Sreelekshmi R, Siril E A
Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram 695581 India.
3 Biotech. 2021 Feb;11(2):95. doi: 10.1007/s13205-021-02645-7. Epub 2021 Jan 27.
The effective reversion of hyperhydricity (HH) in L. facilitated efficient in vitro production of hyperhydricity-free plantlets. Under routine sub-culture practice, the problem of HH arises after third sub-culture in agar (0.85%) gelled Murashige and Skoog (MS) medium containing 2.5 µM 6-benzyladenine (BA). To confirm the role of ethylene on hyperhydricity induction, an ethylene releasing compound ethephon (5 µM) was used in combination with 2.5 µM BA and demonstrated 100% HH with reduced stomatal aperture. Supplementation of 10 µM silver nitrate (AgNO) to 2.5 µM BA containing medium resulted HH reversion with reduced shoot number (19.0); however, addition of 5 µM cobalt chloride (CoCl) produced highest microshoots (202.0). The combination effect of AgNO (10 µM), CoCl (5 µM), and BA (2.5 µM) showed complete HH reversion and upheld normal microshoots (55.0) with reduced relative water content (78.3%). The Ag and Co salts regulate ethylene biosynthesis and thereby 50% reductions in HO content characterized by formation of green healthy shoots with proper stomatal morphology. The gene expression profile of 1-Amminocyclopropane-1-carboxylase synthase () and 1-Amminocyclopropane-1-carboxylic acid oxidase (1) showed reduced expression after the retroversion of microshoots in anti-ethylene reversion medium compared to hyperhydric shoot. In vitro raised shoots were rooted (93.3%) ex vitro by 10 mM IBA treatment and 92.2% plants were survived. The genetic stability of micropropagated plants were analyzed and proved that addition of low levels of heavy metal salt in the medium does not cause any variation in banding pattern. The protocol forwards a novel method to revert HH of in vitro cultures by adopting intermittent exposure of anti-ethylene compounds added in the medium and the procedure can be applied to many other plants facing similar HH problems.
The online version contains supplementary material available at 10.1007/s13205-021-02645-7.
解除石蒜的玻璃化现象（HH）有效促进了无玻璃化苗的高效离体生产。在常规继代培养操作中，在含有2.5 μM 6-苄基腺嘌呤（BA）的琼脂（0.85%）固化的Murashige和Skoog（MS）培养基中，第三次继代培养后出现了玻璃化问题。为了证实乙烯在玻璃化诱导中的作用，将乙烯释放化合物乙烯利（5 μM）与2.5 μM BA联合使用，结果显示100%出现玻璃化，气孔孔径减小。在含有2.5 μM BA的培养基中添加10 μM硝酸银（AgNO）可使玻璃化现象逆转，但苗数减少（19.0）；然而，添加5 μM氯化钴（CoCl）产生的微芽数量最多（202.0）。AgNO（10 μM）、CoCl（5 μM）和BA（2.5 μM）的组合效应显示玻璃化现象完全逆转，维持了正常的微芽数量（55.0），相对含水量降低（78.3%）。银盐和钴盐调节乙烯生物合成，从而使过氧化氢含量降低50%，其特征是形成具有适当气孔形态的绿色健康芽。与玻璃化苗相比，在抗乙烯逆转培养基中微芽逆转后，1-氨基环丙烷-1-羧酸合酶（）和1-氨基环丙烷-1-羧酸氧化酶（1）的基因表达谱显示表达降低。通过10 mM IBA处理，离体培养的芽在离体条件下生根率为（93.3%），植株存活率为92.2%。对微繁殖植物的遗传稳定性进行了分析，证明在培养基中添加低水平的重金属盐不会导致条带模式出现任何变化。该方案提出了一种通过间歇性暴露培养基中添加的抗乙烯化合物来逆转离体培养物玻璃化现象的新方法，该方法可应用于许多面临类似玻璃化问题的其他植物。
在线版本包含可在10.1007/s13205-021-02645-7获取的补充材料。
