巴西橡胶成龄无性系茎段的试管微繁技术研究

Micropropagation of Rubber Tree(Hevea brasiliensis) by Employing Mature Stem as Explants

巴西橡胶成龄无性系茎段组织培养在生产中有很大的潜在应用价值,但一直是橡胶树组织培养的难点,至今国内外鲜有成功的报道。本研究以在中国热带地区大面积推广种植的巴西橡胶树优良品种热研7-33-97不同生长时期成龄树茎段为外植体,进行无性系试管微繁技术研究。研究结果表明不同生长时期的茎段在自然条件中受污染程度不同,茎段真菌污染率为稳定期>淡绿期>古铜期,细菌污染率为淡绿期>稳定期>古铜期。不同生长时期的成龄树茎段需要通过不同的灭菌方法才能显著提高外植体的利用率,外植体灭菌方法显著影响橡胶成龄树茎段的组织培养效率;与稳定期相比,古铜期和淡绿期茎段在组织培养过程中诱导丛生芽萌芽快、萌芽多,是较优的外植体材料;古铜期和淡绿期茎段在激素配比为4.0～5.0mg/L 6-BA+0.5mg/LGA3的条件下能很好的诱导抽出丛生芽;丛生芽在激素配比为2.0mg/L 6-BA+0.5mg/L NAA,1.0mg/L 6-BA+1.0mg/LKT+0.5mg/L NAA或0.5mg/L 6-BA+1.5mg/LKT+0.5mg/L NAA的条件下能很好的诱导丛生芽抽出健壮的芽条;丛生芽抽出的健壮芽条在激素配比为0.5mg/LIBA+0.5mg/LIAA的条件下能较好的抽根成苗。外植体生长时期、激素种类和浓度配比都是影响巴西橡胶成龄无性系茎段的试管微繁的重要因素。

Tissue culture of Brazil mature-type asexual system stems holds a substantial potential value of application for production,which has been a difficulty for rubber(Hevea brasiliensis) tissue culture for a long time.And there is seldom successful report about it in the world yet.In this paper,we carry out micropropagation of rubber tree by using mature-type stems of Reyan 7-33-97,which is an excellent cultivar of Brazil rubber widely planted in tropical area,in different growth stage as explants.The results show that the stem segments in different growth stages have different contamination levels under normal conditions.Among them,stem fungal contamination rate at stable stage is the highest,light green stage takes the second place,while that at bronze stage is the lowest.and stem bacterial contamination rate at light green stage is highest,stable stage takes second place,while bronze stage has the lowest.Sterilization methods for mature stem at different growth period is different in order to significantly improve the utilization of the explant,howhever methods for explant sterilization significantly affect the tissue culture efficiency of mature rubber stems.Compared with stems at stable stage,adventitious buds in-duced by stems at bronze stage and light green stage sprout faster and germinated more during tissue culture.So the stems at these two stages are suitable as explants.Moreover,stems at bronze stage and light green stage maintained on medium supplemented with 4.0～5.0 mg/L 6-BA,0.5 mg/L GA3 can well induce multiple adventitious buds.Robust shoots were obtained from multiple adventitious buds cultured on medium supplemented with 2.0 mg/L 6-BA,0.5 mg/L NAA,1.0 mg/L 6-BA,1.0 mg/L KT and 0.5 mg/L NAA,or supplemented with 0.5 mg/L 6-BA,1.5 mg/L KT and 0.5 mg/L NAA.These shoots take roots and developed into seedlings on the medium supplemented with 0.5 mg/L IBA and 0.5 mg/L IAA.The growth stage of explants,hormone kinds and concentrations are the key factors of Brazil rubber micropropagation by using mature stem as explants.