Pullman G S, Zhang Y, Phan B H
Institute of Paper Science and Technology, 500 10th Street, Atlanta, GA 30318, USA.
Plant Cell Rep. 2003 Sep;22(2):96-104. doi: 10.1007/s00299-003-0674-x. Epub 2003 Jul 19.
Somatic embryogenesis (SE), the most promising technology for the large-scale production of high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and the uniformity of future U.S. forests. To be successful for commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine ( Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation percentages of loblolly pine, Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco], and Norway spruce ( Picea abies L., Karst.) were improved through the use of brassinolide. Brassinosteroids, which include brassinolide, are a relatively new group of natural plant growth regulators that are found in many plant species. They have been shown to have diverse, tissue-specific, and species-specific effects, including the stimulation of cell elongation and ethylene production and increasing resistance to abiotic stress. In our media, brassinolide was effective at concentrations ranging from 0.005-0.25 micro M. Using control medium (no brassinolide) and brassinolide-supplemented (0.1 micro M) medium, we achieved improved initiation percentages in loblolly pine, Douglas-fir, Norway spruce, and rice-15.0% to 30.1%, 16.1% to 36.3%, 34.6% to 47.4%, and 10%, respectively. Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of loblolly pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation. Initiation percentages in loblolly pine were improved through the combination of modified 1/2-P6 salts, 50 mg/l activated carbon (AC), adjusted levels of Cu and Zn (to compensate for adsorption by AC), 1.5% maltose, 2% myo-inositol (to raise the osmotic level, partially simulating the megagametophyte environment), 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid, 0.63 mg/l 6-benzylaminopurine, 0.61 mg/l kinetin, 3.4 mg/l silver nitrate, 10 micro M cGMP, 0.1 micro M brassinolide, and 2 g/l Gelrite. Across 12 open-pollinated families of loblolly pine, initiation percentages ranged from 2.5% to 50.7%, averaging 22.5%.
体细胞胚胎发生（SE）是先进育种和基因工程项目中大规模生产高价值针叶树最具前景的技术，有望在提高美国未来森林的生产力、可持续性和一致性方面发挥重要作用。为成功实现商业应用，SE技术必须适用于多种基因多样的树木。在我们主要关注的火炬松（Pinus taeda L.）中，理想基因型的起始培养往往很困难。通过使用油菜素内酯提高了火炬松、花旗松[Pseudotsuga menziesii (Mirb.) Franco]和挪威云杉（Picea abies L., Karst.）的起始培养率。油菜素内酯类化合物包括油菜素内酯，是一类相对较新的天然植物生长调节剂，存在于许多植物物种中。它们已被证明具有多种、组织特异性和物种特异性的作用，包括刺激细胞伸长和乙烯生成以及增强对非生物胁迫的抗性。在我们的培养基中，油菜素内酯在0.005 - 0.25微摩尔浓度范围内有效。使用对照培养基（不含油菜素内酯）和添加了油菜素内酯（0.1微摩尔）的培养基，我们分别使火炬松、花旗松、挪威云杉和水稻的起始培养率得到了提高——分别提高了15.0%至30.1%、16.1%至36.3%、34.6%至47.4%和10%。油菜素内酯使火炬松胚性组织的重量增加了66%，并刺激了火炬松和花旗松中更难培养的家系的起始培养，从而在一定程度上弥补了起始培养中的基因型差异。通过改良的1/2 - P6盐、50毫克/升活性炭（AC）、调整铜和锌的水平（以补偿AC的吸附）、1.5%麦芽糖、2%肌醇（提高渗透压，部分模拟大配子体环境）、500毫克/升酪蛋白氨基酸、450毫克/升谷氨酰胺、2毫克/升α - 萘乙酸、0.63毫克/升6 - 苄基腺嘌呤、0.61毫克/升激动素、3.4毫克/升硝酸银、10微摩尔环鸟苷酸、0.1微摩尔油菜素内酯和2克/升结冷胶的组合，提高了火炬松的起始培养率。在12个火炬松开放授粉家系中，起始培养率在2.5%至50.7%之间，平均为22.5%。
