Standardization and Development of Improved Tissue Culture Techniques for Genotype Co 94012 in Sugarcane

Abstract

Sugarcane is a major cash crop in India and is affected by various pests and diseases. Also, it requires more irrigation as compared to other crops to grow. The genetic improvement by conventional breeding is still a challenge due to long crop cycle and poor genetic diversity. To overcome these constraints, genetic transformation and CRISPR mediated gene editing in plants can prove as beneficial tools. To develop traits in sugarcane using these technologies requires highly reproducible and very efficient somatic embryo based protocol in popular cultivars. In this study, we aimed to develop high efficiency, reliable and reproducible somatic embryos based tissue culture system amenable for Agrobacterium or biolistic-mediated genetic transformation in popular cultivars of sugarcane. The improved protocol can be used for generation of transgenic or genome edited plants in desired numbers. In the present investigation, we used high yielding sugarcane var. Co 94012 for development of high efficiency callus based regeneration protocol. Leaf disc from two months old shoots were used to induce the callus. Embryogenic calli were obtained using initial boost of 3mg/lit, 2, 4 D followed by subsequent subculture at 2 mg/lit 2, 4 D. Regeneration response was 100 percent on MS media with 1 mg/lit BAP. These regenerated shoots were elongated on MS media enriched with 0.2 mg/lit BAP for two to three weeks. Furthermore, Cv. (cultivar) Co 94012 showed 100 percent rooting on MS media with 1 mg/lit NAA. Root initiation was observed within 8 to 10 days. 100% survival of transplanted plants was observed. This is the first report of sugarcane regeneration and development of plants through callus using var. (variety) Co 94012.