【作者】 王克臣；

【导师】 李明；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2008， 博士

【摘要】 亚麻(Linum usitatissimum L.)是一种重要的经济作物,亚麻茎杆可以剥取高档纤维用于纺织业,籽粒可以榨制富含高不饱和脂肪酸的营养保健油品。黑龙江省是我国纤维亚麻的主要产区,随着人们生活水平的提高,发展亚麻生产显得越来越重要。本文选择特点各异的国产品种黑亚14和引进品种Opaline为代表,对纤维亚麻在器官发生和体细胞胚胎发生过程中的组织细胞学、生理生化特性、内源激素含量变化、RAPD标记进行了研究,以阐述其发生机理。研究结果如下:1.对亚麻器官发生途径的培养条件进行了优化,试验得出了不同基因型无菌苗的最佳消毒时间,10-15日龄的无菌苗活性较好,亚麻下胚轴是诱导愈伤组织最理想的取材部位,平放外植体对出愈最有利。器官发生途径的初始愈伤组织诱导的最佳培养基为MS+4mg/L IAA +2mg/L KT;诱导不定芽发生的最佳培养基为B5+2.5mg/L KT +1mg/L IAA;出苗后最佳生根诱导培养基为1/2B5+0.3mg/L IBA。初始愈伤组织诱导周期为20d左右,呈淡绿色,质地致密坚硬。不定芽诱导周期为30d,IAA与KT是诱导亚麻愈伤组织出芽的关键物质。本实验中基因型间生长趋势差异明显,添加1.5g/L的活性炭可降低愈伤组织褐化率。2.以体胚发生为目的的亚麻初始愈伤组织的诱导,需要高浓度的2,4-D和较低浓度6-BA,但是过高浓度的2,4-D会抑制出愈和愈伤组织的生长,最佳诱导培养基为MS+2mg/L 2,4-D +0.5mg/L 6-BA;体细胞胚胎诱导培养基为MS+0.5mg/L NAA +0.5mg/L 6-BA;胚性愈伤组织大多呈黄白色,小颗粒状、松散易剥离,表面湿润。基因型对体胚发生影响明显,本实验只在H14体胚诱导中得到了球形胚和心形胚。另外光的变化对亚麻体胚发生没有影响。3.组织细胞学研究表明,亚麻胚性愈伤组织细胞规则,细胞核大,细胞质浓,非胚性愈伤组织细胞体积大,细胞核小,细胞质稀薄。体细胞胚发生经历了胚性细胞团、2细胞、3细胞、多细胞原胚、球形胚、心形胚发育阶段,但是没有更远的发展。PAS反应中,H14外植体下胚轴的横切可见到皮层内侧有淀粉粒分布,OP外植体子叶的横切可观察到栅栏组织细胞有淀粉粒分布;H14和OP初始愈伤组织外缘淀粉粒较多。H14和OP胚性愈伤组织胚性细胞含有大量的淀粉粒,与细胞的进一步分裂和分化提供能量有关。4.透射电镜观察表明,初始愈伤组织细胞有非常明显的中央大液泡,细胞质被中央大液泡挤压成一薄层分布于细胞的边缘,细胞核被大液泡挤压,位于细胞的一角,但仍可观察到核仁的存在。胚性愈伤组织细胞壁与初始愈伤组织细胞壁相比加厚,细胞质浓厚,充满整个细胞,细胞核大,核仁明显,在靠近细胞膜的胞质区域里有较多的淀粉粒,还可观察到其内含有淀粉粒的叶绿体。非胚性愈伤组织细胞几乎完全为中央大液泡所占据,细胞质被挤压在四周,很难观察到细胞核和其它细胞器。褐化组织细胞器崩解基本没有细胞器。扫描电镜观察表明,亚麻初始愈伤组织表面较质密,细胞之间排列紧密,没有空隙;胚性愈伤组织表面较光滑,细胞形成团粒结构,形成团粒的细胞之间结合紧密,团粒与团粒之间结合松散,可观察到大的空隙,细胞形状较规则。非胚性和褐化愈伤组织表面形状不规则,有较多的覆盖物,呈片状,细胞形状不规则,体积较大。5.在胚性愈伤中的CAT、POD、可溶性蛋白、核酸和可溶性糖含量均高于非胚性愈伤组织,这些物质含量和组分的变化可能与胚性的维持和细胞的旺盛分裂分化有关。CAT、可溶性糖在无菌苗含量最高,CAT、核酸、可溶性蛋白、可溶性糖在褐化组织中最低,可以说明褐化愈伤组织的生理状态。POD在初始愈伤中含量最高,这与机械性损伤有关;SOD在无菌苗和不定芽期含量高,初始愈伤组织中的含量是最低的,与其抗氧化能力密切相关;可溶性糖等指标在原胚和球形胚时期均呈上升趋势,为体胚发生作好物质和能量上的准备。6.SDS-PAGE结果显示,非胚性愈伤组织蛋白染色非常浅,蛋白代谢非常弱。亚麻体胚发生各时期的主要蛋白质组分相似,体现了体胚蛋白质组分的稳定性。H14各个阶段的蛋白质范围约为24-94kD,OP各个阶段的蛋白质范围约为20-100kD。在亚麻体细胞胚胎发生早期,共出现了9条POD同工酶酶带,其中有6条带是在亚麻体胚发生早期各阶段陆续出现的,可以作为体细胞胚发生的生化标记。淀粉同工酶在亚麻体胚发生早期出现的条带很少,酶带在非胚性和褐化组织中较多,而且淀粉同工酶的条带数也因品种不同而不同。亚麻早期体胚发生过程中,各个阶段EST同工酶酶带颜色由深到浅,这与POD同工酶酶带颜色和淀粉同工酶酶带颜色的变化相反。7.内源激素水平和激素间的平衡对亚麻形态建成具有重要的调节作用。胚性愈伤组织中的IAA含量高于非胚性愈伤组织,而且在原胚期和球形胚时期都保持了较高的水平,褐化组织中的IAA含量最低。ABA在外植体中的含量最高,在原胚和球形胚时期较低,低含量水平的ABA有利于胚的形成。ZR的峰值出现在初始愈伤中,胚性愈伤中的ZR含量低于非胚性愈伤。胚性愈伤中的IAA/ZR远高于非胚性愈伤,这可能有利于胚性能力的表达。8.本实验中,亚麻染色体数2n=32,继代5次以后变异率为2.5%,继代9次后变异率为4.3%,既有数量变异,也观察到有染色体结构的变异。经RAPD标记,发现引物S10、S21、S24和S48扩增产物的电泳结果中,褐化组织比无菌苗及其它时期少一条带,说明在继代过程中(到褐化组织时),亚麻的基因组DNA发生了变异。更多还原

【Abstract】 Flax (Linum usitatissimum L.) is one of important economic crops, from flax stalk we can obtain high-grade fiber for textiles, and from seeds we can obtain high-grand oil which have rich unsaturated fatty acids. Heilongjiang province is one main production area of fibre flax in China. Along with the improvement of people’s living standard, it is gradually important to develop flax production. Two varieties, H14(domestic) and Opaline(foreign) were selected as test materials to study on organization cytology, characteristics of physiology and biochemistry, changes of endogenous hormones content and RAPD markers during organogenesis and somatic embryogenesis of flax, and explain its occurrence mechanism. Research results are as follows.1. Culture conditions for organogenesis in flax were optimized, we obtained optimal sterilization time of aseptic plantlet with different genotype, aseptic plantlet with 10-15d had good activity. Furthermore, flax hypocotyl was optimal material for