苜蓿花药悬浮培养体系的建立及其影响因素

苜蓿(Medicago sativa L.)花药悬浮培养体系的建立可为苜蓿体细胞杂交、遗传转化以及获得苜蓿单倍体,建立苜蓿加倍单倍体群体(DH)提供快捷有效的途径。采用固-液培养基结合的方式建立苜蓿花药悬浮培养体系,通过探索体系建立的基础条件及其影响因素,可建立适宜的苜蓿花药悬浮培养体系。结果表明,苜蓿悬浮培养体系中,基本液体培养基为NB培养基,培养液最佳组合为NB+2,4-D 0.3 mg·L^-1+NAA 0.3 mg·L^-1,蔗糖2%;建立苜蓿花药悬浮细胞系,应选取淡黄色或乳白色,结构疏松,颗粒状胚性愈伤组织为悬浮培养的基础材料;苜蓿花药悬浮细胞系继代周期为6~8 d;苜蓿花药悬浮培养时初始接种量为2g/30mL时悬浮细胞PCV、鲜重以及干重的增殖倍数最高,增殖倍数均超过4倍;植物生长调节剂2,4-D的添加浓度以0.3 mg·L^-1为宜;糖作为组织培养过程中唯一的碳源,在苜蓿细胞悬浮培养体系中应有一定的浓度范围。因此,苜蓿花药悬浮培养中,蔗糖浓度为2%-3%时效果最好。

Establishment of Alfalfa Anther Suspension Culture System and the Influencing Factors

Establishment of alfalfa anther suspension system will offer an effective approach for alfalfa somatic hybridization and genetic transformation,acquisition of alfalfa haploid,and creation of double haploid(DH).The objectives of this research were to established alfalfa suspension system and explore the basic condition and influencing factors.Combination of solid and liquid culture mediums was chosen to establish this system.The results show that NB was the basic liquid medium for the establishment of alfalfa suspension cell culture system and the best liquid medium combination was NB+2,4-D 0.3 mg/L+NAA 0.3 mg/L and sugar concentration was 2%.Embryogenic callus characterized with straw yellow or ivory yellow,graininess,loose structure should be chosen as basic material to alfalfa anther suspension cell system and the subculture cycle was 6-8 d.When the initial inoculation dosage was 2 g/30 mL,the proliferation multiples of Packed Cell Volume(PCV),wet weight,and dry weight of suspension cell was the highest and all of them were above 4 times.When establishing alfalfa suspension cell system,the ideal concentration of 2,4-D was 0.3 mg/L.Being the only carbon source in tissue culture,sugar should have a given concentration range during the establishment of alfalfa suspension cell culture system.And in alfalfa anther suspension culture,sugar concentration at 2%~3% had the best effect.