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1

In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana

100%
Rathore N. S., Rathore N., Shekhawat N. S.
Acta Physiologiae Plantarum
|
2013
|
tom 35
|
nr 09
We developed a micropropagation protocol for Cleome gynandra, a C4 model plant with medicinal importance. Surface-sterilized nodal segments obtained from 1 to 2-month-old field grown plant were used as explants for culture establishment and plant regeneration. Multiple shoots differentiated through bud breaking on Murashige and Skoog (MS) medium with different concentrations of benzyladenine (BA) and kinetin (Kin). The optimum shoot differentiation occurred on medium with 1.5 mg l-1 BA. Out of various concentrations and combinations of cytokinins and auxins, MS medium containing 0.5 mg l-1 BA and 0.1 mg l-1 IAA (indole-3-acetic acid) was found best for shoot multiplication. However, the differentiated shoots exhibited hyperhydration, leaf curling and early leaf fall during subculturing. To overcome these problems, regenerated shoots were transferred to the modified MS medium with reduced nitrates (825 mg l-1 NH4NO3 and 950 mg l-1 KNO3) and 100 mg l-1 (NH4)2SO4. The micropropagated shoots were rooted (i) in vitro on one-fourth strength of MS salts with 0.25 mg l-1 each of IBA (indole-3 butyric acid) and NOA (2-naphthoxyacetic acid) ? 100 mg l-1 activated charcoal, and (ii) ex vitro, by treating the shoot base(s) with 200 mg l-1 of IBA for 3 min and transferred to soilrite moistened with one-fourth strength of MS macro salts in culture bottles. The plants were hardened in the greenhouse with 85 % survival rate. Micromorphological studies of the plants were conducted during hardening with reference to development and changes in vein spacing, glandular trichome and stomata. In comparison to leaves under in vitro condition, higher density of veins and glandular trichomes was observed in the leaves of hardened plants. In addition, stomata became functional during hardening which were non-functional under in vitro condition.
2

In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs

100%
Lodha D., Patel A. K., Rai M. K., Shekhawat N. S.
Acta Physiologiae Plantarum
|
2014
|
tom 36
|
nr 11
Ephedra foliata, (Gymnosperm) is a pharmaceutically important plant known for the last 5,000 years and has a number of medicinal properties. We describe here for the first time, a method for plant regeneration from callus established from axillary buds as explant, with the aim of optimizing alkaloids production in vitro. The tissue cultures initiated are being maintained for the last 3 years on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 0.5 mg l⁻¹ each of 2, 4-D and Kin. Maintained callus cultures exhibited regeneration potential and maximum number (23.5 ± 0.44 shoots per culture vessel) of shoots with an average height (4.94 ± 0.23 cm) was achieved on MS medium containing combination of 0.25 mg l⁻¹ each of Kin, BA and 0.1 mg l⁻¹ of NAA. About 84.9 % regenerated shoots were rooted under ex vitro conditions on Soilrite®, if their base was treated with 500 mg l⁻¹ of IBA for 5 min. The rooted plantlets were successfully acclimatized under greenhouse conditions with ≈80 % survival rate. We analyzed alkaloid contents of tissue culture raised plants/callus as affected by the different concentrations and combination of two additives, i.e., L-phenylalanine and IBA. The alkaloid production was higher in the in vitro grown cultures than field-grown plants. Highest alkaloid content was recorded in callus culture on M5 medium having 0.5 mg l⁻¹ each of 2, 4-D and Kin, 100 mg l⁻¹ L-phenylalanine and 5 mg l⁻¹ IBA. The present protocol may be applicable for the large-scale cultivation of E. foliata and selection of cell line having higher secondary metabolite contents of this pharmaceutically important threatened plant species.
3

Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis

88%
Rathore N. S., Rai M. K., Phulwaria M., Rathore N., Shekhawat N. S.
Acta Physiologiae Plantarum
|
2014
|
tom 36
|
nr 02
A newly developed and novel DNA marker technique, i.e. start codon targeted (SCoT) polymorphic markers that target plant gene regions were used to assess genetic stability of in vitro raised plants of Cleome gynandra multiplied by enhanced bud proliferation from nodal segments. Seven randomly selected micropropagated plants, following at least 2 months of growth in the greenhouse along with mother plant were subjected to molecular analysis. Of 24 primers screened, 15 primers produced unambiguous and reproducible bands. All 15 primers generated a total of 65 fragments, with a mean of 4.3 ranging 2–7 per primer. No polymorphism was detected in regenerated plants and the mother plant, revealing the genetic fidelity of the in vitro raised plantlets. To verify the results of SCoT analysis, random amplified polymorphic DNA (RAPD) markers were also used for the assessment of genetic fidelity of tissue culture raised plants. The monomorphic banding pattern in micropropagated plants and the mother plant obtained from SCoT and RAPD analysis confirms the genetic stability of the in vitro raised plants and demonstrates the reliability of our micropropagation system for C. gynandra, an important C₄ plant.
4

An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers

88%
Phulwaria M., Patel A. K., Rathore J. S., Ram K., Shekhawat N. S.
Acta Physiologiae Plantarum
|
2014
|
tom 36
|
nr 05
An improved micropropagation method has been developed for Salvadora oleoides, a valuable tree species of alkaline and arid regions. Nodal explant obtained from a mature tree (30- to 35-year-old) responded optimally (80.0 %) on BAP (2.0 mg l⁻¹) and produced (4.56 ± 0.52) shoots. Shoots were further multiplied by subculturing the in vitro excised shoots and transferring them to MS medium containing either BAP (0.0–2.0 mg l⁻¹) alone or in combination with lower concentrations of an auxin (IAA or NAA 0.05–0.4 mg l⁻¹). Among all the PGRs combination tested, MS medium supplemented with BAP (0.5 mg l⁻¹) and IAA (0.1 mg l⁻¹) formed the maximum number of shoots (68.40 ± 2.74 per culture bottle) with an average height (6.59 ± 0.30 cm), after 6 weeks of culture. Rooting in regenerated shoots was achieved by ex vitro methods and about 92.5 % of shoots were rooted with 5.25 ± 0.64 roots per shoot and an average length of 2.76 ± 0.53 cm after 3 weeks of incubation in the green house. More than (80 %) of hardened plantlets survived in the field conditions. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD and ISSR. Of the 10 RAPD primers finally selected, a total of 42 bands (out of 43) were monomorphic and one polymorphic, whereas from 10 ISSR primers selected, all the 43 bands were monomorphic revealing a high level of genetic homogeneity in the regenerated plants and the donor plant. In the present investigation, we achieved significantly more number of shoots during multiplication, which are higher than all previous reports and further evaluated the genetic fidelity of protocol for the first time in S. oleoides, which concludes the clonal (true-to-type) nature of micropropagated plantlets.
5

An improved micropropagation of Terminalia bellirica from nodal explants of mature tree

76%
Phulwaria M., Rai M. K., Harish, Gupta A. K., Ram K., Shekhawat N. S.
Acta Physiologiae Plantarum
|
2012
|
tom 34
|
nr 1
An efficient and improved in vitro propagation method has been developed for Terminalia bellirica, a medicinally important tree from nodal explants of 10-yearold mature tree. Shoot multiplication was influenced not only by cytokinin types, their concentrations and their interaction with auxin but also by successive transfer of mother explants for different passages, subculture of excised shoots on fresh medium and different medium composition. MS medium containing 2.22 µM BAP was found to be the best for shoot multiplication in a single step. After excision of newly formed shoots, mother explants successively transferred to the same medium produced maximum shoots per explant after IV passage. Further enhancement in morphogenetic response occurred when excised shoot clumps (2–3 shoots) were subcultured on MS medium supplemented with 2.22 µM BAP, 1.16 µM Kn and 0.57 µM IAA. Half-strength MS medium supplemented with 24.60 µM IBA and 100 mg l⁻¹ AC was most effective for rooting of the shoots. To reduce labor, cost and time, an experiment on ex vitro rooting was also carried out and it was observed that highest percent shoots rooted ex vitro when treated with 2,460 µM IBA for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. In comparison to plantlets developed from in vitro rooted, percent survival of plants those rooted ex vitro was significantly higher. Use of ex vitro rooting technique for plant production serves as a more economical option; therefore, present method can be used for large-scale commercial production of this medicinally important tree.
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