气动生物反应器油松胚性愈伤增殖体系的建立研究

Enhancement of embryogenic callus proliferation in Chinese pine(Pinus tabuliformis) by airlift bioreactor

【目的】大量具有活性的胚性愈伤组织的增殖可为油松体细胞胚胎发生研究和植株再生提供充足的材料,锥形瓶悬浮培养体系下胚性愈伤组织的增殖率低且易酸化坏死,建立气动生物反应器油松胚性愈伤增殖体系可促进油松胚性愈伤组织的增殖。【方法】本研究以油松愈伤组织为材料,利用L9(34)正交试验设计,探究了生物反应器中愈伤接种量、新旧培养基的配比和激素浓度3个因素对愈伤组织增殖的影响,建立了增殖体系。而后在相同条件下与锥形瓶悬浮培养进行对比。【结果】结果表明:气动生物反应器中每100 mL液体培养基内接种胚性愈伤10 g,2,4-D 0.2 mg/L,保留20%的旧培养基时能使胚性愈伤增殖率达到最高,可达216.18%;【结论】与锥形瓶悬浮培养体系相比,ALB系统在一周内胚性愈伤生长速度更快,为锥形瓶悬浮培养体系的3.15倍;显微观察显示增殖的胚性愈伤组织稳定且优质。该研究为基于油松体胚体系的大规模扩繁提供技术支持。

[Objective]The proliferation of a great number of active embryogenic callus can provide sufficient materials for somatic embryogenesis and plant regeneration of Pinus tabuliformis.However,the yield of flask suspension proliferation is limited and easy acidification necrosis.The establishment of airlift bioreactor system for embryogenic callus multiplication of P.tabuliformis can promote the proliferation of embryogenic callus of P.tabuliformis.[Method]In this study,P.tabuliformis callus was used as material,and L9(34)orthogonal design was used to investigate the effects of three factors on callus proliferation in bioreactor,including inoculation volume,ratio of old and new media and hormone concentration.Then the suspension culture in conical flask was compared with that in conical flask under the same conditions.[Result]The highest embryogenic callus proliferation rate,216.18%was obtained using 10 g embryogenic callus,0.2 mg/L 2,4-D and 20%old culture medium inoculated in 100 mL liquid medium in the ALB.[Conclusion]Compared with conical flask suspension culture,the growth rate of embryogenic callus in ALB system was 2.15 times faster in a week.Microscopic observation shows that the proliferated embryogenic callus is stable and high quality.This study provides technical support for large-scale propagation of P.tabuliformis based on somatic embryo system.