响应面法优化麻疯树花粉离体萌发培养基的研究

麻疯树因其种子含油率较高,种子油提炼的生物柴油可部分替代汽油,而成为一种极具潜能的能源作物,但由于产量低,麻疯树在热带、亚热带的发展受到极大限制。杂交育种是提高产量的重要手段,杂交亲本花粉生活力的高低直接影响到育种的成效。因此,寻求麻疯树离体花粉萌发的最适培养基配方,探明花粉萌发培养基中各主要培养基成分间的交互作用对生产上麻疯树杂交结实率和种子产量的提高具有重要意义。该研究以麻疯树开花初期雄花上花药刚散粉时的成熟花粉粒为材料,采用Box-Behnken设计(Box-Behnken design,BBD)的响应面法,对麻疯树花粉离体萌发培养基中各主要培养基成分的浓度配比及各主要培养基成分的交互作用进行了研究。以花粉萌发率为响应指标,建立了4种营养成分(蔗糖、硼酸、硝酸钙、硝酸钾)与花粉萌发率的响应面模型,并对各主要培养基成分的浓度配比进行了优化。通过R软件进行响应面分析的结果表明:4因素对花粉萌发率的影响顺序为蔗糖硼酸硝酸钙硝酸钾;蔗糖与硼酸、蔗糖与硝酸钙、蔗糖与硝酸钾之间的交互作用显著。响应面建模优化后的最佳培养基为13.77%蔗糖+32.14 mg·L~(-1)硼酸+22.21 mg·L~(-1)硝酸钙+19.95 mg·L~(-1)硝酸钾+200 mg·L~(-1)硫酸镁,在此条件下的理论萌发率为99.73%。采用此培养基成分配比得到麻疯树花粉离体试验萌发率为98.97%,与理论响应值相吻合,同时也表明利用BBD设计的响应面模型进行麻疯树花粉离体萌发培养条件优化方法的有效性。

Optimal medium for pollen germination of Jatropha curcas in vitro using response surface methodology

Jatropha curcas is likely to become one of the key energy crops in the world because of high oil content in seed which can be refined into high quality biodiesel. J. curcas widely distributes and cultivates in many tropics and sub-tropics areas of China. Due to inadequate pollination and very low productivity, J. curcas was a wild or semi-wild tree for a long time. Effective pollination is a prerequisite for fruit-set and seed-set of J. curcas. Information on the most suitable ingredient concentration ratio of B-K medium which was widely used to pollen culture in vitro for pollen germination and inactions among ingredients in vitro is required to increase rate of hybrid seed set and seed production. Gathered mature pollen grains when petals opened and the anther just began scattering pollen in primary flowering time to do pollen germination test. Ingredient concentration ratio was optimized by response surface methodology(RSM)based on the Box-Behnken design(BBD). The primary variables chosen were sucrose, H₃BO₃, Ca(NO₃)₂·4H₂O and KNO₃. A total number of 30 runs were tested. We constructed a response surface model between the pollen germination rates and four ingredients for the culture medium in vitro by R 3.0 software. The effect order of 4 factors to influence germination rate were: sucrose > H₃BO₃ >Ca(NO₃)₂·4H₂O>KNO₃. There were significant interactions of pollen germination between sucrose and H₃BO₃, sucrose and Ca(NO₃)₂·4H₂O, sucrose and KNO₃. The optimal ingredient concentration ratio which supported the germination of pollen and growth of pollen tubes was 13.77% sucrose + 32.14 mg·L^-1 H₃BO₃+ 22.21 mg·L^-1 Ca(NO₃)₂·4H₂O + 19.95 mg·L^-1 KNO₃ + 200 mg·L^-1 Mg(SO₄)₂·4H₂O. Under this condition, the germination rate theoretically achieved 99.73%. The verification test were made corresponding to the optimal ingredient concentration ratio, the results of pollen germination experiments showed that pollen germination rates of 98.97%, the proportion matched theoretical response value well. It can also verify the effectiveness of the BBD-based response surface model and analysis technique in obtaining optimal ingredient concentration ratio for pollen germination.