Solórzano-Cascante Paúl, Sánchez-Chiang Neiva, Jiménez Víctor M
Centro para Investigaciones en Granos y Semillas, Universidad de Costa Rica, San Pedro, Costa Rica.
Instituto de Investigaciones Agrícolas, Universidad de Costa Rica, San Pedro, Costa Rica.
Front Plant Sci. 2018 Dec 4;9:1769. doi: 10.3389/fpls.2018.01769. eCollection 2018.
A protocol to propagate papaya hybrid plants through indirect somatic embryogenesis was developed considering the effect of explant type, culture system, particular cytokinins and encapsulation, in different stages of the process. Optimal 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations for non-embryogenic callus formation ranged between 9.0 and 27.1 μM in half-cut seeds, while higher concentrations were harmful. Non-embryogenic callus was also obtained with 22-158 μM 2,4-D from hypocotyl segments. Callus with embryogenic structures was only obtained in half-cut seeds cultured in the darkness on half-strength Murashige and Skoog culture medium supplemented with 2,4-D, while hypocotyl segments and isolated zygotic embryos failed to produce this type of callus regardless of the 2,4-D and sucrose (30 and 70 g l) concentrations tested in this study. Both, embryogenic callus development and quantity of somatic embryos formed per embryogenic callus, which ranged between 11 and 31 units after 14 months, required 2,4-D, but without any effect of the concentration. Histological studies confirmed the multicellular origin of the somatic embryos. In further steps, liquid medium induced over four times more somatic embryos than agar-gelled medium and showed significantly higher production of globular somatic embryos (85 vs. 57%). Both, 6-benzyladenine (BA) and meta-topolin (Mtop) stimulated sprouting (40-45%) of the somatic embryos (development of shoots only) in concentrations of up to 2.7 and 10 μM, respectively. Sprouting probability showed a 2nd order polynomial trend despite the range of concentration used for each cytokinin. This is the first report about the positive effect of Mtop on the apical shoot development of somatic embryos known to the authors. Radicle growth was observed in 5% or less of the cultivated embryos, regardless of the BA concentration. Finally, all encapsulation conditions tested (2.5, 3.5, and 4.5% sodium alginate, combined with 50 and 100 mM CaCl) reduced sprouting of somatic embryos when compared to the non-encapsulated ones, whereas capsule hardness showed low correlation with embryo sprouting. Embryos were further cultivated until they became plantlets approximately 5 cm long. They were acclimatized and afterward planted in the field, where they flowered and produced fruit.
考虑到外植体类型、培养系统、特定细胞分裂素和包封在该过程不同阶段的影响，制定了一种通过间接体细胞胚胎发生来繁殖番木瓜杂交植物的方案。在半切种子中，形成非胚性愈伤组织的最佳2,4-二氯苯氧乙酸（2,4-D）浓度范围为9.0至27.1μM，而较高浓度则有害。从下胚轴切段中使用22 - 158μM的2,4-D也可获得非胚性愈伤组织。仅在补充了2,4-D的半强度Murashige和Skoog培养基上黑暗培养的半切种子中获得具有胚性结构的愈伤组织，而下胚轴切段和分离的合子胚无论在本研究中测试的2,4-D和蔗糖（30和70 g/l）浓度如何，均未能产生这种类型的愈伤组织。胚性愈伤组织的发育以及每个胚性愈伤组织形成的体细胞胚数量（14个月后在11至31个单位之间）都需要2,4-D，但不受浓度影响。组织学研究证实了体细胞胚的多细胞起源。在后续步骤中，液体培养基诱导产生的体细胞胚比琼脂凝胶培养基多四倍以上，并且球形体细胞胚的产量显著更高（85%对57%）。6-苄基腺嘌呤（BA）和间-拓扑替林（Mtop）分别在高达2.7和10μM的浓度下刺激体细胞胚的发芽（仅芽的发育，发芽率为40 - 45%）。尽管每种细胞分裂素使用的浓度范围不同，但发芽概率呈现二阶多项式趋势。这是作者所知的关于Mtop对体细胞胚顶芽发育有积极影响的首次报道。无论BA浓度如何，在5%或更少的培养胚中观察到胚根生长。最后，与未包封的胚相比，所有测试的包封条件（2.5%、3.5%和4.5%的海藻酸钠，与50和100 mM的CaCl₂组合）均降低了体细胞胚的发芽率，而胶囊硬度与胚发芽的相关性较低。胚进一步培养直至长成约5厘米长的小植株。它们经过驯化，然后种植在田间，在那里开花结果。
