Research Article |
Corresponding author: Soom Nath Raina ( soomr@yahoo.com ) Academic editor: Elena Mikhailova
© 2020 Rakesh Kr. Thakur, Vijay Rani Rajpal, Satyawada Rama Rao, Apekshita Singh, Lata Joshi, Pankaj Kaushal, Soom Nath Raina.
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:
Thakur RKr, Rajpal VR, Rao SR, Singh A, Joshi L, Kaushal P, Raina SN (2020) Induction and evaluation of colchitetraploids of two species of Tinospora Miers, 1851. Comparative Cytogenetics 14(2): 211-229. https://doi.org/10.3897/CompCytogen.v14i2.33394
|
Autotetraploidy, both natural and/or induced, has potential for genetic improvement of various crop species including that of medicinal importance. Tinospora cordifolia (Willdenow, 1806) Miers, 1851 ex Hooker et Thomson, 1855 and T. sinensis (Loureiro, 1790) Merrill, 1934 are two diploid species, which are dioecious, deciduous and climbing shrubs with high medicinal importance. Among the three methods used for induction of polyploidy by colchicine treatment, it was cotton swab method which successfully induced the polyploidy in both species. The morphological and cytogenetical features of the synthetic tetraploids were compared with their diploid counterparts. The tetraploids were morphologically distinct from diploid plants. They exhibited larger organs, such as stem, leaves, inflorescence, fruits, flowers and seeds. The tetraploids were characterized by the presence of low quadrivalent frequency and high bivalent average. Unequal distribution of chromosomes at anaphase I was found in 60% cells. The present study provides important information on the superiority of autotetraploids as compared to diploid counterparts in both species.
colchicine treatment, cytogenetics, flow cytometry, morphology, polyploidy, Tinospora cordifolia, Tinospora sinensis
Polyploidy, the presence of more than two sets of chromosomes, has played a pivotal role in the diversity, evolution, genetic improvement and speciation of both wild and cultivated plants (
Genus Tinospora includes 34 species distributed widely throughout the tropical and subtropical parts of Asia, Africa and Australia. Many of them are well known for their medicinal importance (
The present study deals with the induction, for the first time, of autotetraploidy in T. cordifolia and T. sinensis and their morphological and cytogenetical features in comparison to their diploid counterparts.
The stem cuttings and seeds of two plants (one male and one female) of T. cordifolia were collected from Central Institute of Aromatic and Medicinal Plants (CIMAP), Lucknow, India. The two plants (one male and one female) of T. sinensis were collected from surrounding forests of Shivaji University, Kolhapur, Maharashtra, India. The authenticity of the plant material of T. cordifolia and T. sinensis was duly verified by taxonomists at CIMAP and Department of Botany, Shivaji University, respectively. The voucher specimens were deposited in herbarium of Department of Botany, North Eastern Hill University, Shillong, India and accession numbers were obtained. The accession numbers allocated by the herbarium are NEHU-12091, NEHU-12092 for T. cordifolia and NEHU-12093 and NEHU-12094 for T. sinensis.
Colchicine treatment was given to 2600 seeds/seedlings/vegetative buds of T. cordifolia and T. sinensis (Table
Frequency of induced tetraploidy by colchicine treatment in Tinospora cordifolia and T. sinensis.
