Shiwali Sharma

Synseed technology—A complete synthesis

Progress in biotechnological research over the last two decades has provided greater scope for the improvement of crops, forest trees and other important plant species. Plant propagation using synthetic seeds has opened new vistas in the field of agriculture. Synseed technology is a highly promising tool for the management of transgenic and seedless plant species, polyploid plants with elite traits and plant lines that are difficult to propagate through conventional propagation methods. Delivery of synseeds also alleviates issues like undertaking several passages for scaling up in vitro cultures as well as acclimatization to ex vitro conditions. Optimization of synchronized propagule development followed by automation of the whole process (sorting, harvesting, encapsulation and conversion) can enhance the pace of synseed production. Cryopreservation of encapsulated germplasm has now been increasingly used as an ex vitro conservation tool with the possible minimization of adverse effects of cryoprotectants and post-preservation damages. Through synseed technology, germplasm exchange between countries could be accelerated as a result of reduced plant quarantine requirements because of the aseptic condition of the plant material.

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.

Efficient Micropropagation of Spilanthes acmella (L.) Murr.: A Threatened Medicinal Herb

The present study describes an efficient and reproducible protocol for micropropagation of S. acmella . Shoot tips taken from 3 week-old aseptic seedlings were cultured on Murashige and Skoog (MS) semi-solid medium supplemented with different concentrations of TDZ. Among various concentrations, 0.25 µM TDZ was found to be optimum for shoot regeneration as it induced a maximum of 30.0 shoots per explant however with retarded growth (1.0 cm). Among different volumes of culture media, 15 ml liquid culture medium favored best response wherein a maximum of 80.2 shoots per explant with an average shoot length of 7.0 cm were induced after 6 week of subculturing. Successful in vitro rooting was induced on 2.5 µM NAA containing half-strength MS medium. Almost 96% rooted plants successfully transferred and acclimatized ex vitro under green house conditions. Morphological and physiological parameters compared with the in vivo -grown seedlings of the same age appeared to be ‘normal’ in respect to the fundamental characteristics examined.

Enhanced multiplication and improved ex vitro acclimatization of Decalepis arayalpathra

The proposed work describes a protocol for high-frequency in vitro regeneration through nodal segments and shoot tips in Decalepsis arayalpathra, a critically endangered medicinal liana of the Western Ghats. Nodal segments were more responsive than shoot tips in terms of shoot proliferation. Murashige and Skoog’s (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA) was optimum for shoot initiation through both the explants. Among different combinations of plant growth regulators and growth additive screened, MS medium added with 5.0 μM BA + 0.5 μM indole-3-acetic acid + 20.0 μM adenine sulphate effectuated the highest response: 11.8 shoots per nodal segment and 5.5 shoots per shoot tip with mean shoot length of 9.2 and 4.8 cm, respectively. Half-strength MS medium with 2.5 μM α-naphthalene acetic acid was optimum for in vitro root induction. The plantlets with the well developed shoot and root were acclimatized in Soilrite™ with 92 % survival rate in the field conditions. During acclimatization, chlorophyll content, net photosynthetic rate, stomatal conductance, and transpiration rate were gradually changed in dependence of formation of new leaves. Further, the changes in activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) as well as activity of carbonic anhydrase were also observed: a continuous rise in SOD activity, but a rise and fall in the activities of CAT, APX, and GR were also noticed. Maximum fresh mass (3.1 g plant-1), dry mass (0.35 g plant-1) of roots and 2-hydroxy-4-methoxybenzaldehyde content of 9.22 μg cm-3(root extract) were recorded after 8 weeks of acclimatization.