callus induction, and the better position is horizontal onto the medium. The prime medium of initial callus induction was MS+4mg/L IAA +2mg/L KT, and the best medium of adventitious buds induction during organogenesis was B5+2.5mg/L KT +1mg/L IAA. The best rooting induction medium was 1/2B5+0.3mg/L IBA. Induction duration was 20d or so, and initial callus were weak green, compact and stiff. The adventitious buds induction period was about 30d. IAA and KT were key factor to induce bud of flax callus. There are different in growth of callus among genotypes. 1.5g/L activate carbon could lower the browning ratio of callus.2. For somatic embryogenesis,the initial callus induction of flax needed high concentration 2,4-D and low concentration 6-BA. but overabundance 2,4-D could inhibit induction and growth of callus, the prime induction medium was MS+2mg/L 2,4-D +0.5mg/L 6-BA. The induction medium of somatic embryogenic was MS+0.5mg/L NAA +0.5mg/L 6-BA. Mostly embryogenic callus were yellow-white, granule, incompact and wetting surface. Genotype had evident effects on somatic embryogenesis. We only gained globular and heart-shaped embryo in H14. Changes of light had not effect on somatic embryogenesis in flax.3. It was showed by cytology method that flax embryogenic callus consisted of regular shaped cells with dense cytoplasm, large nucleus while the nonembryogenic callus cells with large volume, small nucleus and shin cytoplasm. Embryogenic cell mass, 2-cell, 3-cell, proembryogenic masses, initial spherical embryo and hearted embryo were observed, but Somatic embryogenesis had not further development. It showed that the medial cortex had starch grains distribution in PAS reaction from cross section of H14 hypocotyls. Starch grains distribution in palisade tissue cells of cross section of OP cotyledon were observed. There were plenty of starch grains in outside cell of initial callus of H14 and OP. Embryogenic cell of H14 and OP accumulated large number of starch grains which might supply energy for cell division and differentiation. 4. It was showed by transmission electron microscope that the initial callus cells had large central vacuole, which squeezed its cytoplasm to a thin layer around the brim of cell. Nuclear distributed the corner of cells because of being squeezed by large vacuole, however we found the presence of nuclear. Compared embryogenic callus with initial callus its cell wall became thick, it had dense cytoplasm which was full of the whole cell, big nucleus with obvious nucleolus. Many starch grains and chloroplasts including starch grains distributed on the cytoplasm area of cell membrane. The cells of nonembryogenic callus were almost occupied by central large vacuole, and its cytoplasm was extruded. Nucleus and other organelles were hardly observed. Browning organelle had breakdown and had not organelle at all. As showed as followed by Scanning electron microscope that flax initial callus owned dense surfaces and there was little gap among cells, which shared silk-shaped attachment in the cell surface. On the contrary, embryogenic callus had smooth surfaces, and the embryogenic cells were closely conjuncted to form cell masses in which loosely connection and large empty were observed. The cells of cell masses were regular- shaped, and many of them exposed on the surface of embryogenic callus. Nonembryogenic