Mahendra Phulwaria

The role of abscisic acid in plant tissue culture: a review of recent progress

Abscisic acid (ABA) plays a significant role in the regulation of many physiological processes of plants. It is often used in tissue culture systems to promote somatic embryogenesis and enhance somatic embryo quality by increasing desiccation tolerance and preventing precocious germination. ABA is also employed to induce somatic embryos to enter a quiescent state in plant tissue culture systems and during synthetic seed research. Application of exogenous ABA improves in vitro conservation and the adaptive response of plant cell and tissues to various environmental stresses. ABA can act as anti-transpirant during the acclimatization of tissue culture-raised plantlets and reduces relative water loss of leaves during the ex vitro transfer of plantlets even when non-functional stomata are present. This review focuses on the possible roles of ABA in plant tissue culture and recent developments in this area.

High frequency plantlet regeneration from nodal segment culture of female Momordica dioica (Roxb.)

An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L−1 6-Benzylaminopurine (BAP) + 0.1 mg L−1 Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L−1 BAP + 0.1 mg L−1 IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L−1 BAP + 0.1 mg L−1 IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L−1 Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.

A genetically stable rooting protocol for propagating a threatened medicinal plant—Celastrus paniculatus

Nodal segments, obtained from 12 years-old mature plant, were used as explants for in vitro propagation of Celastrus paniculatus, an important medicinal plant of India. Shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoot clumps on MS medium supplemented with various concentrations of BAP alone or in combination with auxin (IAA or NAA). In vitro raised shoots were rooted under ex vitro condition. Genetic fidelity of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD).

Conservation genetics of endangered medicinal plant Commiphora wightii in Indian Thar Desert.

To ascertain the conservation priorities and strategies for Commiphora wightii, an endangered medicinal plant of Indian Thar Desert, genetic diversity was estimated within and among different populations. The total of 155 amplification products were scored using ten each of RAPD and ISSR primers, exhibiting an overall 86.72% polymorphism across 45 individuals representing eight populations. The cumulative data of two markers were used to compute pair-wise distances. The Neighbor-Joining tree revealed high genetic differentiation among populations except Kiradu population. Neis gene diversity (h) ranged between 0.082 and 0.193 with total diversity at species level is 0.294. Shannons information index (I) ranged between 0.118 and 0.275 with an overall diversity of 0.439. Analysis of molecular variance showed more diversity among population level (56.65%) than at within population level (43.35%). The low gene flow value (Nm=0.349) and high coefficient of genetic differentiation (GST=0.589) and high fixation index (FST=0.566) demonstrated elevated genetic differentiation among the population and can be predicted that these populations are not in Hardy-Weinberg proportions. Principal Co-ordinate Analysis confirms that Akal population has become phylogenetically more distinct and less diverse than the rest of the samples. Mantels test revealed no correlation between genetic and geographical distances of populations (R(2)=0.122). Overall highest diversity was observed in the population of Machiya Safari Park and Kiradu, while lowest in Akal population, later may constitute an evolutionary significant unit, having merit for special management.

Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment.

Isolation of intact and pure genomic DNA (gDNA) is essential for many molecular biology applications. It is difficult to isolate pure DNA from mature trees of hot and dry desert regions because of the accumulation of high level of polysaccharides, phenolic compounds, tannins etc. We hereby report the standardized protocol for the isolation and purification of gDNA from seven ecologically and medically important tree species of Combretaceae viz. Anogeissus (Anogeissus sericea var. nummularia, Anogeissus pendula, and Anogeissus latifolia) and Terminalia (Terminalia arjuna, Terminalia bellirica, Terminalia catappa and Terminalia chebula). This method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin-column. gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is also critical. The quality of DNA was determined by the A(260)/A(280) absorbance ratio. gDNA was also observed for its intactness by running on 0.8% agarose gel. The suitability of extracted DNA for PCR was tested by amplification with RAPD primers, which was successful. Further, rbcLa (barcoding gene) was amplified and sequenced to check the quality of extracted gDNA for its downstream applications.

Micropropagation of Salvadora oleoides—An Oil Yielding Tree of Arid Forests

Salvadora oleoides is an ecologically important multipurpose tree of the arid forest that occurs in saline areas of northwest India. The seed of this plant yields non-edible commercially usable oil. Poor seed germination, low seed viability, and increasing industrialization are some of the constant factors which significantly affect the status of the natural population of this plant. Therefore, there is a great need to develop an efficient propagation system using the tissue culture technique. In the present communication, we demonstrate the development of an in vitro propagation system for S. oleoides. Multiple shoots were induced from nodal segments harvested from about 25- to 30- yr-old lopped trees of S. oleoides on MS medium + 0.1 mg L−1 NAA (Naphthalene acetic acid) + 2.5 mg L−1 BA (6-Benzylaminopurine) + additives. The shoots were multiplied by (a) repeated transfer of the mother explants on MS medium + 1.0 mg L−1 BA + 0.1 mg L−1 NAA + additives and (b) subculturing of shoot on MS + 1.0 mg L−1 BA + additives. About 84% shoots rooted ex vitro on soilrite within 3–4 weeks when base (4–5 mm) of shoots was treated with 100 mg L−1 of IBA (Indole-3-butyric acid) for 5 min. The plantlets were hardened successfully in the greenhouse and transferred to the pots and field. To the best of our knowledge, this is the first report of a regeneration protocol for S. oleoides from explants obtained from mature trees. Use of the ex vitro rooting technique for plant production serves as a more economical option as it reduces labor, cost, and time. We suggest that the methods developed and described in this article can be used for large-scale plant production and conservation of germplasm of this tree species.