Soumendra K. Naik

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.).

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.

High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Punica granatum L.)

A complete protocol is presented for in vitro regeneration of pomegranate (Punica granatum L.), a tropical fruit tree, using cotyledonary nodes derived from axenic seedlings. Shoot development was induced from cotyledonary nodes on Murashige and Skoog (1962) (MS) medium supplemented with 2.3‐23.0 mM benzyladenine (BA) or kinetin (Kn). Both type and concentration of cytokinin significantly influenced shoot proliferation. The maximum number of shoots (9.8 shoots/explant) was developed on a medium containing 9.0 mM BA. Shoot culture was established by repeatedly subculturing the original cotyledonary node on a fresh batch of the same medium after each harvest of the newly formed shoots. In vitro raised shoots were cut into nodal segments and cultured on a fresh medium for further multiplication. Thus, from a single cotyledonary node about 30‐35 shoots were obtained in 60 days. Shoots formed in vitro were rooted on half-strength MS supplemented with 0.054‐5.4 mM naphthaleneacetic acid (NAA). However, a medium containing 0.54 m MN AA resulted in the highest per cent rooting of shoots and significantly higher number of roots than other concentrations. Plantlets were successfully acclimated and established in soil. # 2000 Elsevier Science B.V. All rights reserved.

Shoot organogenesis and plantlet regeneration from hypocotyl-derived cell suspensions of a tree legume, Dalbergia sissoo Roxb.

SummaryA complete and efficient protocol is presented for plant regeneration from cell-suspension cultures of Dalbergia sissoo Roxb., an economically important leguminous tree. Factors influencing callus initiation, establishment of cell-suspension culture, callus formation from embredded microcolonies, and shoot organogenesis from suspension-derived callus were identified. Of the two different auxins tested, callus induction was better on a medium containing naphthalene acetic acid (NAA). The percentage of callus induction increased considerably when NAA at 2.0 mg l−1 (10.8 μM) was added in conjunction with 0.5 mg l−1 (2.2 μM) N6-benzyladenine (BA). Of the three different explants evaluated for callus induction, hypocotyl segments were most responsive. Friable hypocotyl-derived callus from the second subculture passage was used to initiate the cell-suspension culture. Optimum growth of the cell suspension was observed in MS medium supplemented with the same growth regulators as described above for callus induction, with an initial inoculum cell density of 1%. The plating efficiency of the microcolonies was greatly influenced by harvesting time and the gelling agent used for plating. Efficiency was highest (93%) with cells harvested at their exponential growth phase and plated in 1.2 g l−1 Phytagel. Shoot organogenesis from callus cultures was higher on a medium supplemented with a combination of BA and NAA than on BA alone. Seventy-one per cent of cultures exhibited shoot-bud differentiation on a medium containing 3.0 mg l−1 (13.3 μM) BA and 0.5 mg l−1 (2.7 μM) NAA. Regenerated shoots were rooted on half-strength MS medium containing 1 mg l−1 each of indole-3-acetic acid (5.7 μM), indole-3-butyric acid (4.9 μM) and indole-3-propionic acid (5.3 μM). Plantlets were acclimated and established in soil.

High-frequency plant regeneration by in vitro shoot proliferation in cotyledonary node explants of grasspea (Lathyrus sativus L.)

SummaryMultiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N6-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl−1 (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl−1 (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.

Search for antisickling agents from plants

The sickle cell disease is fatal in nature. Thousands of children are dying off due to this health problem throughout the globe. Due to the rapid development of diagnosis and clinical managements such patients are living up to a respectable age. But as there is no permanent cure the patients are suffering from bone and joint pain, jaundice, hepato-splenomegaly, chronic infections etc. The main physiological complicacy is due to the polymerization of sickle hemoglobin (HbS), (sickling process) inside the red blood cell (RBC) of these patients during deoxygenating state. The change of RBC from spherical to sickle shape is due to the polymerization of mutant hemoglobin (HbS) inside the RBC and membrane distortion during anoxic condition. The mechanism and the process of sickling are very complex and multifactor in nature. To get rid from such complicacies it is necessary to suitably and accurately stop the sickling of RBC of the patients. The potential anti-sickling agents either from natural sources and/or synthetic molecules may be helpful for reducing the clinical morbidity of the patients. A lot of natural compounds from plant extracts have been tried by several workers in recent past. Most of the studies are based on in vitro red cell sickling studies and their mode of action has not been properly understood. Although, few studies have been in vivo in nature pertaining to transgenic sickle animal model, there is paucity of data on the human studies. The result of such studies although has shown some degree of success, a promising anti-sickling agent is yet to be established.

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production

Abstract Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as “Indian Ginseng”, is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.

