Omirulleh S, Abrahám M, Golovkin M, Stefanov I, Karabaev M K, Mustárdy L, Mórocz S, Dudits D
Institute of Plant Physiology, Hungarian Academy of Sciences, Szeged.
Plant Mol Biol. 1993 Feb;21(3):415-28. doi: 10.1007/BF00028800.
A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the beta-glucuronidase (GUS) gene, under the control of the doubled enhancer element (the -208 to -46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to -389 bp from ATG) promoter of wheat, alpha-amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10-20 resistant calli, or GUS-expressing colonies after treatment of 10(6) protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80-90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.
已开发出一种适用于玉米的可重复且高效的转化系统，该系统基于将DNA直接导入胚性原生质体，并从原生质体衍生的转基因愈伤组织再生出可育植株。将含有β-葡萄糖醛酸酶（GUS）基因的质粒DNA导入HE/89基因型悬浮培养的原生质体中，该质粒DNA受来自花椰菜花叶病毒35S启动子的双增强子元件（-208至-46 bp上游片段）控制，并与小麦α-淀粉酶基因的截短启动子（距ATG最多-389 bp）相连。构建的转化载体携带新霉素磷酸转移酶（NPTII）或草丁膦乙酰转移酶（PAT）基因作为选择标记。所应用的DNA导入方案在处理10⁶个原生质体后至少产生了10 - 20个抗性愈伤组织或GUS表达菌落。微愈伤组织的活性GUS染色使得从GUS染色组织再生出芽成为可能。80 - 90%的卡那霉素或PPT抗性愈伤组织显示出GUS活性，并且从140多个克隆中再生出了转基因植株。Southern杂交和PCR分析均表明转化体的基因组DNA中存在导入的外源基因。由组织特异性单子叶植物启动子和病毒增强子元件组成的嵌合启动子在玉米植株中指定了与双子叶植物和其他单子叶植物中的完整花椰菜花叶病毒35S启动子所确定的相似表达模式。在老叶中发现GUS比活性最高，而在幼叶、茎和根中的活性逐渐降低。GUS的组织化学定位揭示了启动子在叶表皮、叶肉和维管束以及根的皮层和维管束柱中的功能。在根中，分生组织顶端区域和维管组织染色强烈。选择的转化体生长至成熟，并在杂交后获得了具有GUS活性分离的第二代幼苗。如Southern杂交所示，表达GUS的分离株也携带NPTII基因。
