Aastha Sahai

Histology of organogenesis and somatic embryogenesis in excised root cultures of an endangered species Tylophora indica (Asclepiadaceae)

This paper reports an efficient regeneration protocol through parallel organogenic and embryogenic pathways from green root segments (GRSs) of Tylophora indica (Burm.f) Merrill. GRSs explants from one year old in vitro cultures were cultured on Murashige and Skoog (MS) medium containing various cytokinins. Five µmol/L of 6-benzyladenine (BA) was most responsive for organogenesis in 1.5 cm long GRSs. Repeated subculture on medium containing both BA (5 µmol/L) and 1-naphthleneacetic acid (NAA) (0.1 µmol/L) promoted multiplication and proliferation of direct shoot buds (46.80 ± 0.96) and callus mediated somatic embryogenesis (18.07 ± 0.33). Germinated embryos isolated from callus were transferred onto maturation medium consisting of half-strength MS medium either devoid of plant growth regulators (PGRs) or with various concentrations of gibberellic acid (GA). Microshoots were excised during subculture and transferred onto root induction medium, thus ensuring a continuous supply of germplasm. Morphogenic variations were noticed in types of roots induced on various auxins. Regenerated plantlets and emblings hardened best on vermiculite with a survival rate of 90% and 70% respectively. However, the emblings were healthier in comparison to the regenerated plants. Histological analysis showed the origin and development of organogenesis.

Antimicrobial Potential of Balanites Aegyptiaca (L.) Del, Stevia Rebaudiana(Bert.) Bertoni, Tylophora Indica (Burm.f.) Merrill, and Cassia Sophera(Linn.)

Background: Despite the medicinal importance of Balanites aegyptiaca (L.) Del, Stevia rebaudiana (Bert.) Bertoni, Tylophora indica (Burm.f.) Merrill and Cassia sophera (Linn.), the antimicrobial potential of these plants has not been explored extensively, especially those of Cassia sophera. Methods: Both aqueous and alcoholic extracts of various parts of respective plants, and their in vitro raised calli were tested for antimicrobial activity by agar well diffusion method against a range of gram-positive and gram-negative bacteria and several fungal species. The calli of Balanites aegyptiaca and Tylophora indica were only tested as calli of Stevia rebaudiana and Cassia sophera could not be generated due to failure of regeneration in our laboratory. The extracts were also tested against genetically characterized bacterial isolates harboring bla (antibiotics resistance) genes. Minimal inhibitory concentrations (MICs) of the extracts were determined by broth microdilution method. Results: The extracts from all the four plants showed significant antibacterial activity against gram-negative bacteria, including, Salmonella typhi, Salmonella paratyphi A, Escherichia coli, Klebsiella species, Proteus vulgaris, Citrobacter spp., Salmonella typhimurium, Pseudomonas aeruginosa and Vibrio cholerae. Among all the tested plants, only B. aegyptiaca showed significant antibacterial activity against the tested gram-negative bacteria harboring bla genes including, Escherichia coli (bla ampC ), Klebsiella spp. (bla CTX-M ), Klebsiella spp. (bla SHV ), Escherichia coli (bla SHV+CTX- M ) and Citrobacter spp. (bla ampC+SHV ). Extracts of B. aegyptiaca and S. rebaudiana also showed activity against gram- positive bacteria whereas the extracts from Tylophora indica and Cassia sophera did not. Extracts of Balanites aegyptiaca, Tylophora indica, and Stevia rebaudiana demonstrated potential antifungal activity whereas extracts of Cassia sophera din not demonstrate the antifungal activity. Conclusions: This is among the premier reports describing the antimicrobial potential, especially through calli extracts, of Tylophora indica (Burm.f.) Merrill and Balanites aegyptiaca (L.) Del and in particular against gram-negative bacteria harboring bla genes. This is also a premier report looking extensively for the antimicrobial potential in Cassia sophera.

Phytoactive Compounds from In Vitro Derived Tissues

Plants are valuable sources for the discovery of new products of medicinal importance. Several distinct chemicals and compounds derived from plants are being used as important drugs all around the world. The evolving commercial importance of secondary metabolites in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of plant tissue culture technology. Plant tissue culture techniques are found to have potential as a supplement to traditional agriculture in industrial production of bioactive compounds which is an alternative to produce the desirable medicinal compounds from plants. Phytoactive compounds have been isolated from a number of plants cultured in vitro mainly from callus cultures viz. Cardiospermum halicacabum, Cassia fistula, Pisum sativum, Centella asiatica. Some of those compounds are of great medicinal value and are used in recovery of fatal diseases like cancer such as Ajmalicine and Taxol. Discoveries of cell cultures capable of producing specific medicinal compounds have accelerated in the last few years viz. Datura metel, Catharanthus roseus, Chlorophytum borivilianum, Bacopa monieri. Some of the medicinal compounds localized in morphologically specialized tissues or organs of native plants have been produced in culture systems not only by inducing specific organized cultures, but also by undifferentiated cell cultures. Due to these advances, researches in the area of tissue culture technology for the production of plant chemicals have bloomed beyond expectations.

