Krishnan Subramanian Radhesh, Priya Arumugam Mohana, Ramesh Manikandan
Department of Biotechnology, Alagappa University, Karaikudi, 630 003, Tamil Nadu, India.
Bot Stud. 2013 Dec;54(1):47. doi: 10.1186/1999-3110-54-47. Epub 2013 Oct 21.
Cereal crops are the major targets for transformation mediated crop improvement and IR36 is an early maturing, high yielding, insect and disease resistant rice variety however, it is abiotic stress sensitive. Hence, development of an efficient and reproducible micropropagation system via somatic embryogenesis and Agrobacterium tumefaciens mediated transformation is prerequisite to develop abiotic stress tolerant IR36. Further, Genetic stability of analysis of plantlets through RAPD and ISSR and Ploidy level through Flow cytometry (FCM) measurement of 2C DNA content is necessary for future application of transformed IR36.
In this study, Mature seeds inoculated on (Murashige and Skoog) MS medium with 11.31 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.3 μM Kinetin (Kn) had highest callus induction frequency (98%). The highest regeneration frequency (80%) was observed in MS + 13.28 μM Benzyladenine (BA) with 8.06 μM α-naphthalene acetic acid (NAA). Randomly Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Flow Cytometry (FCM) analysis showed no significant variation in the 2C DNA (0.81 pg/2C) content and Ploidy level between wild type IR36 and in vitro maintained rice lines. Of the various OD bacterial culture, an optimum OD of 0.4 and inoculation duration of 10 min resulted in efficient Agrobacterium-mediated transformation. β-glucuronidase activity was maximum in callus (99.05%).
These results described here confirm the reliability of this protocol for micropropagation and delivery of desirable gene using A. tumefaciens into indica rice.
谷类作物是通过转化介导进行作物改良的主要目标，IR36是一种早熟、高产、抗病虫害的水稻品种，然而，它对非生物胁迫敏感。因此，通过体细胞胚胎发生和根癌农杆菌介导的转化建立一个高效且可重复的微繁殖系统是培育耐非生物胁迫IR36的先决条件。此外，通过RAPD和ISSR分析植株的遗传稳定性以及通过流式细胞术（FCM）测量2C DNA含量来确定倍性水平对于转化后的IR36的未来应用是必要的。
在本研究中，接种在含有11.31 μM 2,4-二氯苯氧乙酸（2,4-D）和0.3 μM激动素（Kn）的（Murashige和Skoog）MS培养基上的成熟种子具有最高的愈伤组织诱导频率（98%）。在含有13.28 μM苄基腺嘌呤（BA）和8.06 μM α-萘乙酸（NAA）的MS培养基上观察到最高的再生频率（80%）。随机扩增多态性DNA（RAPD）、简单序列重复区间（ISSR）和流式细胞术（FCM）分析表明，野生型IR36和离体保存的水稻品系之间的2C DNA（0.81 pg/2C）含量和倍性水平没有显著差异。在各种OD值的细菌培养物中，最佳OD值为0.4且接种持续时间为10分钟可实现高效的农杆菌介导转化。β-葡萄糖醛酸酶活性在愈伤组织中最高（99.05%）。
此处描述的这些结果证实了该方案用于微繁殖以及使用根癌农杆菌将所需基因导入籼稻的可靠性。
