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Nirmal Joshee*, Bipul K. Biswas and Anand K. Yadav

Centella asiatica L. (Apiaceae family), also called `Indian Pennywort,' is a prostrate, faintly aromatic, and stoloniferous perennial herb with long petiolated leaves. In the Ayurvedic medicine, it is reputed as a nervine tonic along with antibacterial, antifeedant, antileprotic and wound healing properties. Centella contains glycosides, indocentelloside, brahmoside, and asiaticoside. Its leaves are rich in carotenoids and vitamins B and C. In vitro culture techniques which offer a viable tool for mass propagation of plants have recently become increasingly popular for conservation of rare, endangered and threatened medicinal plants germplasm. Centella tissue culture has been reported to experience high incidences of microbial contamination which drastically reduces survival of explants. Thus, the main purpose of this study was to develop an efficient micropropagation technique for Centella asiatica to reduce explant contamination and rapidly disseminate superior clones for research and production. Here we present induction and further development of somatic embryos, using Centella stolons as explants. Somatic embryos were induced in response to 2,4-D shock on MS medium. Initially, somatic embryos appeared as highly nodular callus and eventually developed into somatic embryos that exhibited globular, heart shaped and cotyledonary stages. After auxin shock, cultures were regularly transferred to MS basal medium where somatic embryos completed various developmental stages and then germinated to give rise to new plantlets. In this presentation, we will demonstrate complete protocols for the successful sterilization of Centella explants prepared from plants that had abundance of fungal and bacterial contamination.

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Bipul K. Biswas*, Nirmal Joshee and Anand K. Yadav

Guava (Psidium guajava L.), also called `apple of tropics,' is immensely nutraceutical and horticulturally important. Being a tropical plant, it cannot stand temperatures below 25° F and needs frost protection to grow in temperate regions. To adapt in cold climate, cold hardy guava cultivars are needed. Conventional ways are uneconomic in time and efforts. Still, transgenic plants developed using biotechnological approaches of tissue culture and rDNA technology, appear to have great potential. Thus, protocols for in vitro propagation of guava were developed via organogenesis and somatic embryogenesis using nodal explants from mature trees and young zygotic embryos, respectively. Nodal explants induced multiple shoots when cultured on MS medium fortified with KIN, BAP and Ad.S. Adding a (NO3)2 to medium was useful to prevent in vitro shoot tip browning of adventitious shoots. Rocker liquid culture greatly increased growth of multiple shoots compared to the agar-based medium. It appears to be a good tool for woody plant tissue culture. Induction of somatic embryos in guava was also achieved on MS medium supplemented with IAA auxin. About 80% to 90% somatic embryos germinated normally. To achieve Agro-bacterium-mediated gene transfer in guava, on-going co-cultivation of organogenic tissues of guava is to optimize protocols for freeze tolerance gene (CBF1, CBF2, CBF3) transfer. Plasmid vectors containing selectable markers (nptII gene for antibiotic selection and GUS reporter gene as scorable gene mediated selection), with CaMV 35S promoter gene has been introduced into guava tissues and the resultant plants showed antibiotic resistance. Details of the experimental procedures and up-to-date results will be discussed.

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Nirmal Joshee, Bipul K. Biswas and Anand K. Yadav

Past research experience with Centella asiatica micropropagation suggests a very high rate of contamination during the culture establishment stage. We demonstrate protocols for successful sterilization of Centella explants prepared from field-grown plants with an abundance of fungal and bacterial contamination. Sequential steps during sterilization and explant preparation process included a dip for 30 s in 70% ethyl alcohol, weak bleach treatment for 12 min, and a 60-min soak in plant preservative mixture before establishing cultures. We also report a reproducible system for somatic embryogenesis in Centella using leaf and stolon tip explants collected from naturally growing populations. Somatic embryos were induced within 3 to 4 weeks of culture in the dark on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Initial embryogenic mass appeared as nodular callus, which eventually developed into actual somatic embryos exhibiting globular, heart-shaped, and cotyledonary stages. Leaves produced embryogenic calli at 2.26 and 4.52 μm 2,4-D, whereas stolon tips were responsive only in the 9.04 μm 2,4-D treatment. Withdrawal of 2,4-D/growth regulators from the induction medium resulted in the maturation and further development of the embryos into plantlets. Regular subculturing of the embryogenic calli into MS medium sustained their regenarability for more than 1 year. Somatic embryos were individually encapsulated in sodium alginate and calcium chloride-based encapsulation matrix to produce artificial or synthetic seeds (synseeds). Synseeds with 2% sodium alginate were found best for the survival and germination recorded after their storage at 5 to 8 °C for 30 and 60 days. We report protocols for C. asiatica to reduce explant contamination before establishment of cultures on somatic embryo induction medium and efficient somatic embryogenesis to facilitate conservation and mass production of elite germplasm. This may further assist rapid dissemination of superior clones needed for research and commercial production.

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Bipul Biswas, Nirmal Joshee, Ashish Yadav and Anand K. Yadav

Phalsa[Grewia asiatica (L.) Tiliaceae] is an exotic fruit with good nutraceutical values. It cannot be grown in temperate climates with severe winters. Therefore, genetic improvement of phalsa for cold tolerance is essential. In order to apply biotechnology through genetic transformation to enhance cold hardiness, a reliable and rapid micropropagation system is needed. Thus, developing the most dependable micropropagation protocols for phalsa was the primary goal of this research. Phalsa explants prepared from different tissues, including leaf, nodes, internode, and zygotic embryos, were collected from mature trees growing in the specialty plants house, cultured on MS medium supplemented with various cytokinins alone or along with auxins and incubated under a 10-hour photoperiod at ambient temperature. In vitro propagation of phalsa tissues through both organogenesis and somatic embryogenesis was achieved. Of these, single shoots were developed from nodal explants as a result of budbreak on MS medium supplemented with BAP, kinetin, and zeatin separately. Somatic embryos were developed from the zygotic embryos when cultured on MS medium with 0.023 μm BA + 0.022 μm zeatin, for 2 weeks following a pulse treatment on NN medium supplemented with 5% sucrose, 0.11 μm BAP, 0.22 μm 2,4-D, and 29.20 μm L-glutamine. Somatic embryogenesis was also observed on modified basal medium supplemented with 13% sucrose, 58.40 μm L-glutamine, and 1.75 μm IAA. Enormous callusing was a major problem for in vitro studies with this species, irrespective of media composition. Further studies for multiple shoot development and higher frequency of SE induction are under way.