Species | Seed treatment method | Bud treatment method | Cotton swab method | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Concentration of colchicine (%) | No. of seeds treated | Duration of treatment (in h) | No. of days* | No. of plantlets survived | No. of vegetative buds treated | Duration of treatment (in h) | No. of days* | No. of vegetative buds survived | No. of colchitetra–ploids | No. of seedlings treated | Durati–on of treatment (in h) | No. of days* | No. of seedlings survived | Colchitetraploids** | Percentage colchi–tetraploids | |||
No. | Gender | |||||||||||||||||
M | F | |||||||||||||||||
Tinospora cordifolia | 0.10 | 150 | 12 | 1 | 0 | 100 | 18 | 3 | 0 | 0 | 100 | 12 | 2 | 86 | 0 | – | – | 0 |
100 | 18 | 3 | 76 | 0 | – | – | 0 | |||||||||||
0.15 | 150 | 24 | 2 | 0 | 100 | 18 | 3 | 0 | 0 | 100 | 12 | 2 | 70 | 4 | 3 | 1 | 4 | |
100 | 18 | 3 | 65 | 7 | 5 | 2 | 7 | |||||||||||
100 | 24 | 4 | 23 | 2 | 2 | 0 | 2 | |||||||||||
100 | 30 | 5 | 20 | 0 | 0 | 0 | 0 | |||||||||||
0.20 | – | – | – | – | 100 | 18 | 3 | 0 | 0 | 50 | 12 | 2 | 9 | 1 | 1 | 0 | 2 | |
50 | 18 | 3 | 0 | 0 | 0 | 0 | ||||||||||||
Total | 300 | 0 | 300 | 0 | 0 | 700 | 349 | 14 | 11 | 3 | ||||||||
T. sinensis | 0.10 | 150 | 12 | 1 | 0 | 100 | 18 | 3 | 0 | 0 | 100 | 12 | 2 | 70 | 0 | 0 | 0 | 0 |
100 | 18 | 3 | 63 | 0 | 0 | 0 | 0 | |||||||||||
0.15 | 150 | 24 | 2 | 0 | 100 | 18 | 3 | 0 | 0 | 100 | 12 | 2 | 60 | 3 | 3 | 0 | 3 | |
100 | 18 | 3 | 45 | 5 | 5 | 0 | 5 | |||||||||||
100 | 24 | 4 | 31 | 0 | 0 | 0 | 0 | |||||||||||
100 | 30 | 5 | 15 | 0 | 0 | 0 | 0 | |||||||||||
0.20 | – | – | – | – | 100 | 18 | 3 | 0 | 0 | 50 | 12 | 2 | 0 | 0 | 0 | 0 | 0 | |
50 | 18 | 3 | 0 | 0 | 0 | 0 | 0 | |||||||||||
Total | 300 | 0 | 300 | 0 | 0 | 700 | 284 | 8 | 8 | 0 |
a. Seed treatment: Seeds of T. cordifolia and T. sinensis were immersed in 0.1% and 0.15% aqueous colchicine (Sigma-Aldrich) for 12 h and 24 h. After the treatment, the seeds were thoroughly washed in double distilled water and sown in pots with soil.
b. Vegetative bud treatment: Sterilized cotton balls immersed either in 0.1 or 0.15, or 0.2 % colchicine were placed on the growing buds of T. cordifolia and T. sinensis of ~ one year old rooted stem cuttings for 6 h each for 3 consecutive days.
c. Cotton swab method: Seeds were germinated in pots containing loamy soil and the protruding apical meristem tips between two cotyledonary leaves of ~ 5 days old seedlings were immersed in 0.1 or 0.15, or 0.2 % colchicine with the help of cotton swab soaked in colchicine, for 6 h each for 2, 3, 4 or 5 consecutive days. The colchicine solution was intermittently dropped on the swab to maintain the same colchicine concentration.
The colchicine treatment in all the three methods were carried out in growth chamber maintained at 27 °C, 60% humidity and photoperiod of 12 h duration. Treatment with distilled water of seeds/buds/apical meristem served as control. The pots containing treated and control seedlings/stem cuttings were transferred to glass house one month after treatment.
