Research Article |
Corresponding author: Rakesh Kumar Chahota ( rkchahota@yahoo.com ) Academic editor: Ilya Gavrilov-Zimin
© 2015 Savita Rani, Tilak Raj Sharma, Rakesh Kapila, Rakesh Kumar Chahota.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rani S, Sharma TR, Kapila R, Chahota RK (2015) Identification of new cytotypes of Valeriana jatamansi Jones, 1970 (Valerianaceae) from North-Western Himalayan region of India. Comparative Cytogenetics 9(4): 499-512. https://doi.org/10.3897/CompCytogen.v9i4.8875
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Valeriana jatamansi, a medicinally important species of the family Valerianaceae, has been cytologically studied in different geographical areas of North-Western Himalayan region of India. The tetraploid cytotype with chromosome numbers 2n=32 is in conformity with the earlier reports of the species from different parts of the world. An octoploid cytotype (2n=64) makes a new addition for the species on a worldwide basis, whereas the diploid cytotype (2n=16) is new to India have been reported for the first time in India. These cytotypes (2n=16, 32, 64) show significant variations with respect to morphology as well as geographical distribution in the Western Indian Himalayas. Further, anomalous populations have been marked with meiotic abnormalities in the form of cytomixis, chromosomal stickiness, unoriented bivalents, formation of laggards and bridges resulting in abnormal microsporogenesis, and production of heterogeneous-sized fertile pollen grains along with reduced pollen fertility.
Valeriana jatamansi , cytotypes, meiotic abnormalities, morphovariants, western Himalayas
The genus Valeriana Linnaeus, 1753 belongs to the family Valerianaceae which comprises 250 species of perennial herbs (
Valeriana jatamansi Jones (= Valeriana wallichii de Candolle, 1830) popularly known as Indian valeriana (English), Mushkibala (Kashmiri/ Hindi), Sugandhwala or Tagar (Sanskrit), grows wild in the temperate Himalayan region between 1000 and 3000 m altitude. The species generally grows on sloppy, moist places, damp woods, ditches and along the streams. The species grows particularly in the understory of Quercus leucotrichophora Camus, 1938 – Pinus roxburghii Sargent, 1897 mixed forests and on grassy habitats of Himalayas. Its occurrence can be observed in different geographical areas and the species possesses diverse morphological and genetic features, affecting its levels of active ingredients. V. jatamansi is a perennial herb with thick root stocks, horizontal thick descending fibers with pubescent stem, radical leaves often 1-3 cm in diameter, deeply cordate and usually acute toothed margins. Flowers white, stamens- 3, ovary -3 celled, stigma shortly 2-3 fid and fruits oblong lanceolate and compressed crowned by persistent pappus calyx. The flowering and fruiting time for the species is March–June. The mode of propagation is both sexual through seeds and asexual through rhizome.
The species is being exploited for its roots and rhizomes which contain valepotriates (
Therefore, keeping in view the economic importance, threatened status of the species, and cytological variability within the species, at present cytomorphological studies have been carried out on a population basis from different areas of the North-Western Himalayas. The present study also discusses the impact of cytomixis on meiotic behavior and reduced pollen fertility and formation of heterogeneous-sized pollen grains in the species.
Material. Material for cytological studies in the form of buds was collected from different parts of the North-Western Himalayas. The propagating material of these plants was also collected and planted in the experimental fields of Chaudhary Sarwan Kumar Himachal Pradesh Agricultural University, Palampur, India in a Randomized Complete Block design with two replications. The plant to plant distance was kept at 5 cm while row to row distance was kept at 50 cm.
Different qualitative morphometric characters were studied for each cytotype to have proper insight on morphological variation present in these cytotypes. For stomatal studies, mature leaves were treated with 10% aqueous solution of potassium hydroxide (KOH) at room temperature for 10–15 min and then epidermal peels so obtained were stained with 10% saffanin in 90% ethanol. In order to reveal the significant difference in the stomata and pollen grain size of diploid, tetraploid and octaploid cytotypes, the t-test was been performed.
