Rice production of mutants in plant research (Sallaud

Rice is the major food for more than 3.2 billion people in the world and India occupied the second place in production of rice. With the present increased world population, the production falls short of more than 60% to satiate world’s hunger (FAO 2009). Due to genome sequence availability, relatively small genome size (390Mb) and economic importance, this crop selected as a model crop among monocots and developed mutant population was widely used as a source material in functional genomics studies (Goff et al. 2002). With the availability of complete rice genome sequence, the focus has moved from structural genomics study to functional genomics study. In insertional mutagenesis known DNA segment such as jumping gens or transposable elements are integrated into plant genome randomly. Integration within the gene resulting loss-of-functional mutation or nearby target gene will gives gain-of-functional mutation. For the functional characterization of genes, compared to loss-of-function mutations (recessive), gain-of-function mutations (dominant) will produce some kind of new phenotype. Majorly T-DNA and transposon tagging (Tos17, En/Spm and transposon (Ac-Ds) tagging) are the methods used for production of mutants in plant research (Sallaud et al. 2003). McClintock (1949) identified the transposable genetic elements like Ac and Ds in Maize (Zea mays) and further studies on these transposable elements led to the cut and paste transposition. Four copies of CaMV35S enhancer (4XEn) element insertion into plant genome and over expression of endogenous genes located in either orientation at the place of insertion gives dominant gain-of-function mutations, that is still widely accepted today (Manimaran et al. 2017). In the transposon based mutated plants have numerous novel insertions in every plant progeny and cover whole genome with mutations with limited starter lines. From a single Ac-Ds based transgenic line recovery of more independent stable Ds lines are produced and identification of flanking regions nearby enhancer element (Kuromori et al. 2004). Through the Ac and Ds transposon activation tagging system, several important genes and their function have identified in various plant species such as Arabidopsis (Raina et al. 2002)  poplar (Fladung and Polak 2012) tomato (Carter et al. 2013) and in rice (Lo et al. 2015). Ac-Ds based Activation tagging strategies are used to produce collection of morphologically different dominant mutants and cloning of corresponding genes are the important sources for analysis of functional genomics in rice (Qu et al. 2008).

Most of the genetic transformation approaches deploys through Agrobacterium mediated, because of its several advantages like low-copy number of gene of insert, easy to handle and efficiency and integration of foreign DNA more predictably (Gelvin et al. 2010). Explants such as calli derived from mature seeds, mature seeds, calli derived from immature seeds, shoot apex and calli derived for leaf base (Sahoo and Tuteja 2012). However, scutellum derived embryogenic calli was reported as the most suitable source for genetic transformation in indica varieties (Kumar et al. 2005). For callus induction, indica rice was less responsive than the japonica rice (Khanna and Raina 1998).  Lower regeneration frequency in indica rice remains a limiting factor in genetic transformation experiments. Partial desiccation(physical) of embryogenic calli promote embryo differentiation and lead to higher regeneration in soybean (Rance et al. 1994), sugarcane (kaur and gosal 2009) and rice (Ikram-ul-Haq et al. 2009). Present study we report that development of activation tagged lines based on  Ac-Ds transposon system in the indica rice cv BPT-5204, which is a high yielding and widely cultivated in India for its fine grain and best cooking quality, but the variety is susceptible to biotic and abiotic stresses. Partially desiccated calli originating from scutellar region of rice seeds are used as explants for Agrobacterium mediated transformation, resulted in increased transformation efficiency and a large number of Ds as well as Ac-Ds transgenic rice lines were obtained using this strategy.

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