tantray and Raghbir Chand Gupta

Department of Botany, Faculty of Life Sciences, Punjabi University, Patiala, Punjab-147002, India

Received February 15, 2019; accepted March 7, 2019

Summary The present communication reports the meiotic analysis on three wild populations of Rorippa palus- tris collected from Ladakh division, Jammu & Kashmir and records the existence of first ever intraspecific eu- ploid cytotypes, the diploid (2n=14) and hexaploid (2n=42). Beside chromosome number, the two cytotypes also differ in morphometric parameters such as flower color, height, size of leaf and number of leaves per plant. The 6x grow much taller and robust compared to those of 2x and size of stomata and pollen grains are also significantly larger in the 6x compared to the 2x. Both cytotypes exhibit normal meiotic behavior characterized by normal chromosome pairing, regular segregation of chromosomes and nearly perfect pollen fertility.

Key words Rorippa palustris, Morphology, Meiosis, Cytotype, Intraspecific euploidy, Chromosome number.

Rorippa palustris (L.) Besser., (Brassicaceae) is an Materials and methods annual or perennial glabrous or hairy tall herb grow- ing in marshy areas between 2000–3300 m. The spe- Collection of plant samples cies is widespread and native to parts of and, Materials for male meiotic studies were collected much of and the . To assess from Suru Valley (PUN 62553) and Drass Valley (PUN the cytological status of R. palustris, field surveys 62554, 62555) within elevations of 3100–3250 min La- were conducted in the Suru Valley and Drass Valley of dakh division of J&K. Cytologically examined speci- Ladakh division in Jammu and Kashmir. Earlier chro- mens were deposited in the Herbarium, Department of mosomal studies in the species by Naqshi and Javeid Botany, Punjabi University, Patiala (PUN). (1976) from India and several cytologists from outside of India Jonsell (1968), Javurkova-Kratochvilova and Cytological studies and pollen fertility Tomsovic (1972), Löve (1982), Zhukova (1982) and For the investigation of gametic chromosome number, Mulligan (1984) were confined to merely counting the meiotic course and pollen fertility, young and develop- chromosome number and no attempt has been made to ing floral buds were fixed in a freshly prepared Carnoy’s correlate the extent of morphological variability among fixative (6 ethanol : 3 chloroform : 1 glacial acetic acid) different cytotypes and their relative distribution pat- for 24 h subsequently transferred in 70% ethanol and terns. Looking at its wider distribution and variations stored in a refrigerator until analysis. Meiocyte prepara- in habitat, cytomorphological studies were undertaken tions were made by squashing the developing anthers on an individual plant basis in respect to phytogeogra- in 1% acetocarmine. A total of 50–100 meiocytes were phy and cytology from cold deserts of Ladakh division. examined in each case for determining the chromo- Consequent to accession based analysis, plants with two some number and detailed meiotic course at diakinesis, chromosome numbers, 2n=14 and 2n=42 were detected. M I, A I/T I and sporad stage. Pollen fertility was esti- Therefore, the present investigations were carried out mated through stainability tests for which anthers from with the following aims, (i) to reveal the exact chromo- fully developed and opened flowers were squashed in a some number on individual plant basis (ii) to analyze the glycerol–acetocarmine (1 : 1) mixture. Well-filled pol- meiocytes for the meiotic course including microsporo- len grains with stained cytoplasm were taken as fertile genesis and pollen fertility (ii) to pinpoint the morpho- while shriveled or unstained/partly stained cytoplasm metric parameters which could be employed to segregate was scored as sterile. the variants. Morphometric analysis * Corresponding author, e-mail: [email protected] The morphometric parameters were analyzed in the DOI: 10.1508/cytologia.84.207 specimens examined cytologically. Trichomes and sto- 208 N. A. Khan et al. Cytologia 84(3)

Fig. 1. Meiotic figures in Rorippa palustris. (A) A PMC showing seven bivalents at diakinesis. (B) A PMC with seven bivalents at M I. (C) A PMC at A I with 7 : 7 equal segregation of chromosomes. (D) A PMC at diakinesis with 21 bivalents. (E) A PMC with 21 bivalents at M I. (F) A PMC at A I with 21 : 21 equal segregation of chromosomes. Pollen grains of 2x (G) and 6x (H) cytotypes. Scale bar=10 µm.

Table 1. Morphological comparison of 2x and 6x cytotypes of Rorripa palustris.