High frequency shoot proliferation from cotyledonary node of Lawsonia inermis L. and validation of their molecular finger printing

An efficient and reproducible protocol for in vitro plant regeneration was developed for Lawsonia inermis L. using cotyledonary node explant derived from axenic seedlings. Highest shoot proliferation frequency (ca 96.6%) was achieved on Murashige and Skoog’s, 1962 (MS) basal medium supplemented with 8.88 μM 6-Benzyladenine (BA) + 2.68 μM Napthalene acetic acid (NAA). Up-scaling of shoots was carried out using in vitro nodes on MS medium supplemented with 4.44 μM BA. So overall, an average of 238 shoots was produced at 75 days. Of the four different forms of cotyledonary node explants evaluated, highest shoot multiplication was observed in cotyledonary node explant with two whole cotyledons. In vitro regenerated shoots were best rooted (ca 34.3 roots / shoot) on ½ MS medium devoid of any growth regulator. The plantlets were successfully acclimated in sand:soil:: 1:1and established in the garden soil. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed a homogeneous amplification profile for all micropropagated plants validating the genetic fidelity of the in vitro-regenerated plants and supporting the regeneration protocol for economic commercial exploitation.

Biotechnological Intervention Through Tissue Culture in Hedychium coronarium: A Potential Anticancer Plant

Hedychium coronarium is a valuable medicinal plant and commonly known as butterfly ginger, belonging to family Zingiberaceae. It has enormous medicinal values in traditional system of medicine and also has the potential to be used in modern medicine. A number of bioactive compounds with different pharmaceutical properties including anticancer activity have already been isolated from this plant. Realizing its medicinal potential, there is an increasing demand for this plant species. This has exposed the plant to unsustainable harvesting. Thus the species requires conservation-friendly approaches in its use. Regeneration of plants with uniform biochemical and genetic makeup through plant tissue culture is vital for conservation of such plant species and sustainable development. Till date several tissue culture-mediated plant regeneration protocols and assessment of biochemical and clonal fidelity of the regenerated plants have been reported on H. coronarium. The aim of this chapter is to present a comprehensive account of the tissue culture-mediated biotechnological intervention in H. coronarium and also to summarize the different works on genetic diversity of this plant species for its conservation strategies.

In Vitro Secondary Metabolite Production Through Fungal Elicitation: An Approach for Sustainability

Being sessile, plants produce an array of secondary metabolites in response to different biotic and abiotic stimuli to survive under adverse conditions. Many of these secondary metabolites are valued for their medicinal properties and are known for their usage to cure diseases since ancient time. Even after the discoveries of synthetic drugs, the interest for drugs derived from medicinal plants is gaining importance as they are safe and devoid of any side effects. A number of important medicinal plants are yet to be cultivated for commercial purposes and are collected from wild habitat, posing a threat to their existence. To protect the biodiversity and achieve the sustainability, development of alternative strategies for production of secondary metabolites is necessary. Plant cell, tissue and organ culture including hairy root cultures have shown great potential for production of secondary metabolites. However, sometimes, these cultures fail to synthesise adequate secondary metabolites compared to plants growing under natural condition. The production of secondary metabolites can be enhanced by using different elicitors, either biotic or abiotic, which act as stress agents and enhance the production of secondary metabolites in plant callus/cell suspension culture, root culture, shoot culture and hairy root culture. Fungal elicitor (including yeast extract) is one of the preferred elicitors used for this purpose. In this chapter, attempts have been made to provide a comprehensive account of the strategies used for increasing the production of secondary metabolites in different in vitro culture system using fungal elicitors. A brief account of elicitors, elicitations and general mechanism of elicitations has also been provided.

Comparative thin-layer chromatographic studies and development of a high-performance thin-layer chromatography method for the quantification of lawsone in natural and micropropagated plant parts of Lawsonia inermis L.

A simple, sensitive, and precised high-performance thin-layer chromatography (HPTLC) method has been developed for the analysis of lawsone in natural vis-à-vis micropropagated plant parts of Lawsonia inermis L. Separation of the components was perfectly achieved on high-performance thin-layer chromatography (TLC) plates using optimized tertiary mobile phase of benzene–ethyl acetate– acetic acid (7.5:2.5:0.1, v/v). Densitometric scanning was performed before derivatization of the plate in absorption/reflection mode, and lawsone was quantified at its maximum absorbance of wavelength of 275 nm. Linearity of the method was obtained in the concentration range of 50 to 350 ng spot−1 with a correlation coefficient (r2) of 0.9999, indicating good relationship between concentrations in opposition to the peak area. The limit of detection and limit of quantification were found to be 16 and 50 ng spot−1, respectively. The obtained recovery ranges from 95.09% to 96.90% with an average value of 96.02% proved the excellent accuracy of the method. The developed method was found to be highly sensitive, and the mobile phase enables outstanding separation of lawsone from other components present in the mixture. The International Conference on Harmonization (ICH) guidelines were followed for validation of the HPTLC method in terms of precision, repeatability, and accuracy. The maximum content of lawsone was reported in the leaves of the micropropagated plant.