Genotype-dependent responses during in vitro seed germination and establishment of Balanites aegyptiaca (L.) Del. – an endangered agroforestry species

The present study reports variation in in vitro germination responses between two genotypes of Balanites aegyptiaca (L.) Del. an endangered agroforestry species. Fruits were collected from arid and semi-arid zones of Jodhpur (J) and Ahmedabad (A) respectively. Mature seeds of both the germplasms were isolated and subjected to in vitro germination on Murashige and Skoog medium supplemented with gibberellic acid (GA). Marked differences were observed in germination frequency of both genotypes at variable concentrations of GA. Whereas ‘A’ acquired 100% germination frequency on medium containing 1 μM GA, ‘J’ required 2.5 μM GA. Morphogenic variations for seedling traits were also recorded in relation to genotype for shoot, internode and root length along with number of nodes/shoot and leaf size and color. ‘A’ seedlings appeared healthy as compared to ‘J’ seedlings. These morphogenic variations were studied in correlation with the native environmental conditions of both germplasms. In vitro germinated seedlings were hardened and acclimatized in ex situ conditions and survivability was checked during in vitro hardening and ex situ acclimatization in relation to required humidity and shade density respectively.

Hairy Root Culture: An Efficient System for Secondary Metabolite Production

Plants are a potential source for the discovery of new products of medicinal value and served as lead compounds for drug development. Tremendous efforts have been made to commercialize production of plant metabolites employing plant cell culture in bioreactors, but very few have achieved commercial success. Hairy root culture is an unsurpassed choice for bio-processing system for various root associated pharmaceuticals due to fast growth rate, easy culture, genetic manipulations and most important biochemical stability of neoplastic roots. It serves as a model system for plant metabolism and physiology and utilized as a technical alternative to plant cell suspension culture. Moreover, hairy root culture plays a significant role in design the principle for plant metabolic engineering, germplasm conservation, expression of foreign protein and phytoremedaition. However, its global utilization requires attempts on the establishment of effective and economical scaled up culture that can reduce the consumption, but obtain the biggest benefits.

In Vitro Conservation Protocols for Some Endangered Medicinal-Plant

India is one of the twelve megabiodiversity centres of the world with a rich diversity of plant species. Plants are useful as medicines, ornamentals, perfumery, timber, trade etc. Majority of the world population rely on traditional medicines derived from plants resulting into the increasing demand for medicinal plants. Indiscriminate collection, intensive grazing and habitat destruction are some of the major factors resulting in the depletion of valuable plant species from the nature leading to their mass extinction. The growing demand of plants is putting a heavy strain on the existing resources causing a number of species to be either threatened or endangered category. The gradual decline in the natural population of plant species demands conservation efforts so as to ensure continuous and ample supply of the valuable plant materials which are in great demand by the various pharmaceutical industries. One of the possible methods of protection of endangered plant species is multiplying and conservation of plants through in vitro cultures. The development of reliable tissue culture protocols are of great importance for the conservation of rare and endangered plant species by virtue of producing uniform planting material. In recent years, there has been an increased interest in in vitro culture techniques which offer a viable tool for mass multiplication and germplasm conservation of rare, endangered and threatened medicinal plants. The present study highlighted some of the important in vitro protocols developed for selected endangered plant species of India like Commiphora wightii, Tylophora indica, Eremostachys superba, Gloriosa superba, Tecomella undullata etc.

Plant Edible Vaccines: A Revolution in Vaccination

Plants have been used as a source for many pharmaceutical since long. However, utilization of plant systems for production of edible vaccines has been a comparatively recent phenomenon. There are several potential advantages of plant derived vaccines over other conventional systems of vaccine production such as mammalian or avian cell culture. The cost of vaccines is one factor preventing further use of vaccination, leaving hundreds of thousands of children susceptible to preventable diseases. Especially for developing world this novel technique proved to be a boon for its low cost of production, convenient administration, easy storage and negligible chances of infection whereas the conventional system of vaccine production limits the applicability of vaccines in many parts of the world. These vaccines are prepared by introducing selected desired genes into plants and inducing these genetically modified plants to manufacture the encoded proteins. Transgenic plants may provide an ideal expression system, in which transgenic plant material can be fed directly as oral dose of recombinant vaccines. Expression of vaccines in plant tissue eliminates the risk of contamination with animal pathogen, provides a heat stable environment and enables oral delivery thus eliminating infection related hazards. Identification of transgenic material, containment of the transgenes and control of recombinant protein may be potential problems for large scale production of vaccines in plants. Factors like scaling up production as well as distribution and handling of transgenic plant material must comprise the future consideration in this field.

In Vitro Clonal Propogation of Coleus forskohlii via Direct Shoot Organogenesis from Selected Leaf Explants

Present study provides an easy and efficient protocol for large scale clonal propagation of Coleus forskohlii, a threatened medicinal plant of commercial importance. Basal leaf lamina excised from upper three nodes of shoot was used as explant and its size, position, orientation and season of collection were initially optimized to select the most responsive explant condition. Enhanced shoot production and proliferation has been achieved on medium containing 2 μM BA + 0.1 μM NAA wherein, a highest number of 35 shoots/explant were produced. The regenerated shoots of varied length (3–5 cm) were transferred to root induction medium comprising of IBA, NAA and IAA (1–5 μM) in half-strength MS medium to determine the most suitable shoot length for proper root induction. Rooted plantlets were acclimatized in field conditions after proper hardening. Histological analysis was also carried out to confirm the nature of origin of shoot buds from leaf explants.