Enhancing somatic embryogenesis of Malaysian rice cultivar MR219 using adjuvant materials in a high-efficiency protocol

Enhancing somatic embryogenesis of Malaysian rice cultivar MR219 using adjuvant materials in a high-efficiency protocol R. Abiri 0 1 2 3 4 5 6 M. Maziah 0 1 2 3 4 5 6 N. A. Shaharuddin 0 1 2 3 4 5 6 Z. N. B. Yusof 0 1 2 3 4 5 6 N. Atabaki 0 1 2 3 4 5 6 M. M. Hanafi 0 1 2 3 4 5 6 M. Sahebi 0 1 2 3 4 5 6 P. Azizi 0 1 2 3 4 5 6 N. Kalhori 0 1 2 3 4 5 6 A. Valdiani 0 1 2 3 4 5 6 0 Young Researchers and Elite Club of IAU , Kermanshah , Iran 1 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences , Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor DE , Malaysia 2 Editorial responsibility: Xu Han 3 Department of Biology, Faculty of Science, University Putra Malaysia (UPM) , 43400 Serdang, Selangor , Malaysia 4 IAU of Tehran Science and Research Branch , Tehran , Iran 5 Institute of Tropical Agriculture , Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor DE , Malaysia 6 Institute of Bioscience , Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor DE , Malaysia Enhancing of the efficient tissue culture protocol for somatic embryos would facilitate the engineered breeding plants program. In this report, we describe the reproducible protocol of Malaysian rice (Oryza sativa L.) cultivar MR219 through somatic embryogenesis. Effect of a wide spectrum of exogenesis materials was assessed in three phases, namely callogenesis, proliferation and regeneration. Initially, rice seeds were subjected under various auxin treatments. Secondly, the effect of different concentrations of 2,4-D on callus induction was evaluated. In the next step, the efficiency of different explants was identified. Subsequently, the effects of different auxins, cytokinins, L-proline, casein hydrolysate and potassium metasilicate concentrations on the callus proliferation and regeneration were considered. For the callogenesis phase, 2 mg L-1of 2,4-D and roots were chosen as the best auxin and explant. In the callus proliferation stage, the highest efficiency was observed at week eight in the MS media supplemented with 2 mg L-1 of 2,4-D, 2 mg L-1 of kinetin, 50 mg L-1 of L-proline, 100 mg L-1 of casein hydrolysate and 30 mg L-1 of potassium metasilicate. In the last phase of the research, the MS media added with 3 mg L-1 of kinetin, 30 mg L-1of potassium metasilicate and 2 mg L-1 of NAA were selected. Meanwhile, to promote the roots of regenerated explants, 0.4 mg L-1 of IBA has shown potential as an appropriate activator. Callogenesis; Proliferation; Potassium metasilicate and regeneration; Root explants - The production of high productive engineered plants needs appropriate strategies. The genetic transformation mechanism involves pivotal steps such as preparation of the initial conditions, usage of an efficient DNA delivery method, establishment of effective growth and selection of medium as well as maintenance of transformants (Abiri et al. 2015). Notwithstanding improvement of gene transformation efficiency in some plant species, the pre- and post-transformation conditions of Indica rice have been a matter of concern (Visarada and Sarma 2002). The efficiency ratio of engineered plants is severely genotype dependent (Yinxia and Te-chato 2012). On the other hand, presenting protocols of pre-transformation phases for the same species have revealed the vast effects of other factors on the plant’s adaptation to in vitro experiments. In this regard, size, source and age of explants, seasonal variation, oxygen gradient, intensity as well as quality of light, temperature and ploidy level are effective endogenous or exogenous factors which may change the genotype feedback in different experiments (Aggarwal et al. 2012). Therefore, the evaluation of useful endogenous or exogenous factors for differentiation and regeneration of Indica rice in vitro are pre-requirements in genetic transformation programs (Haque et al. 2003). Plant tissue culture is a symphony of art and science, which develops genetic diversity, produces virus-free plants and improves micropropagation under aseptic conditions in the short term (Birch 1997). Plant cells possess high plasticity potential for cell differentiation. Stresses such as pathogen infection or wounding may lead to the production of tumours or callus. The history of the first callus growth traced back to 1979 when Neely described a massive and disorganized cell mass in debarked trees (Neely 1979). Embryogenic calli, rather than direct tissues such as immature inflorescences, shoot spices, leaves and roots, is an effective and safe tool for regeneration of wild and modified plants in vitro conditions (Benlioglu et al. 2015). Interestingly, calli are divided to various subgroups according to their microscopic traits. For instance, calli with some organ regeneration are named embryonic, shooty or rooty calli, whereas calli without organ regeneration are called compact or friable callus (Ikeuchi et al. 2013). Callogenesis and growth highly depend on genotype, basal salt mediu (...truncated)