Biotechnological strategies for the conservation of medicinal and ornamental climbers

Since time immemorial human beings are utilizing plants, apart from food and shelter, as medicine to cure ailments and ornamentals as aesthetic value. The recognized plants for medicinal uses mostly belong to tree, shrub and herbs, but there is another group of plants categorized as CLIMBERS . The climbing habit is a key innovation in angiosperms evolution. Climbing plant taxa have greater species richness than their non-climbing sister groups. Although it is considered as highly diversified clades but a much neglected group of the plants. In contrast with either erect or prostrate species, which occupy a narrow range of the light, climbers may use a very broad range of light availability. With the occupation of such an expanded ecological niche-ranging from forest floor to understory to forest canopy- a greater exposure to different pollinator, fruit/seed dispersers and herbivores would be granted. Almost one-third of the plant families includes climbers and contributes significantly to the function of any forest system. Climbers are not well investigated by the researchers, however considerable information has been gathered in this book, which delineates for the researchers and readers to exploit medicinal and ornamental climbers for their benefit. As concerted efforts have not been made to popularize climbers for medicinal and aesthetic uses, the contributions made in this book will provide a platform to move ahead for better utilization of climbers in the service of human beings. This book offers an insightful look on different biotechnological interventions for the conservation of medicinal and ornamental climbers. The book starts with a discussion on the evolution and diversification of climbers among the angiosperms. Thereafter chapters describe various approaches of conservation, biotechnological strategies like micropropagation, synseed production, genetic transformation for the quality improvement, production of bioactive compounds under in vitro conditions. This book also provides a compilation of standardized in vitro micropropagtion protocols for some threatened climbers. It also includes chapters on various molecular markers and their application in medicinal and ornamental climbers for their desired improvement. The book provides an essential information for advanced students, teachers and research scientists it the field of plant biotechnology, pharmaceutical industries and medical sciences

Historical Perspective and Basic Principles of Plant Tissue Culture

In 1902 Gottlieb Haberlandt proposed the idea to culture individual plant cells on artificial nutrient medium. Although he failed to culture them due to poor choice of experimental materials and inadequate nutrient supply, he made several valuable predictions about the nutrients’ requirement for in vitro culture conditions, which could possibly induce cell division, proliferation and embryo induction. Tissue culture has now become a well-established technique for culturing and studying the physiological behaviour of isolated plant organs, tissues, cells, protoplasts and even cell organelles under precisely controlled physical and chemical conditions. Micropropagation is one of the most important applications of plant tissue culture. It provides numerous advantages over conventional propagation like mass production of true-to-type and disease-free plants of elite species in highly speedy manner irrespective of the season requiring smaller space and tissue source. Therefore, it provides a reliable technique for in vitro conservation of various rare, endangered and threatened germplasm. Micropropagation protocols have been standardized for commercial production of many important medicinal and horticultural crops. Somatic embryogenesis is an extremely important aspect of plant tissue culture, occurring in vitro either indirectly from callus, suspension or protoplast culture or directly from the cell(s) of an organized structure. Advantages of somatic embryogenesis over organogenesis include several practical means of micropropagation. It reduces the necessity of timely and costly manipulations of individual explants as compared to organogenesis.

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.

Tylophora indica (Burm. f.) Merrill: Medicinal Uses, Propagation, and Replenishment

Tylophora indica is an important medicinal and endangered climber of Asclepiadaceae family, commonly called as “antmool.” This perennial climber is indigenous to India and mainly occurs on plains, hilly slopes, and the outskirts of the forests of eastern and southern India. The plant is used by the common people in certain regions of India for the treatment of various diseases including bronchial asthma, cancer, dysentery, hay fever, inflammation, rheumatism, arthritis, and dermatitis. The plant has also been employed to cure cold, psoriasis, whooping cough, seborrhea, anaphylaxis, respiratory infections, and leucopenia and is an inhibitor of the Schultz-Dale reaction. The active principle of this plant lies in its leaves and roots which are known to exhibit emetic, cathartic, laxative, expectorant, diaphoretic, and purgative properties. The presence of pharmacologically active alkaloids, viz., tylophorine, tylophorinine, tylophorinidine, imparts medicinal value to the plant. Tylophorine exhibits strong anti-inflammatory activity, while tylophorinidine is a potent antitumor alkaloid of Tylophora. Indiscriminate collection of this plant from its natural habit, low seed germination potential, difficulty in propagation through vegetative cuttings have resulted in the rapid depletion of the natural strands of this plant thus giving it an endangered status. Micropropagation or plant tissue culture approaches are one of the potent alternative strategies for conservation as well as utilization of this valuable germplasm. Various biotechnological approaches have also been employed for improvement of this plant species using genetic transformation and somaclonal variation for the commercial exploitation of valuable alkaloids. The present chapter deals with the literature concerning the medicinal importance of Tylophora, its pharmacological properties, micropropagation protocols combined with genetic engineering specifically transformation technology for large-scale production of tylophora, and its improved germplasm with high-volume production of medicinally important alkaloids.

