Hatzilazarou Stefanos, Kostas Stefanos, Nendou Theodora, Economou Athanasios
Department of Horticulture, School of Agriculture, Aristotle University, 54124 Thessaloniki, Greece.
Polymers (Basel). 2021 May 20;13(10):1666. doi: 10.3390/polym13101666.
The present study demonstrates the potential of the alginate encapsulation of shoot tips and nodal segments of Ellis, the short-term cold storage of artificial seeds and subsequent successful conversion to desirable, uniform and genetically stable plantlets. Shoot tips and first-node segments below them, derived from shoots of in vitro cultures, responded better than second-to-fourth-node segments on agar-solidified Murashige and Skoog (MS) nutrient medium and thus, they were used as explants for alginate encapsulation. Explant encapsulation in 2.5% sodium alginate in combination with 50 mM of calcium chloride resulted in the production of soft beads, while hardening in 100 mM of calcium chloride formed firm beads of uniform globular shape, suitable for handling. The addition of liquid MS nutrient medium in the sodium alginate solution doubled the subsequent germination response of the beads. The maintenance of alginate beads under light favored their germination response compared to maintenance in darkness. Encapsulated shoot tip explants of gardenia, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in their regeneration response (73.3, 68.9, 53.3%, respectively), whereas, non-encapsulated explants (naked), stored under the same time durations of cold conditions, exhibited a sharp decline in regeneration response up to entirely zeroing (48.9, 11.1, 0.0%, respectively). Shoots, derived from 12-week cold-stored encapsulated explants, were easily rooted in solid MS nutrient medium with the addition of 0.5 μM of Indole-3-acetic acid (IAA) and after transplantation of the rooted plantlets individually to pots containing a peat-perlite (3:1, /) substrate, they were successfully acclimatized in the greenhouse under the gradual reduction of 75 or 50% shading with survival rates of 95-100%. The genetic stability of the acclimatized plantlets was assessed and compared with the mother plant using inter simple sequence repeat (ISSR) markers. ISSR analysis confirmed that all regenerated plantlets were genetically identical to the mother plant. This procedure of artificial seed production could be useful for the short-term storage of germplasm and the production of genetically identical and stable plants as an alternative method of micropropagation in .
本研究证明了对埃利斯的茎尖和节段进行藻酸盐包封、人工种子的短期冷藏以及随后成功转化为理想、均匀且基因稳定的植株的潜力。源自离体培养芽的茎尖及其下方的第一节段，在琼脂固化的Murashige和Skoog（MS）营养培养基上的反应优于第二至第四节段，因此，它们被用作藻酸盐包封的外植体。将外植体包封在2.5%的海藻酸钠中并结合50 mM的氯化钙，会产生软珠，而在100 mM的氯化钙中硬化则会形成形状均匀的坚硬球状珠，便于处理。在海藻酸钠溶液中添加液体MS营养培养基，使珠子随后的萌发反应加倍。与在黑暗中保存相比，在光照下保存藻酸盐珠有利于其萌发反应。将栀子的包封茎尖外植体在4℃下保存4、8或12周，其再生反应逐渐下降（分别为73.3%、68.9%、53.3%），而在相同冷藏时间下保存的未包封外植体（裸体外植体），其再生反应急剧下降直至完全为零（分别为48.9%、11.1%、0.0%）。源自冷藏12周的包封外植体的芽，在添加0.5 μM吲哚-3-乙酸（IAA）的固体MS营养培养基中很容易生根，将生根的植株单独移植到含有泥炭 - 珍珠岩（3:1，体积比）基质的花盆中后，它们在温室中通过逐渐减少75%或50%的遮荫成功驯化，成活率为95 - 100%。使用简单重复序列区间（ISSR）标记评估驯化植株的遗传稳定性并与母株进行比较。ISSR分析证实，所有再生植株在基因上与母株相同。这种人工种子生产方法对于种质的短期保存以及作为一种替代微繁殖方法生产基因相同且稳定的植株可能是有用的。
