N. S. Shekhawat

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.

Micropropagation of Arnebia hispidissima (Lehm). DC. and production of alkannin from callus and cell suspension culture

Alkannin, a red-purple dye and bioactive compound found in the roots of Arnebia hispidissima has antibiotic and anti-inflammatory properties and is also used in cosmetic and textile industries at a large-scale. In the present communication, we demonstrate the establishment of callus and cell suspension culture of A. hispidissima with the aim of optimizing the production of alkannin. Highest alkannin content was recorded in cell suspension and callus culture established on M-9 medium. Production of alkannin was influenced by the different culture medium. Evaluation of alkannin content of roots of field-grown plants and in vitro grown cell, tissue and organ showed that alkannin production was higher in all in vitro grown culture systems (cell suspension, callus and roots) than the roots of field-grown plants. The present investigation may be applicable in designing systems for the large-scale cultivation of A. hispidissima cell suspensions for the production of alkannin.

Assessment of Genetic Stability and Instability of Tissue Culture-Propagated Plantlets of Aloe vera L. by RAPD and ISSR Markers

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.

Recent advances in genetic engineering for improvement of fruit crops

Fruits are one of the major sources of vitamins, essential nutrients, antioxidants and fibers in human diet. During the last two–three decades, genetic engineering methods based on the use of transgenes have been successfully adopted to improve fruit plants and focused mainly on enhanced tolerance to biotic and abiotic stresses, increased fruit yield, improved post harvest shelf life of fruit, reduced generation time and production of fruit with higher nutritional value. However, the development of transgenic fruit plants and their commercialization are hindered by many regulatory and social hurdles. Nowadays, new genetic engineering approaches i.e. cisgenesis or intragenesis receive increasing interest for genetic modification of plants. The absence of selectable marker gene in the final product and the introduced gene(s) derived from the same plant or plants sexually compatible with the target crop should increase consumer’s acceptance. In this article, we attempt to summarize the recent progress achieved on the genetic engineering in fruit plants and their applications in crop improvement. Challenges and opportunities for the deployment of genetic engineering in crop improvement programs of fruit plants are also discussed.

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 Woody Plants

Micropropagation protocols for cloning of mature trees of Balanites aegyptiaca, the Hingota (Balanitaceae); Citrus limon, the Nimbu (Rutaceae) and Syzygium cuminii, the Jamun (Myrtaceae) have been developed. In order to harvest responsive nodal explants the mother tree(s) were pruned during the winter. Fresh shoot sprouts derived from the trees were used as explants. The nodal explants produced multiple shoots in vitro by activation of axillary meristems on MS medium + 0.45 µM BAP. Shoots were further multiplied in culture by (i) repeated transfer of the mother explants and (ii) the subculturing of the nodal segments of in vitro differentiated shoots. Shoots multiplication in Citrus limon could be achieved by amendment of the nutrient medium. The in vitro cloned shoots of the three species were rooted in vitro and ex vitro. Ex vitro root induction was followed to produce plants. Micropropagated plants were hardened in the green house. The hardened and acclimatized plants were transferred to pots and subsequently to field. The cloned plants are growing normal. The protocols defined are reproducible. These can be used for mass multiplication of selected clones and genetic improvement of these species.

Micropropagation of Vegetable Rennet (Withania coagulans [Stocks] Dunal)—A Critically Endangered Medicinal Plant

Withania coagulans (Stocks) Dunal (Solanaceae), popularly called vegetable rennet, is a critically endangered and highly valued medicinal plant. Overexploitation and reproductive failure forced the plant species toward the verge of complete extinction. We describe here the development of a simple, rapid, and cost effective in vitro micropropagation system for W. coagulans for mass-scale production of true-to-type plantlets using nodal shoot segments. Exactly 95.5 ± 0.34% explants responded within 8–10 days (d) and produced multiple shoot buds (4.1 ± 0.10 shoots of 2.95 ± 0.15 cm length) on 0.8% agar-gelled Murashige and Skoogs (MS) basal medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), and additives (100 mg L−1 L-ascorbic acid, 25 mg L−1 each citric acid, adenine sulphate, and L-arginine). The shoots in cultures were multiplied by repeated transfer on MS medium with 4.44 μM BAP, 0.57 μM IAA, and additives. Further cultures were multiplied on a large-scale through the subculturing of shoot clumps differentiated in vitro, on MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and additives. Maximum number (19.1 ± 0.28) of healthy (6.15 ± 0.25 cm) and viable shoots differentiated on this medium. The microshoots were rooted both in vitro and ex vitro. Exactly 67.3 ± 1.01% microshoots rooted in vitro within 25–30 d on agar-gelled half-strength MS salts supplemented with 29.52 μM indole-3-butyric acid (IBA) and 200 mg L−1 of activated charcoal (AC). Alternatively, 73.8 ± 0.65% cloned shoots rooted on sterile soilrite (soilless compost and soil conditioner) under ex vitro conditions after pulse treatment with 2.46 mM IBA for 300 s. The clones of W. coagulans were hardened in a greenhouse within 40–45 d by slow and gradual exposure of plantlets from high relative humidity (RH; 70–80%) and low (26 ± 2°C) temperature to low RH (40–50%) and high (34 ± 2°C) temperature. The hardened plantlets were transferred to soil and stored in agro-net house with more than 90% survival rate. Replacement of pure and laboratory grade sucrose with commercial grade sugar, use of less expensive commercial grade agar-agar in culture medium, higher rate of shoot proliferation, single step ex vitro rooting, and hardening of plantlets in the greenhouse are advantageous features of the protocol. The micropropagation protocol defined here is reproducible, easy to follow, and would be helpful in large-scale restoration programs through true-to-type mass-multiplication of W. coagulans.