Stomatal analysis was conducted in 633 plants of T. cordifolia and T. sinensis which survived after treatment and were transferred to glass house. Lower epidermal peel of the control and colchicine treated plants were mounted side by side on the same slide in drops of water and covered with coverslips (24 mm × 24 mm). Stomata cells of the control and the treated plants were observed under a microscope for obtaining data on the comparative size and number of stomata per unit area by Q CAPTURE PRO 5.0 software (QImaging, Surrey, Canada). Initially, the treated plants with distinct increase in size of stomata and low number of stomata per unit area were earmarked as tetraploids (Table
In T. cordifolia, 14 plants which showed distinct increase in stomatal size and 41 randomly chosen treated plants which had no change in the stomata size, as well as 20 control plants after 45 days in glass house were transferred to experimental field containing loamy soil. In T. sinensis, 8 plants with distinct increase in stomatal size and 7 treated plants with no change in stomatal size, along with 10 control plants were transferred to experimental field.
Comparison of average morphological/micro and macroscopic characters of diploid and colchitetraploids of Tinospora cordifolia and T. sinensis.
Characters | Tinospora cordifolia | Tinospora sinensis | ||||||
---|---|---|---|---|---|---|---|---|
Ploidy | Diploid (2n=2x=26) | Colchitetraploid (2n=4x=52) | Diploid (2n=2x=26) | Colchitetraploid(2n=4x=52) | ||||
Gender | Male | Female | Male | Female | Male | Female | Male | Female |
No. of plants | 3 | 3 | 11 | 3 | 3 | 3 | 8 | 0 |
Thickness of stem (cm, circumference, 90 cm above the ground) | 2.45 ± 0.13a | 4.2 ± 0.45a | 5.14 ± 0.88a | 5.56 ± 0.91a | 2.27 ± 0.21 | 3.98 ± 0.33 | 2.43 ± 0.20 | – |
Length of leaf (cm) | 4.81 ± 0.20a | 4.57 ± 0.08a | 7.50 ± 0.42a | 7.32 ± 0.12a | 6.02 ± 0.27a | 6.17 ± 0.29 | 6.50 ± 0.18a | – |
Width of leaf (cm) | 5.67 ± 0.22a | 5.27 ± 0.21a | 7.00 ± 0.40a | 8.0 ± 0.18a | 5.67 ± 0.22a | 6.10 ± 0.41 | 6.55 ± 0.16a | – |
Length of petiole (cm) | 4.28 ± 0.31a | 3.6 ± 0.33a | 3.78 ± 0.71a | 4.16 ± 0.24a | 4.95 ± 0.36 | 5.20 ± 0.21 | 5.40 ± 0.16 | – |
Number of stomata per unit area (/mm2) | 75.14 ± 11.76a | 62.00 ± 4.65a | 45.00 ± 9.21a | 43.66 ± 5.57a | 70.20 ± 10.05a | 62.00 ± 4.46 | 38.00 ± 7.97a | – |
Length of stomata (µm) | 23.82 ± 1.09a | 23.03 ± 0.40a | 33.22 ± 1.13a | 37.88 ± 0.60a | 23.63 ± 1.03a | 23.03 ± 0.40 | 36.95 ± 1.13a | – |
Width of stomata (µm) | 21.10 ± 0.73a | 18.58 ± 0.76a | 26.99 ± 0.85a | 25.97 ± 2.13a | 18.95 ± 1.03a | 16.92 ± 0.35 | 28.15 ± 0.61a | – |
Length of Inflorescence (cm) | 2.97 ± 0.30a | 3.35 ± 0.10a | 5.15 ± 0.19a | 5.9 ± 0.25a | 2.97 ± 0.30a | 3.12 ± 0.30 | 5.15 ± 0.19a | – |
Flowering period | February–March | February–March | February–March | February–March | February– March | February–March | March | – |
Number of fruits per inflorescence | – | 13.5 ± 1.40a | – | 10.3 ± 0.66a | – | – | – | – |
Fruit size(mm) | – | 2.59 ± 0.13 | – | 2.74 ± 0.13 | – | – | – | – |
Seed weight (g/10 seeds) | – | 0.45 ± 0.03a | – | 0.70 ± 0.12a | – | – | – | – |
Pollen grain size (um) | 16.22 ± 0.66a | – | 28.56 ± 1.13a | – | 16.87 ± 0.67a | – | 19.98 ± 0.85a | – |
Pollen stainability % | 90 | – | 60 | – | 90 | – | 60 | – |
Seed germination % | – | 50 | – | 15 | – | – | – | – |
