Jin Hong, Jia Jing-Fen, Hao Jian-Guo
Provincial Key Laboratory of Biotechnology, Northwest University, Xi'an 710069, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Mar;20(2):221-6.
An efficient protocol for plant regeneration from protoplasts of the methionine resistant variant of Astragalus melilotoides was established. The friable calli induced from internode segments of variant plants were used for protoplast preparation. The protoplasts were isolated through enzyme digestion. Calli were formed after sustained divisions of protoplasts. High frequency of shoot differentiation was obtained from the protocalli on differentiated medium. The effects of different media, culturing methods and plating densities on protoplast divisions and plant regeneration were studied. The results show that agarose-beads culture method, KM8p medium supplemented with 1.0 mg/L 2,4-D, 0.5mg/L 6BA, 0.3 mol/L mannitol, 2% (W/V) sucrose and 500 mg/L casein hydrolysate at a plating density of 3 x 10(5)/mL are the appropriate conditions for protoplast division of the methionine resistant cell line. The division frequency is over 38%. The protoplast-regenerated plants still preserve resistance to methionine and ethionine.This research builds up the foundation for the resistant cell line as a parent of somatic hybridization.
建立了一种从草木樨抗蛋氨酸变异体原生质体高效再生植株的方案。从变异植株的节间段诱导出的易碎愈伤组织用于原生质体制备。通过酶解分离原生质体。原生质体持续分裂后形成愈伤组织。从分化培养基上的原生愈伤组织获得了高频芽分化。研究了不同培养基、培养方法和接种密度对原生质体分裂和植株再生的影响。结果表明，琼脂糖珠培养法、添加1.0mg/L 2,4-D、0.5mg/L 6-BA、0.3mol/L甘露醇、2%（W/V）蔗糖和500mg/L水解酪蛋白的KM8p培养基，接种密度为3×10⁵/mL，是抗蛋氨酸细胞系原生质体分裂的适宜条件。分裂频率超过38%。原生质体再生植株仍保留对蛋氨酸和乙硫氨酸的抗性。本研究为将抗性细胞系作为体细胞杂交亲本奠定了基础。