For meiotic studies, flower buds were collected from plants growing under natural conditions from selected areas of the Western Himalayas. These flower buds were collected from 15 randomly selected plants of each population and fixed in Carnoy’s fixative (6:3:1 ethanol/chloroform/acetic acid v/v/v) for 24 h. Flower buds were washed and preserved in 70% ethanol at 4 °C until used. Smears of appropriate-sized flower buds were made, using the tandard acetocarmine technique (
Detailed meiotic studies were carried out on 22 populations of V. jatamansi collected from different localities with altitude ranging from 764 to 3647 m above mean sea level in the North-Western Himalayan region of India. The data regarding locality with altitude, latitude and longitude, present meiotic chromosome number, ploidy level and meiotic course of the presently worked out populations have been presented in Table
Information about, locality, latitude and longitude, altitude, meiotic chromosome number, ploidy level and meiotic course of Valeriana jatamansi.
S. No. | Locality with latitude and longitude, altitude | Present meiotic chromosome number (2n) | Ploidy level | Meiotic in meters |
---|---|---|---|---|
District Chamba (Himachal Pradesh) | ||||
P-1 | Bhali mata/ 32°37'N, 76°0’E/1,900 | 32 | 4x | Abnormal |
P-2 | Salooni/32°43'N,76°03'E/1,900 | 16 | 2x | Normal |
P-3 | Chamba/ 32°33'N, 76°07'E/ 1,200 | 32 | 4x | Abnormal |
P-4 | Leg Valley/31°58'N, 77°06'E/ 1,720 | 32 | 4x | Normal |
P-5 | Tisa/32°32'N, 76°08'E/ 1,220 | 32 | 4x | Normal |
P-6 | Dehgram/32°41'N, 76°08'E/ 2,165 | 32 | 4x | Abnormal |
District Shimla (Himachal Pradesh) | ||||
P-7 | Kandi/32°36'N, 76°02'E/ 854 | 32 | 4x | Normal |
P-8 | Rampur/ 31°58'N,77°06'E/ 1,350 | 16 | 2x | Normal |
P-9 | Pander/ 31°26'N,77°03'E/ 2,236 | 32 | 4x | Normal |
P-10 | Shimla I/ 31°6'N, 77°10'E/ 2,202 | 32 | 4x | Abnormal |
P-11 | Shimla II/31°18'N, 77°20'E/ 1,820 | 32 | 4x | Normal |
District Mandi (Himachal Pradesh) | ||||
P-12 | Rewalsar/31°38'N, 76°50'E/ 1,360 | 32 | 4x | Normal |
P-13 | Rakni/ 31°24'N, 77°07’E/1,649 | 32 | 4x | Normal |
P-14 | Prashar / 31°45'N, 77°6'E / 2,200 | 32 | 4x | Abnormal |
P-15 | Mandi I/ 31°42'N, 76°51'E/ 764 | 32 | 4x | Normal |
P-16 | Mandi II/31°31'N, 76°59'E/ 945 | 32 | 4x | Abnormal |
District Kullu (Himachal Pradesh) | ||||
P-17 | Kullu I/ 32°09'N,77°02'E/ 3,647 | 64 | 8x | Abnormal |
P-18 | Sojha/31°42'N, 77°54'E/ 2,692 | 32 | 4x | Normal |
P-19 | Jalori Pass/ 31°32'N, 77°22'E/ 3,134 | 64 | 8x | Normal |
P-20 | Kullu II/ 32°08'N, 77°04'E/ 2,734 | 32 | 4x | Abnormal |
P-21 | Parvati valley/32°01'N, 77°20'E/1,640 | 32 | 4x | Normal |
P-22 | Manikaran /32°08'N, 77°26'E/ 1,820 | 32 | 4x | Normal |
Morphological variation was assessed on 24 vegetative and reproductive characters of different cytotypes of V. jatamansi (Table
Detailed morphological comparison of three cytotypes of Valeriana jatamansi.