The material for which flow cytometric analysis was carried out was used as a diploid control for colchicine treated (70) plants transferred to experimental field. Healthy young leaves (ca. 2 cm2) each from the sample and internal standard were chopped together with sharp razor blade for isolation of nuclei, stained in extraction and staining buffer (2 ml) containing 100 mM Tris HCl, 85 mM NaCl, 5 mM MgCl2, 0.1% Triton X 100 and 1µg/ml DAPI (4’,6-diamidino-2-phenylindole) pH 7.0. The solution was filtered through 30 µm nylon mesh and analysed in flow cytometer (FCM) (BD FACS Canto 11, BD Biosciences, San Jose, CA) equipped with software CA3 2.14/2004. Minimum 3000 nuclei were analysed per run. Coefficient of variation of G0/G1 peak up to about 4% was only accepted. Each sample was repeated at least thrice for ploidy estimation. Pennisetum squamulatum Fresenius, 1837 (2C = 7.26 pg) (
The data for morphological analysis was taken two years after field transplant of the control and colchicine treated plants. As mentioned before, at the time of colchicine treatment, the seedlings treated with distilled water instead of aqueous colchicine were grown to maturity. They served as control plants. Six control and 14 tetraploid plants of T. cordifolia and six control and 8 tetraploid plants of T. sinensis were evaluated for morphological features (Table
For meiotic studies, young flower buds of appropriate size were fixed at least for 24 h in freshly prepared acetic-ethanol (1:3) mordanted with saturated FeCl3 solution. A saturated solution of FeCl3 was prepared by dissolving substantial amount of FeCl3 in 10 ml of distilled water. A small drop of FeCl3 solution was added to 100 ml of acetic-ethanol mixture. The acetocarmine moderated with FeCl3 increases the intensity of the stain in chromosomes. Before the anthers of appropriate size were used for meiotic analysis, they were hydrolysed in 1N HCl at 60 °C for 10 min and then stained in Feulgen solution. The stained anthers were subsequently squashed in 1% iron-aceto-carmine to observe various stages of male meiosis. Photomicrographs were taken using Olympus CX40 Microscope fitted with 01-GO-3, QIMAGING camera. Twenty five meiocytes each showing metaphase I and anaphase I stages were analysed in each of the two diploid T. cordifolia and the two T. sinensis plants. The same number of meiocytes were analysed in three colchitetraploids each of T. cordifolia and T. sinensis.
For pollen stainability, pollen grains about to dehisce anthers of the diploid and confirmed autotetraploids were separately immersed in a drop of 1:1 ratio of 1% acetocarmine and glycerine on the microslide and covered with a cover slip (22 mm × 22 mm). They were kept as such for 2 h at room temperature. The slide was then observed under the microscope for the number of pollen grains with intense stain and pollen grains with no stain or less stain. Those pollen grains which were intensely stained and circular were taken as fertile pollen, and those with less stain and crinkled shape were considered sterile. Approximately 500 pollen grains both for diploid and autotetraploid plants were analysed for pollen stainability for each species.
The SPSS ver. 22 statistical software (IBM SPSS Amos™ 22; IBM Corp. Released 2013) was used to assess the variation of phenotypic traits within and between the populations of diploid and colchitetraploid using t-test and one-way ANOVA.
Thirteen hundred seeds/seedlings/vegetative buds each of T. cordifolia and T. sinensis were treated with three different concentrations (0.1, 0.15 and 0.2%) of aqueous colchicine for 6 or 12 h each for 2, 3, 4 or 5 days (Table
Fourteen plants in T. cordifolia and 8 plants in T. sinensis which were given colchicine treatment, and which exhibited distinct increase in the size of stomata (Figs
Comparison between diploid (left) and colchitetraploid (right) T. cordifolia for a, b stomata c leaf d seed e male inflorescence f female inflorescence g, h pollen and i fruit. Scale bars: 10 µm.
