Aslam Mehtab Muhammad, Karanja Joseph K, Zhang Qian, Lin Huifeng, Xia Tianyu, Akhtar Kashif, Liu Jianping, Miao Rui, Xu Feiyun, Xu Weifeng
Center for Plant Water-Use and Nutrition Regulation, College of Life Sciences, Joint International Research Laboratory of Water and Nutrient in Cops, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
Sanming Academy of Agriculture Sciences, Sanming, Fujian 350002, China.
Plants (Basel). 2020 Mar 3;9(3):318. doi: 10.3390/plants9030318.
The tissue culture regeneration system of has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L + NAA 0.1 mg L) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L + NAA 0.1 mg L + BAP 1.67 mg L), and (KT 2.0 mg L + NAA 0.1 mg L) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L + KT 0.1 mg L (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes.
由于其基因型依赖性反应和低再生效率阻碍了基因工程的应用，一直被认为是难培养的材料。建立可重复的植物再生方案是成功应用基因工程的先决条件。本研究的目的是开发一种标准化、高效的方案，以成功地从白羽扇豆子叶节诱导芽。在本研究中，使用5日龄无菌培养的幼苗制备三种外植体（半子叶节、HCN；全子叶节、WCN；传统子叶节、TCN），在含有四种碳水化合物源（蔗糖、果糖、麦芽糖和葡萄糖）的四种浓度的M519培养基（M519、1/2 M519、1/4 M519）上培养，并用激动素（KT）和萘乙酸（NAA）的各种组合刺激以直接再生芽。以HCN为外植体，在1/2 M519培养基（KT 4.0 mg/L + NAA 0.1 mg/L）上获得了80%的高频率芽再生。有趣的是，（KT 4.0 mg/L + NAA 0.1 mg/L + 苄氨基嘌呤1.67 mg/L）和（KT 2.0 mg/L + NAA 0.1 mg/L）的组合显示出相似的60%的芽再生频率。添加0.25 g/L活性炭（AC）不仅降低了褐化效应，还促进了芽的伸长。在所有碳水化合物源中，蔗糖与HCN的再生频率最高。此外，培养28天后，在含有1.0 mg/L吲哚乙酸（IAA）+ 0.1 mg/L KT的1/2 M519培养基上记录到80%的生根频率。本研究描述了一种高效且成功的方案，用于从不同子叶节直接再生白羽扇豆植株。