No. | Morphological character | Diploid (2n=16) | Tetraploid (2n=32) | Octaploid (2n=64) |
---|---|---|---|---|
1 | Distribution | Uncommon | Most Common | Uncommon |
2 | Habit | Small sized herb | Medium sized herb | Medium sized herb |
3 | Habitat | Found under shade of Pinus roxburghii | Found in the moist, shady and humus rich places | Found on the slopes in forests |
4 | Plant height (cm) | 14.42±2.84 | 19.46±2.34 | 28.70±1.69 |
Stem | ||||
5 | Surface | Non-glabrous | Non-glabrous | Glabrous |
6 | No. of hairs/ mm2 | - | 12.6±1.3 | |
7 | Length of hairs (cm) | - | - | 2.3±0.38 |
Basal Leaves | ||||
8 | Number of leaves / plant | 22.66±7.76 | 20.77±4.55 | 47.50±10.60 |
9 | Shape | Ovate | Lanceolate | Lanceolate |
10 | Size (cm) | 4.53±0.95×5.83±1.05 | 5.56±0.86×6.98±1.01 | 7.65±0.63×7.26±1.36 |
11 | Surface | Non-glabrous | Non-glabrous | Glabrous |
12 | Leaves Margin | Toothed | Entire | Wavy |
Cauline Leaves | ||||
13 | Number of leaves / plant | 4.00±1.00 | 11.41±2.18 | 21±2.41 |
14 | Size (cm) | 4.23±0.70×4.63±0.64 | 6.53±0.59×7.40±0.74 | 7.41±2.18×21.00±1.41 |
15 | Surface | Non-glabrous | Non-glabrous | Glabrous |
16 | Leaves Margin | Toothed | Entire | Entire |
Stomata | ||||
17 | Size (µm) | 21.66±1.56×18.3±0.36 | 23.82±0.76×19.58±0.67 | 26.74±0.35×20.55±0.48 |
18 | Stomatal frequency on upper/ lower surface of leaf (mm2) | 5.41±0.45/3.25±0.36 | 5.48±0.48/7.45±0.45 | 3.25±0.36/3.78±0.11 |
19 | Stomatal index of upper/ lower surface of leaf (µm) | 22.28/12.45 | 23.45/13.24 | 24.50/14.32 |
Inflorescence | ||||
20 | Length (cm) | 9.9±1.8 | 10.2±3.5 | 10.7±1.8 |
21 | Diameter (cm) | 2.69±0.23 | 2.39±0.28 | 2.34±0.30 |
22 | Number of flowers/plant | 9.5±1.58 | 11±2.16 | 12.2±1.0 |
Flower | ||||
23 | Petal size (mm) | 4.77±0.59 | 4.06±0.8 | 4.15±0.91 |
24 | Sepal size (mm) | 3.01±0.39 | 2.99±0.40 | 3.38±0.5 |
Pollen size | ||||
25 | Pollen size (µm) | 37.24±1.15×39.06±0.79 | 41.05 ± 0.52×44.55 ± 0.59 | 45.24±0.57×46.19±0.38 |
Based on x=8 (
a (Diploid cytotype) PMC at Anaphase-I showing 8:8 distribution of chromosomes b (Diploid cytotype) PMC at Metaphase-I showing 8II bivalents c (Tetraploid cytotype) PMC at Anaphase-I showing 16:16 distribution of chromosomes d PMC at Metaphase-II showing 16:16 distribution of chromosome e (Octaploid cytotype) PMC at Anaphase-I showing 32:32 distribution of chromosomes f (Diploid cytotype) Three PMCs showing transfer of chromatin material (arrowed) g A group of PMCs (arrowed) involved in cytomixis at telophase-I h (Tetraploid cytotype) Size difference and direct connection between two PMCs during chromatin material (arrowed) i PMC at metaphase-I showing unoriented bivalent (arrowed) j PMC at metaphase-I showing chromatin stickiness k PMC at anaphase-I showing chromatin bridges (arrowed) l PMC at anaphase-II showing unequal distribution of chromosomes (arrowed) m–n PMC at telophase-I showing chromatin chromosomal laggards (arrowed) o Diad p Triad q–r Tetrad with micronuclei (arrowed) s Polyad t–v Fertile, sterile and Heterogeneous sized fertile pollen grains. Bar = 10 µm.
A perusal of cytological literature reveals that majority of the species in the genus have been worked out showing 2n=14, 16, 28, 32, 42, 48, 56, 60, 64, 72, 80, 90 and 96. The genus is dibasic with x=7, 8 as has been suggested by
In the North-Western Himalayas, the distribution pattern of euploid cytotypes shows definite relation to altitudinal variations (Table
The meiotic course was found to be abnormal in eight populations (Table
Data on cytomixis, abnormal meiotic course and pollen fertility in Valeriana jatamansi.