The characteristic feature of all the apical meristems of buds/seedlings treated with colchicine was stunted growth in initial stages and leathery thicker first leaves. After first 3–4 leaves the subsequent leaves in the seedlings showed either normal or thicker, darker and larger leaves. The plants with latter condition on further study were found to be tetraploids. Following cotton swab method, the same morphological condition (normal or thicker, darker and larger leaves) as above was observed in all the colchicine concentrations and duration of treatment.
The colchitetraploids compared to diploid plants were morphologically distinct in several characters (Figs
Flow cytometric panels of T. cordifolia a diploid b diploid and colchitetraploid; T. sinensis c diploid and d colchitetraploid. left panel is reference sample (Pennisetum squamulatum).
Metaphase I and anaphase I in a, b diploid (2n=2x=26) and c–g tetraploid (2n=4x=52) T. cordifolia. Note a 13 II and b 13:13 distribution of chromosomes at anaphase I. Note quadrivalents, trivalents, bivalents and univalents in c (5IV+13II+6I) d (10IV+1III+3II+3I) and e–g 26:26 distribution of chromosomes at anaphase I. Scale bar: 10 µm.
The data pertaining to meiotic analysis of diploids and colchitetraploids of two species T. cordifolia and T. sinensis is given in Tables
Average number and range of chromosome associations at metaphase I in the diploid (2x) and colchitetraploids (4x) Tinospora cordifolia and T. sinensis.
Species | Ploidy | No. of cells analysed | Quadrivalents | Trivalents | Bivalents | Univalents |
---|---|---|---|---|---|---|
Average number and the range | Average number and the range | Average number and the range | Average number and the range | |||
Tinospora cordifolia | 2x | 25 | 12.44; 10–13 | 1.12; 0–6 | ||
4x | 25 | 5.88; 0–10 | 0.16; 0–1 | 12.48; 5–24 | 4.16; 0–16 | |
T. sinensis | 2x | 25 | 12.24; 10–13 | 1.52; 0–6 | ||
4x | 25 | 6.32; 3–10 | 0.24; 0–1 | 11.52; 5–20 | 3.28; 0–7 |
Anaphase I distribution in diploid and colchitetraploids of Tinospora cordifolia and T. sinensis.
Species | Ploidy | No. of Cells analysed | Chromosome distribution at anaphase I | No of cells (%) |
---|---|---|---|---|
Tinospora cordifolia | 2x | 25 | 13:13 | 25(100) |
4x | 25 | 26:26 | 10(40) | |
27:25 | 5(20) | |||
28:24 | 5(20) | |||
24:4U:24 | 5(20) | |||
T. sinensis | 2x | 25 | 13:13 | 25(100) |
4x | 25 | 26:26 | 10(40) | |
27:25 | 5(20) | |||
28:24 | 5(20) | |||
26:2U:24 | 5(20) |
Tinospora cordifolia
Diploid (2n = 2x = 26): In majority of the PMCs observed at metaphase I, thirteen bivalents were regularly observed to occur. Few cells had a mix of both bivalents and univalents. On an average the PMC had 12.44 bivalents and 1.12 univalents. All the cells analysed at anaphase I were characterized by equal distribution (13:13) of chromosomes at two poles.
Colchitetraploid (2n = 4x = 52): The PMCs were characterized by the presence of quadrivalents, trivalents, bivalents and univalents at metaphase I. On an average per cell each PMC had 5.88 IV + 0.16 III+ 12.48 II and 4.16 I. Equal (26:26) distribution of chromosomes at anaphase I was found only in 40% of cells followed by unequal [27:25, 28:24 and 24:4U (Univalents):24] distribution of chromosomes in 60% cells.