Somatic Embryogenesis: A Valuable Strategy for Phyto-Climbing Diversity Conservation

Somatic embryogenesis is the production of embryo-like structures from somatic cells without any gametic fusion. With a low frequency of chimeras, a high number of regenerants, and a limited level of somaclonal variations, somatic embryogenesis is more attractive than organogenesis as a plant regeneration system. Somatic embryos arise both naturally (in vivo) and in culture conditions (in vitro) either direct or through callus (indirect embryogenesis). Secondary embryogenesis is a special case of indirect somatic embryogenesis where secondary somatic embryos are produced through already induced primary embryos as initial explants. Coordinated behavior of morphogenic cells determines single- or multiple-cell origin of somatic embryos. In vitro conditions and plant growth regulators (PGRs) (exogenously added to the culture medium and endogenous PGRs) both are responsible for the induction of embryogenesis. Various extracellular proteins, arabinogalactan proteins, lipochito-oligosaccharides, and genes such as SERK, LEAFY COTYLEDON, BABY BOOM, and WUSCHEL regulate somatic embryogenesis. Being bipolar in nature, somatic embryogenesis is one of the good approaches to speed up the clonal propagation of plants. Synseed preparation is another important aspect of somatic embryos that can be used for germplasm conservation. However, poor germination of embryos is a major limitation of somatic embryogenesis in many plants. The present chapter provides a review on somatic embryogenesis in various medicinal and ornamental climbers.

Synseed Production in Spilanthes mauritiana DC. for Short-Term Storage and Germplasm Exchange

Aims: The present study provides an efficient protocol for short-term storage and germplasm exchange of a potent medicinal herb, Spilanthes mauritiana using encapsulated nodal segments. Study Design: For in vitro conversion of synseeds, 5 beads were placed in each flask having Murashige and Skoog (MS) medium supplemented with different combinations of plant growth regulators (PGRs). While for ex vitro conversion, 5 synseeds per thermocol cups having different planting substrates were directly sown. The data for each experiment were collected after 6 weeks. All the experiments were conducted with a minimum of 20 replicates per treatment and each experiment was repeated thrice. Place and Duration of Study: Plant Biotechnology Lab, Department of Botany, AMU, July 2012 to November 2013. Methodology: Concentration of two different manufacture grade of Na-alginate (purchased from Central Drug House and Loba Chemie) were compared for the production of ideal synseeds. Conversion of synseeds was tested under in vitro and ex vitro conditions. A low temperature storage (4oC) experiment was also carried out to understand the explants’ ability to revive physiological activity leading to plantlet development. Original Research Article British Biotechnology Journal, 4(6): 696-707, 2014 697 Results: A gelling matrix of 4% Na-alginate (CDH) or 3% (Loba Chemie) with 100 mM calcium chloride (CaCl2∙2H2O) was found most suitable for the production of ideal Caalginate bead. However, CDH grade Na-alginate (74.4% conversion) was found to be better than Loba Chemie (62% conversion) in terms of in vitro conversion of synseeds into complete plantlets when cultured on MS basal medium. Supplementation of Plant Growth Regulators (PGRs) to the MS basal medium further enhanced the conversion frequency of the synseeds. Maximum conversion (83%) was recorded on MS basal medium supplemented with 5.0 μM 6-benzyladenine (BA) and 0.5 μM indole-3-acetic acid (IAA). Synseeds, stored at 4oC for 1-8 weeks showed successful sprouting with variable percent in successive weeks of transfer, followed by development into complete plantlets when returned to regeneration medium. Ex vitro conversion of synseeds also occurred when synseeds were sown directly into Soilrite moistened with quarter-strength MS salts. Plantlets regenerated from non-stored and stored synseeds were successfully hardened, acclimatized and established in soil with a success of 90%. While plantlets recovered from direct sowing of synseeds exhibited 80% survivability. Conclusion: Being small in size, synseeds provide an effective tool for conservation, storage and exchange of this valuable medicinal plant species, potentially overcoming many of the difficulties associated with long-distance transport of plant germplasm.