Cytomixis at Meiosis-I/ Meiosis II | Meiotic course showing PMCs with | Pollen grains | |||||
---|---|---|---|---|---|---|---|
Population number | % of PMCs involved | Number of PMCs Involved | Chromosomal stickiness at M-I (%) | Bridges at Meiosis-I/ Meiosis-II (%) | Laggards at Meiosis-I/ Meiosis-II (%) | Micronuclei at T-II (%) | Fertility (%) |
PP-1 | 8.33 (10/120)/ | 2-3 | 4.00 (4/100) | 5.88 (6/102)/ | 2.86 (2/70)/ | 17.47 (18/103) | 66.23 |
7.07 (7/99) | - | 2.63 (2/76) | |||||
PP-3 | 5.78 (7/121)/ | 2-3 | - | 3.80 (4/105)/ | 2.70 (2/74)/ | 18.18 (16/88) | 65.07 |
- | 2.66 (2/75) | ||||||
PP-6 | 4.34 (5/115)/ | 2-3 | - | 2.40 (3/125)/ | 3.30 (4/121)/ | 5.78 (7/121) | 75.78 |
4.59 (4/87) | 1.73 (2/115) | - | |||||
PP-10 | - | - | 4.87 (6/123) | 2.27 (3/132)/ | - | 7.57 (10/132) | 70.32 |
2.60 (3/115) | |||||||
PP-14 | 5.88 (6/102)/ | 2-4 | 9.75 (12/123) | 5.35 (6/112)/ | 5.69 (7/123)/ | 4.46 (5/112) | 71.65 |
6.25 (7/112) | 3.92 (4/102) | 4.83 (6/124) | |||||
PP-16 | - | - | 7.33 (8/109) | - | 3.53 (4/113)/ | - | 77.87 |
- | |||||||
PP-17 | - | - | 8.33 (11/132) | 2.52 (3/119)/ | - | 5.60(7/125) | 79.67 |
- | |||||||
PP-20 | 9.00 (9/100)/ | 2-4 | - | - | 5.73 (7/122)/ | 4.50(5/111) | 79.65 |
8.00 (10/125) | - |
Cytomixis in these populations resulted in the production of hyper- and hypo-ploid PMCs. According to
During present investigations, single or multiple bridges (Fig.
Data on abnormal microsporogenesis in different cytotypes of Valeriana jatamansi marked with abnormal meiosis.
Microsporogenesis Accessions | ||||||||
---|---|---|---|---|---|---|---|---|
Dyads | Triads | Tetrads | Polyads | |||||
WMN | WN | WMN | WN | WMN | WN | WMN | WN | |
PP-1 | 0.99 (1/101) | 0.99 (1/101) | 1.98 (2/101) | 71.28 (72/101) | 21.78 (22/101) | 1.98 (2/101) | - | |
PP-3 | - | - | 2.04 (2/98) | - | 76.53 (75/98) | 21.42 (21/98) | - | - |
PP-6 | 0.96 (1/104) | - | 0.96 (1/104) | - | 70.19 (73/104) | 26.92 (28/104) | 0.96 (1/104) | - |
PP-10 | 2.04 (2/98) | 1.02 (1/98) | 2.04 (2/98) | - | 88.77 (87/98) | 6.12 (6/98) | - | - |
PP-14 | 2.97 (3/101) | 0.99 (1/101) | 3.96 (4/101) | 0.99 (1/101) | 79.20 (80/101) | 9.90 (10/101) | 0.99 (1/101) | 0.99 (1/101) |
PP-16 | - | 0.94 (1/106) | 2.83 (3/106) | 0.94 (1/106) | 84.90 (90/106) | 10.37 (11/106) | - | - |
PP-17 | - | - | 0.98 (1/102) | 0.98 (1/102) | 89.21 (91/102) | 8.82 (9/102) | - | - |
PP-20 | 1.86 (2/107) | 0.93 (1/107) | 3.73 (4/107) | 0.93 (1/107) | 76.63 (82/107) | 14.01 (15/107) | 0.93 (1/107) | 0.93 (1/107) |
The authors are grateful to the Science and Engineering Research Board (SERB) under young Scientist Fellowship Scheme [Registration No. SERB/LS-527/2013] to Dr. Savita Rani. Thanks are also due to the Head, Department of Agricultural Biotechnology, CSK, Himachal Pradesh Agricultural University, Palampur, for necessary laboratory facilities.