Tinospora sinensis
Diploid (2n = 2x = 26): Most of the PMCs observed at metaphase I had thirteen bivalents. A few cells had both bivalents and univalents. The average frequency per cell of chromosome associations was 12.24 II+1.52 I. The presence of univalents in the diploid T. cordifolia and T. sinensis could be due to precocious separation of rod bivalents (
Colchitetraploid (2n = 4x = 52): Most of the PMCs had a mix of quadrivalents, trivalents, bivalents and univalents at metaphase I. On an average, each PMC had 6.32 IV + 0.24 III+ 11.52 II and 3.28 I. Equal distribution (26:26) of chromosomes at anaphase I was recorded only in 40% of cells. The remaining 60% of the PMCs analysed had unequal (27:25, 28:24 and 26:2U:24) distribution of chromosomes.
Metaphase I and anaphase I in a, b diploid (2n=2x=26) and c–f tetraploid (2n=4x=52) T. sinensis. Note a 13 II and b 13:13 distribution of chromosomes at anaphase I. Note quadrivalents, trivalents, bivalents and univalents in c (5IV+1III+10II+9I) d (10IV+5II+2I) and e, f 26:26 distribution of chromosomes at anaphase I. Scale bar: 10 µm.
Among several protocols that have been developed for polyploidy induction, it is the colchicine treatment which has been the most successful procedure for last several decades. However, the induction of polyploidy by colchicine has been most successful in annuals rather than in perennial plants. There are hardly few among vast number of papers published on polyploid induction wherein successful induction in trees, shrubs and perennial climbers such as dioecious Tinospora cordifolia and T. sinensis has been reported (
The success in induction of polyploidy in plants depends on many factors such as, treatment method, concentration of colchicine solution and duration of the treatment. One could see on perusal of earlier literature that optimum colchicine concentration and duration of treatment differs from one species to other (
There is a body of evidence to support that autopolyploidization leads to enhancement of morphological parameters (
The reduction in seed fertility in autotetraploids of T. cordifolia is of little consequence since the species is vegetatively propagated by stem cuttings. The multiplication through seed is rare almost non-existent. The increase in fruit size in autotetraploids, could be due to polyploidy induction and (or) reduce fruit load per plant. What is most important is that it is vegetative organs especially, stem and leaves, and not seeds which are medicinally important. Due to larger vegetative organs such as stem and leaves, the overall secondary metabolites production per unit area will substantially improve in autotetraploids of T. cordifolia and T. sinensis. Further, autotetraploids may positively affect the tolerance to some stresses such as nutrient deficiency, water deficit, temperature, drought, pests and pathogens (
In autotetraploids due to occurrence of sets of 4 homologous chromosomes instead of 2 in diploids, all chromosome associations are expected to be of quadrivalent configuration. That is not, however, always the case in neoautotetraploids. The average number of quadrivalents per cell in T. cordifolia and T. sinensis was 5.88 and 6.32, respectively. The average number of bivalents in T. cordifolia (12.48) and T. sinensis (11.52) outnumbered the frequency of quadrivalents in the two tetraploid species. Such behaviour as in other neoautotetraploids, could be attributed to small size of chromosomes, cryptic structural hybridity and genetic control and (or) points of pairing initiation (
In conclusion, the present results demonstrate that cotton swab method was the best method for inducing polyploidy in the diploid Tinospora cordifolia and T. sinensis. Autopolyploidy of other Tinospora species with medicinal potential may also be induced by this method. The autotetraploids of both species have many morphological features which would establish them as increasingly improved plant materials. The tetraploids can also be utilized for the production of triploids which usually offer heterotic advantage over its parents.
All authors declare that there is no conflict of interests exists. All the authors have contributed substantially to the manuscript and approved the submission.
The authors would like to thank National Medicinal Plants Board (NMPB), Ministry of AYUSH, Government of India for financial support. We also thank the anonymous reviewers and subject editor for comments on the ms and helpful suggestions.