callus and browning callus surface shape was irregular and covered with lamellar. They had different shape with large volume.5. The content of CAT, POD, soluble protein, nucleic acids and soluble sugar in embryogenic callus were higher than in nonembryogenic callus, these substance changes of content and components may be relevanted to maintain of embryogenic and hearty splitting and differentiation of cells. The content of CAT and soluble sugar was maximum in aseptic plantlet, the content of CAT, nucleic acids, soluble protein and soluble sugar callus were all lowest in browning tissue, which showed the physiological state of browning callus. The POD content was highest in initial callus tissue, this was relevant to mechanical damage. The SOD content was high in the aseptic plantlet and adventitious buds, and lowest in initial callus, this was relevant closely to antioxidant ability. The content of soluble sugar ect were gradually increased in proembryogenic and spherical embryogenic, which were prepared for substance and energy of somatic embryogenesis.6. The results of SDS-PAGE indicated that the protein staining of nonembryogenic callus was very light, and the metabolism of protein was very weak. The main protein components of each stage of flax somatic embryogenesis were very similar, which means the stability of somatic embryogenic components. The protein scope of each stage for H14 were 24-94kD, and 20-100kD for OP. It presented 9 POD isozyme strips during the initial somatic embryogenesis, 6 of those were presented continuously during the initial somatic embryogenesis, which were used for the biochemical markers of the initial somatic embryogenesis. The amylase isozyme strips were less in the initial somatic embryogenesis, nonembryogenic and browning callus, and more in nonembryogenic and browning callus, the number of amylase isozyme strips varied with different varieties. The EST isozyme strips color varied from deep to light during the stages of flax initial somatic, which is opposite to the variety of POD isozyme strips and starch isozyme strips.7. The level and the balance of endogenous hormones can adjust to flax morphogenesis. The content of IAA in embryogenic callus was higher than in nonembryogenic callus, and it maintained high level in proembryogenic period and globular embryogenic period, its content was lowest in browning callus. The content of ABA was highest in explant, its content was less in proembryogenic period and globular embryogenic period, low level of ABA was benefited to the embryogenesis. The peak value of ZR content present to initial callus, the ZR content of embryogenic callus was lower than in nonembryogenic callus, its content was the lowest in browning callus. The IAA/ZR ratio of embryogenic callus was far higher than in nonembryogenic callus, it may be benefit to the expression of embryogenic capability.8. The number of flax chromosome were 2n=32, the variation rate was 2.5% after 5 subculture, and 4.3% after 9 subculture in this test. It had quantity variation and the chromosome structure variation. According to RAPD markers, it was found that one strip had lost in browning callus compared with aseptic plantlet and other periods in the amplification product of S10、S21、S24 and S48, it was discerned that the genome DNA of flax had changed during subculture(add to browning callus).更多还原