Anisha Akula

Assessing the Efficiency of RNA Interference for Maize Functional Genomics

A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.

Somatic embryogenesis in clonal neem, Azadirachta indica A. Juss. and analysis for in vitro azadirachtin production

SummaryEfficient and highly reproducible induction of somatic embryogenesis was obtained in four out of seven selected clones of neem, Azadirachta indica A. Juss. This was achieved either directly from root and nodal explants or indirectly from callus cultures initiated from leaf explants excised from 1-yr-old axenic plants. Direct induction of somatic embryogenesis was achieved both from nodal and root segments within 8 wk of culture on MS1 medium without growth regulators. However, the addition of 2.3–4.5 μM thidiazuron and 0.5 μM 2,4-dichlorophenoxyacetic acid into the medium were necessary to induce somatic embryogenesis via callus phase from leaf explants. Repetitive embryogenesis was observed within 3–4 wk following transfer of somatic embryos to a plant growth regulator-free medium. When somatic embryos of nodal and root segments were left on the induction medium without subculturing, approximately 15% of the somatic embryos developed into whole plantlets after passing through a series of developmental stages. Plantlets thus produced were hardy, lush green, and acclimatized casily under greenhouse conditions. However, somatic embryos derived from leaf explants showed low conversion rates (<5%). HPLC analysis revealed no detectable levels of azadirachtin in somatic embryos.

Protocols for Somatic Embryogenesis and Plantlet Formation from Three Explants in Tea (Camellia sinensis (l.) o. kuntze)

Tea plant production via micropropagation is technically possible but its commercial utility in tea industry is severely hampered because of the cost factor. Tea producers still rely on conventional methods of propagation using either single leaf node cuttings or seeds. Clonal variability of rooting response is also a big problem with micropropagated shoots. Another major disadvantage of micropropagation is that the resulting plants will only have adventitious roots and hence are highly susceptible to drought. On the other hand, with the increase in world demand for high quality tea, the demand for clonal planting material is also increasing. Somatic embryogenesis as a tool for in vitro propagation and genetic improvement could play a more significant role in the production of tea. Besides, the recent surge of biotechnological advances like gene cloning and gene transfer offer great promise for rapid improvement of genotypes with desirable traits and integrate well with the technique of somatic embryogenesis. Although somatic embryogenesis has been fully exploited in herbaceous species there remain difficulties with woody species like tea. Unlike micropropagated shoots, the plants derived from somatic embryos have both root and shoot together. Furthermore, the production cost via somatic embryogenesis is potentially cheaper than the micro-cuttings especially if bioreactors and automation are used in the production process. This gives them a decided advantage over the