Synergistic effects of L-glutamine and inorganic nitrogen molar ratios enhance the induction of somatic embryogenesis of Pinus maximinoi H.E. Moore

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS ACS Applied Bio Materials Pub Date : 2024-04-06 DOI:10.1007/s11240-024-02748-y

P. S. Nzama, A. A. Myburg, P. N. Hills

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Abstract

Clonal breeding programs of Pinus maximinoi require the establishment of a robust somatic embryogenesis (SE) protocol to produce enough cell lines to accelerate the effective continuous deployment of elite planting stocks to research and commercial compartments. Somatic embryogenesis was induced from immature zygotic embryo explants enclosed in megagametophytes of P. maximinoi collected from two plantations located in different climatic conditions. Cones were collected during the winter months from July to August and the influence of seed family, cone collection date and culture medium formulation, with emphasis on the organic and inorganic nitrogen supply, were studied. Ammonium to nitrate molar ratios of 1:1 and 1:2 in modified Litvay’s medium (mLV) produced the highest numbers of extrusions, while a 1:4 ratio mostly produced unhealthy, non-embryogenic extrusions. The formation of a tissue showing a rapidly-proliferating, spiky morphotype was produced in a medium supplemented with 1.5 g/L of L-glutamine. Morphologically advanced cultures with nodular structures were produced in megagametophytes from both plantations in a 1:2 NH₄⁺:NO₃⁻ medium regardless of L-glutamine supplementation levels. The optimal medium for P. maximinoi SE induction contained a 1:2 NH₄⁺:NO₃⁻ molar ratio with 1.5 g/L L-glutamine. The synergy between the molar ratio of NH₄⁺:NO₃⁻ and L-glutamine resulted in the highest numbers of extrusions. The overall inductive competence window for somatic embryogenic response in P. maximinoi was determined to be from the second week of July to the first week of August for both plantations. The “peak” period was in the fourth week of July 2022. The success of the SE technology in P. maximinoi seed families is determined by the optimal inductive competence window of the immature megagametophytes enclosing zygotic embryos and the chemical composition of the induction medium in terms of the ammonium to nitrate molar ratio and the concentration of the L-glutamine used.

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左旋谷氨酰胺和无机氮摩尔比的协同效应可促进格大松（Pinus maximinoi H.E. Moore）的体细胞胚胎发生

摘要毛冠松树的克隆育种计划需要建立一套强有力的体细胞胚胎发生（SE）方案，以产生足够多的细胞系，从而加快在研究和商业领域持续有效地部署精英种植种群。体细胞胚胎发生是从位于不同气候条件下的两个种植园中采集的大冠松树巨型叶内的未成熟子胚外植体中诱导出来的。锥果采集于冬季的 7 月至 8 月，研究了种子种类、锥果采集日期和培养基配方的影响，重点是有机氮和无机氮的供应。在改良利特维培养基（mLV）中，铵与硝酸的摩尔比为 1:1 和 1:2，产生的挤出物数量最多，而 1:4 的比例大多产生不健康、无胚胎的挤出物。在添加了 1.5 克/升 L-谷氨酰胺的培养基中，可形成快速增殖的尖刺状组织。在 1:2 NH4+:NO3- 的培养基中，无论补充多少 L-谷氨酰胺，两个种植园的巨型植物都能产生具有结节结构的形态高级培养物。诱导 P. maximinoi SE 的最佳培养基包含 1:2 NH4+:NO3- 摩尔比和 1.5 克/升 L-谷氨酰胺。NH4+:NO3- 与 L-谷氨酰胺摩尔比之间的协同作用可产生最高数量的挤压。经测定，两个种植园中 maximinoi 的体细胞胚胎发生反应的总体诱导能力窗口期均为 7 月第二周至 8 月第一周。2022年7月第四周为 "高峰期"。体细胞胚胎诱导技术在大叶黄杨种子家庭中的成功取决于未成熟巨型叶绿体包围子代胚胎的最佳诱导能力窗口，以及诱导培养基的化学成分（铵与硝酸盐摩尔比和所使用的 L-谷氨酰胺浓度）。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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