In vitro regeneration and development of stress tolerant peanut (arachis hypogaea l.) through agrobacterium-mediated genetic transformation

Abstract

Peanut (Arachis hypogaea L.) is one of the most economically important oilseed crop of the world. Bangladesh has a great opportunity to cultivate peanut in the marginal lands where hardly any other economically important crops are growing. However, in Bangladesh peanut production is hampered due to the incidence of various fungal diseases. The modern techniques of biotechnology such as plant genetic transformation can be applied for its improvement towards the development of disease resistance. The objective of the present investigation was to develop a fungal disease resistant peanut line through Agrobacterium-mediated genetic transformation. As a prerequisite of the transformation protocol, an efficient in vitro regeneration system was established for three locally grown peanut varieties, namely, Dhaka-1, BARI Chinabadam-8 and Binachinabadam4. Four different types of explants, namely, immature leaflet, decapitated half embryo, deembryonated cotyledon and single cotyledon attached decapitated embryo were used for regeneration. Highest percentage (71.8%) of multiple shoot regeneration was achieved when de-embryonated cotyledon explants initially cultured on 88.8 µM BAP, followed by two subsequent cultures on lower concentrations of BAP (66.6 µM BAP and 13 µM BAP) containing medium. It took about 45 days for the regeneration of multiple shoots. Best response towards induction of roots for all the varieties was obtained when regenerated shoots were cultured on half strength of MS medium supplemented with either 2.5 µM IBA or 1.0 µM IAA. Fully developed in vitro regenerated plantlets were successfully established in soil for further growth and development. For optimization of genetic transformation protocol, Agrobacterium strain LBA4404 containing binary vector plasmid pBI121GUS-NPTII harboring GUS (β-Glucoronidase) and nptII (neomycin phosphotransferase) genes (construct I) was used. Transient GUS histochemical assay revealed that among the three explants used maximum transient GUS expression (86.66%) was observed from de-embryonated cotyledon explants of variety Dhaka-1 having an optical density of 0.8 at 600 nm with 10 minutes incubation period. Transformed shoots were cultured on 200 - 250 mg/l kanamycin supplemented medium to select the putatively transformed shoots. Transformation frequency was found to be 0.34% in Dhaka-1 using pBI121GUS-NPTII construct. Agrobacterium strain LBA4404 containing plasmid pCAMBIA2300enh35SAFP harboring AFP (antifungal protein) gene and nptII (neomycin phosphotransferase) (construct II) was used for the integration of gene of interest. De-embryonated cotyledon and single cotyledon attached decapitated embryo explants of variety Dhaka-1and BARI Chinabadam-8 were used for transformation experiments. Among the two explants best response was observed from de-embryonated cotyledon explants. In this case, transformation efficiency was found to be 1.52% in Dhaka-1 and 0.85% in BARI Chinabadam-8. A total 17 putatively transgenic plants (T ) of Dhaka-1 were confirmed through PCR analysis. The integration of nptII gene was further confirmed by Southern blot hybridization in 10 T 0 peanut plants survived through acclimatization and developed till maturation. T 1 0 seeds were collected and raised following the Biosafety guidelines in the double layered insect proof net house for further investigation. Peanut as a grain legume has been considered as recalcitrant towards in vitro plant regeneration as well as for genetic transformation. The results of both in vitro plant regeneration and Agrobacterium-mediated genetic transformation using marker as well as antifungal (gene of interest) gene has pave the way for developing fungal disease resistant peanut plants.