Tippani Radhika, Nanna Rama Swamy, Mamidala Praveen, Thammidala Christopher
1Department of Biotechnology, Kakatiya University, Warangal, Telangana 506 009 India.
3Department of Biotechnology, Telangana University, Nizamabad, Telangana India.
Physiol Mol Biol Plants. 2019 Mar;25(2):569-579. doi: 10.1007/s12298-018-0602-8. Epub 2018 Nov 16.
An efficient plantlet regeneration protocol using immature zygotic embryos (IZEs) via somatic embryogenesis has been developed in Roxb. The regenerated plantlets were evaluated for their genetic stability. IZEs were incubated on Murashige and Skoog (MS) media augmented with 1.07-16.11 μM naphthalene acetic acid (NAA) or 0.90-13.97 μM 2,4-dichlorophenoxyacetic acid. The optimum callus induction (96.6%) was observed on MS medium augmented with 5.37 μM NAA. Induction of somatic embryos (SEs) was observed after sub-culture of calli on medium with decreased concentrations of NAA (0.54-5.37 μM), either alone or 2.69 μM NAA in combination with 2.22-8.90 μM benzyladenine (BA) or 2.32-9.30 μM Kinetin. Maximum number (33.4 ± 0.85) of SEs occurred on MS medium augmented with 2.69 μM NAA + 4.40 μM BA + 3% sucrose. Highest percentage (67.3 ± 0.37) of SEs matured and developed into cotyledonary stage by subsequent subculture on the same medium. SE formation and maturation decreased when sucrose concentrations were higher than 3%. Seventy percent of mature somatic embryos developed into plantlets on half strength MS medium augmented with 5.80 µM gibberellic acid. The various stages of development during somatic embryogenesis include globular, heart, torpedo and mature stages as revealed by the stereomicroscopic and histological studies of explants. Plantlets derived from SEs were successfully acclimatized in the greenhouse with a survival rate of 78%. Among the survived plantlets, 9 plantlets were randomly selected for inter-simple sequence repeat (ISSR) analysis. Of the 13 primers used, 8 produced reproducible and monomorphic bands. ISSR analysis revealed a homogenous amplification profile for all regenerated plantlets analyzed validating the genetic stability of somatic embryo derived plantlets.
已开发出一种利用未成熟合子胚（IZEs）通过体细胞胚胎发生高效再生植株的方案。对再生植株的遗传稳定性进行了评估。IZEs在添加了1.07 - 16.11 μM萘乙酸（NAA）或0.90 - 13.97 μM 2,4 - 二氯苯氧乙酸的Murashige和Skoog（MS）培养基上培养。在添加5.37 μM NAA的MS培养基上观察到最佳愈伤组织诱导率（96.6%）。将愈伤组织继代培养在NAA浓度降低（0.54 - 5.37 μM）的培养基上，单独使用或与2.69 μM NAA和2.22 - 8.90 μM苄基腺嘌呤（BA）或2.32 - 9.30 μM激动素联合使用时，观察到体细胞胚（SEs）的诱导。在添加2.69 μM NAA + 4.40 μM BA + 3%蔗糖的MS培养基上，体细胞胚的数量最多（33.4 ± 0.85）。通过在同一培养基上继代培养，最高比例（67.3 ± 0.37）的体细胞胚成熟并发育到子叶期。当蔗糖浓度高于3%时，体细胞胚的形成和成熟减少。70%的成熟体细胞胚在添加5.80 μM赤霉素的1/2强度MS培养基上发育成植株。通过对外植体的体视显微镜和组织学研究表明，体细胞胚胎发生过程中的各个发育阶段包括球形、心形、鱼雷形和成熟阶段。源自体细胞胚的植株在温室中成功驯化，成活率为78%。在存活的植株中，随机选择9株进行简单序列重复区间（ISSR）分析。在所使用的13种引物中，8种产生了可重复的单态条带。ISSR分析显示，所有分析的再生植株具有均匀的扩增图谱，验证了体细胞胚衍生植株的遗传稳定性。