Cytotypes Parameters Diploid (n=7) Hexaploid (n=21) Habitat Meadows and roadsides Streamsides and grasslands Plant height (cm) 65.93±3.17 98.28±5.25 No. of leaves/plant 18–30 22–34 Leaves Texture Pubescent or glabrous Pubescent Shape Oblong Oblong or elliptic Size (cm) 16.66±1.049×1.91±0.54 23.52±2.67×2.34±0.46 Flower Colour Light yellow Bright yellow Size (mm) 20.0–25.0 22.0–32.0 Stomata Size (µm) 24.52±1.77×21.30±0.685 30.12±1.68×23.20±0.61 Stomatal index 10–13 12–14 Trichome size (µm) 137.67±6.48×32.08±1.41 198.05±10.39×4.59±1.40 Pollen grains Size (µm) 85.08±2.23×68.23±1.13 94.73±2.76×77.63±1.09 Fertility (%) 99 98–100 mata were measured in the abaxial epidermal peels of Photomicrographs mature leaves after treatment with 10% KOH for 24 h Photomicrographs of meiotic chromosome counts, and heated at 70°C. The peels were stained Safranin sporads, pollen grains, stomata, and trichomes were solution (0.1% safranin in 60% ethanol) and mounted on taken using a Nikon Eclipse 80i microscope. a glass slide with a coverslip. Stomatal index was calcu- lated by using the following formula: SI=S/S+E×100. Results Where SI stands for a stomatal index, S is the number of stomata and E is the number of epidermal cells. Cytological analysis including pollen fertility One wild plant collected from Sankoo Village 2019 Intraspecific Polyploidy in Rorippa palustris 209

Fig. 2. Plants of diploid (A) and hexaploid cytotypes (B) growing in wild. Trichomes of 2x (C) and 6x (D) cytotypes. A portion of abaxial epidermal peel showing stomata in diploid (E) and hexaploid (F) cytotypes. Scale bar=10 µm.

(3250 m) possessed light yellow flowers and showed Morphometric analysis a gametic chromosome number of n=7 which was Plants of 2x and 6x cytotypes grow ectopically as ascertained from the presence of seven bivalents at annual herbs along roadsides and in meadows, with 2x diakinesis/M I (Fig. 1A, B) and 7 : 7 equal chromosomal plants noticed to be more common in the area compared segregation at A I (Fig. 1C). Meiotic course includ- to the 6x plants. Besides differing in flower color, the ing sporads was normal resulting in 98% fertile pollen two cytotypes also differ in various morphometric pa- grains. rameters which include plant height, number of leaves/ Two wild plants collected from Drass (3100 m) and plant, size of the leaf, and flower color (Table 1). The Pandrass (3190 m) bore bright yellow flowers. These analysis revealed that 6x plants grow much taller (90.80– plants during meiosis showed a gametic chromosome 105.40 cm, Fig. 2A) compared to 2x (60.50–70.40 cm, count of n=21 as confirmed from the presence of 21 Fig. 2B). The 6x plants also possessed larger-sized leaves bivalents at diakinesis (Fig. 1D) and M I (Fig. 1E) and (18.0–26.2 cm×1.8–3.0 cm) and flowers (22.0–32.0 cm) 21 : 21 chromosomes at poles during A I (Fig. 1F). The compared to those of 2x (15.0–18.0×1.0–2.5; 20.0– further meiotic course was perfectly regular leading to 25.0 cm). These cytotypes also differ at a cellular level fertile pollen grains. which was reflected in the size of stomata, trichomes and 210 N. A. Khan et al. Cytologia 84(3) pollen grains. Leaf trichomes were noticed to be larger assistance under the Departmental Research Support in 6x (182.33–212.20 µm×38.22–42.40 µm) compared Special Assistance Programme I, II and III, the assis- to the 2x (126.46–148.84 µm×30.34–34.10 µm) (Fig. 2C, tance for strengthening of infrastructure for science and D). Stomata were also recorded to be larger sized in 6x technology programme and Junior Research Fellowship (28.02–32.78 µm×22.42–24.04 µm) compared to the 2x to Nissar Ahmad Khan under CSIR (Award letter no. (23.01–26.47 µm×20.87–22.15 µm) (Fig. 2E, F). Pollen 09/140(0173)/2018-EMR-I). The authors are also thank- diameter was also recorded to be larger in the 6x (90.48– ful to the Head Department of Botany, Punjabi Univer- 98.35 µm×76.96–79.38 µm, Fig. 1G) compared to the 2x sity, Patiala and Sophisticated Instrumentation Centre (82.26–88.84 µm×66.33–70.10 µm, Fig. 1H). of the University for providing necessary lab facilities during the work. 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