弗吉尼亚栎不定芽增殖及试管苗再生影响因子研究

目的]探析弗吉尼亚栎组织培养中不定芽增殖和生根的关键影响因子,建立弗吉尼亚栎试管苗再生体系,为弗吉尼亚栎无性系选育提供技术支撑。方法]以弗吉尼亚栎带芽茎段为外植体,采用不定芽增殖途径,研究弗吉尼亚栎组织培养过程中外植体选择、褐化控制以及培养基选择、激素配比对不定芽增殖和生根的影响。结果]表明:向培养基中添加抗坏血酸、硫代硫酸钠和活性炭均显著降低外植体的褐化半径(p<0.05),其中,外植体在含有3.00、5.00 g·L^-1活性碳的培养基中褐化半径最小。基本培养基筛选试验表明:以1/4MS或WPM为基本培养基时,平均芽长和芽数明显优于MS培养基。不定芽增殖最佳培养基为1/4MS+1.20 mg·L^-1 6-BA,培养周期40 d,增殖倍数可达6.6。最佳生根培养基为1/4MS+0.50 mg·L^-1 IBA+0.50 mg·L^-1NAA,生根率达53.33%,且根粗壮,木质化程度较高。组培苗移植到灭菌河沙中,平均成活率达57.78%。结论]培养基成分和激素配比是影响弗吉尼亚栎不定芽增殖和试管苗再生的主要因子,低盐培养基(1/4MS或WPM)不仅可以促进不定芽增殖,且能够减轻外植体褐化;还发现野外取材的外植体极易褐化,而来源于无菌苗的外植体,褐化程度明显低于野外取材的外植体。

Study on the Factors Affecting Adventitious Shoots Proliferation and in vitro Regeneration of Quercus virginiana

Objective] To study the factors that affect the adventitious buds development and rooting process, so as to develop an efficient in vitro propagation methodological system in live oak(Quercus virginiana). Method] Using stem segments as explants, the effects of basal medium, browning control, the origin of explant, and different hormone combinations on cluster buds proliferation and rooting were studied. Result] The results showed that the addition of ascorbic acid, sodium thiosulfate, and powdered activated carbon could effectively reduce the browning range of explants compared with the control(p<0.05), among which 3.00 g·L^-1 and 5.00 g·L^-1 powdered activated carbon were proved to be the most effective. The growth medium screening test showed that more and longer shoots were achieved on the low salt medium with a quarter of Murashige and Skoog(MS) medium or woody plant medium(WPM). And the browning was also alleviated with the low salt medium in this study. The highest proliferation rate(6.6 buds per explant) was achieved on a quarter of MS medium supplemented with 1.20 mg·L^-1 6-benzylaminopurine(BA) after 40 days. In vitro regenerated shoots were rooted on a quarter of MS medium supplemented with0.50 mg·L^-1 indole-3-butyric acid(IBA) and 0.50 mg·L^-1 naphthalene acetic acid(NAA) with the highest rooting rate53.33% and more lignified roots. The plantlet was transferred to sterile sand after 3-5 days acclimation in greenhouse and 57.78% survival rate was observed finally. Conclusion] Based on the repeated experiments during the past three years, it is confirmed that the basal medium composition and the proportion of hormone combination will be the major factors determining the proliferation of adventitious shoots and in vitro regeneration of Q. virginiana. The basal mediums that contain lower concentration of inorganic salt are proved to be favorable not only for the adventitious shoots proliferation, but also for alleviating the browning of explants. It is concluded that the explants from the field are much easier to become brown than that from sterile seedlings.