Kumar Rakesh, Mamrutha Harohalli Masthigowda, Kaur Amandeep, Venkatesh Karnam, Grewal Anita, Kumar Raj, Tiwari Vinod
Biotechnology Engineering, University Institute of Engineering and Technology, Kurukshetra University, Kurukshetra, Haryana 136119 India.
ICAR-Indian Institute of Wheat and Barley Research (IIWBR), Karnal, Haryana 132001 India.
Physiol Mol Biol Plants. 2017 Oct;23(4):945-954. doi: 10.1007/s12298-017-0463-6. Epub 2017 Sep 22.
The availability of reproducible regeneration system through tissue culture is a major bottleneck in wheat improvement program. The present study has considered to develop an efficient callus induction and regeneration system using mature and immature embryos as explants in recently released agronomically superior spring wheat varieties. An efficient sterilization process was standardized using 0.1% HgCl and 70% ethanol for both seeds and embryos. The maximum possible combinations of plant growth regulators (PGRs) were evaluated for their effect on different wheat regeneration processes through tissue culture starting from callus to root induction. Picloram is found as an effective auxin with 87.63-98.67% callus induction efficiency in both explants. Supplementation of CuSO along with 2,4-D, zeatin in regeneration medium significantly enhanced the multiple shoot induction. The shoot development was achieved using full strength Murashige and Skoog's (MS) medium and root induction using half MS medium without PGRs. The optimized medium and method has resulted up to 100% regeneration irrespective of the genotype used with high reproducibility. Thus, the standardized regeneration system can be used in the regeneration of healthy plants from embryos rescued from interspecies crosses, transgenic production, induced mutation breeding and recently developed genome editing techniques for the procreation of wheat plants having novel traits.
通过组织培养获得可重复的再生系统是小麦改良计划中的一个主要瓶颈。本研究旨在利用最近发布的农艺性状优良的春小麦品种的成熟胚和未成熟胚作为外植体，开发一种高效的愈伤组织诱导和再生系统。使用0.1% HgCl₂和70%乙醇对种子和胚进行了有效的消毒处理标准化。评估了植物生长调节剂（PGR）的最大可能组合对从小麦愈伤组织到生根诱导的不同组织培养再生过程的影响。发现毒莠定是一种有效的生长素，在两种外植体中愈伤组织诱导效率为87.63 - 98.67%。在再生培养基中添加硫酸铜以及2,4 - D、玉米素显著提高了多芽诱导率。使用完全强度的Murashige和Skoog（MS）培养基实现芽的发育，使用不含PGR的半强度MS培养基进行生根诱导。无论使用何种基因型，优化后的培养基和方法都能实现高达100%的再生，且具有高重复性。因此，标准化的再生系统可用于从种间杂交拯救的胚、转基因生产、诱变育种以及最近开发的基因组编辑技术中再生健康植株，以培育具有新性状的小麦植株。
