Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D.!Don
Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D.!Don
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DOI：
10.1007/s00425-003-1053-0
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发表时间：
2003-09-01
期刊：
影响因子：
4.3
通讯作者：
Harris, PJ
中科院分区：
文献类型：
--
作者：
Möller, R;McDonald, AG;Harris, PJ
在辐射松愈伤组织培养中诱导了管胞和石细胞的分化。在含有活性炭但不含激素的基本培养基上培养，由木质部条产生的愈伤组织中可分化出石细胞，而由下胚轴切段产生的愈伤组织中不分化出石细胞。下胚轴愈伤组织中分化的管胞次生壁呈螺旋状、梯状、网状或纹孔状，而木质部愈伤组织中分化的管胞次生壁呈网状或纹孔状。加厚的管胞和石细胞壁含有木质素，如与间苯三酚-HCl的红色反应所示。木质素的存在下，在分化的愈伤组织的细胞壁中确认使用裂解气相色谱-质谱法通过检测来自木质素的苯丙素类成分。木质素也检测到使用固态C-13交叉极化/魔角旋转核磁共振光谱和定量为巯基乙酸木质素。从分化的和未分化的愈伤组织分离的细胞壁的单糖分析表明，分化的愈伤组织的细胞壁含有较高比例的葡萄糖和甘露糖，与分化的细胞的次生细胞壁中存在较大比例的葡糖-和/或半乳糖葡糖-甘露聚糖一致。成功地开发了用于未分化的木质部来源的培养物的稳定转化的方案。在用含有来自辐射紫菜的npt II基因的编码区和cad基因的编码区的质粒进行生物射弹粒子轰击后建立转基因细胞系。通过NPTII-酶联免疫吸附试验证实了npt II基因在转基因株系中的表达。cad在转基因株系中的过表达导致肉桂醇脱氢酶（EC 1.1.1.195）表达的下调。
Tracheid and sclereid differentiation was induced in callus cultures of Pinus radiata D. Don by culturing on a basal medium containing activated charcoal but no phytohormones; sclereids differentiated in callus derived from xylem strips, but not in callus derived from hypocotyl segments. The tracheids differentiated in hypocotyl-derived callus had helical, scalariform, reticulated or pitted secondary cell-wall patterns, but those differentiated in xylem-derived callus had a reticulate or pitted pattern. The thickened tracheid and sclereid walls contained lignin as indicated by the red colour reaction given with phloroglucinol-HCl. The presence of lignin in the cell walls of differentiated callus was confirmed using pyrolysis gas chromatography-mass spectrometry by the detection of phenylpropanoid components derived from lignin. Lignin was also detected using solid-state C-13 cross-polarisation/magic-angle spinning nuclear magnetic resonance spectroscopy and quantified as thioglycolic acid lignin. Monosaccharide analyses of the cell walls isolated from differentiated and undifferentiated calli showed that the cell walls of the differentiated calli contained higher proportions of glucose and mannose, consistent with the presence of greater proportions of gluco- and/or galactogluco-mannans in the secondary cell walls of the differentiated cells. A protocol for the stable transformation of undifferentiated, xylem-derived cultures was successfully developed. Transgenic cell lines were established following Biolistic particle bombardment with a plasmid containing the coding region of the nptII gene and the coding region of the cad gene from P. radiata. Expression of the nptII gene in transgenic lines was confirmed by an NPTII-enzyme-linked immunosorbent assay. The overexpression of cad in the transgenic lines resulted in a down-regulation of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) expression.