Chaloushi Babak, Zarghami Reza, Abd-Mishani Cyrus, Omidi Mansour, Agayev Yusif M, Sardood Babak Pakdaman
Department of Biotechnology, Faculty for Agronomical and Animal Sciences, Tehran University, Karaj, Iran.
Pak J Biol Sci. 2007 May 15;10(10):1625-31. doi: 10.3923/pjbs.2007.1625.1631.
Saffron (Crocus sativus L.) is one of valuable and native plants in the land of Iran. By this investigation the best hormonal compositions for callus production from protoplast and for plantlet regeneration from callus were determined. To isolate protoplasts, the embryogenic calli were used. The embryogenic calli were immersed in enzymatic solution to degrade the cell walls. The treated mixture was filtered and then centrifuged at 100 g for 3-5 min and the resulted pellet was rinsed. After one step of washing and another step of centrifugation, the protoplasts were gently mixed with sterile sodium alginate solution and added to MS broth consisting 1% calcium chloride and 0.3 M manitol to form calcium alginates granules. The protoplast-containing granules were exposed to MS broth including 0.3 M manitol and various treatments of two kinds of auxins (2, 4-D and NAA) and three kinds of cytokinins (2ip, Kin, BAP), respectively in four rates of 0.1, 0.2, 0.5, 1.0 mg L(-1) for auxins and in three rates of 0.1, 0.2 and 0.5 mg L(-1) for cytokinins, incubated in dark at 22 +/- 2 degrees C for a period of 30 days. Out of all the treatment of 2, 4-D (1.0 mg L(-1)) and Kin (0.2 mg L(-1)) was the best in callus induction. In order to regenerate plantlets, the resulted calli were transferred to MS broth amended with different rates of ABA (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mg L(-1)) so that they could pass the steps of embryonic maturation. The mature embryos were transferred to MS media with different rates of GA3 (0.0, 5.0, 15.0, 20.0, 25.0 and 30.04 mg L(-1)) to initiate germination. The germinated embryos were then placed in solid MS media with various rates of NAA and 2, 4-D auxins (0.0, 0.5, 1.0 and 2.0 mg L(-1)) and different levels of BAP and Kin cytokinins (0.0, 0.5, 1.0 mg L(-1)). Results from statistical analyses indicated the treatment of NAA and BAP (each 1 mg L(-1)) as the best hormonal treatment for the plantlet regeneration from the domestic saffron calli.
藏红花（番红花）是伊朗本土的珍贵植物之一。通过本研究，确定了用于原生质体愈伤组织诱导及愈伤组织植株再生的最佳激素组合。为分离原生质体，使用胚性愈伤组织。将胚性愈伤组织浸入酶溶液中以降解细胞壁。处理后的混合物过滤后，以100 g离心3 - 5分钟，所得沉淀冲洗。经过一次洗涤和再次离心后，将原生质体与无菌海藻酸钠溶液轻轻混合，并加入含有1%氯化钙和0.3 M甘露醇的MS培养基中以形成海藻酸钙颗粒。将含原生质体的颗粒分别置于含有0.3 M甘露醇及两种生长素（2,4 - D和NAA）和三种细胞分裂素（2ip、Kin、BAP）不同处理的MS培养基中，生长素浓度分别为0.1、0.2、0.5、1.0 mg L(-1)，细胞分裂素浓度分别为0.1、0.2和0.5 mg L(-1)，在22±2℃黑暗条件下培养30天。在所有处理中，2,4 - D（1.0 mg L(-1)）和Kin（0.2 mg L(-1)）组合在愈伤组织诱导方面效果最佳。为使植株再生，将所得愈伤组织转移至添加不同浓度ABA（0.0、0.5、1.0、1.5、2.0和2.5 mg L(-1)）的MS培养基中，使其经历胚胎成熟阶段。将成熟胚转移至添加不同浓度GA3（0.0、5.0、15.0、20.0、25.0和30.04 mg L(-1)）的MS培养基中以启动萌发。然后将萌发的胚置于添加不同浓度NAA和2,4 - D生长素（0.0、0.5、1.0和2.0 mg L(-1)）以及不同水平BAP和Kin细胞分裂素（0.0、0.5、1.0 mg L(-1)）的固体MS培养基中。统计分析结果表明，NAA和BAP（均为1 mg L(-1)）处理是从国产藏红花愈伤组织再生植株的最佳激素处理方法。
