【作者】 郭英华；

【导师】 张振贤；

【作者基本信息】 中国农业大学 ， 蔬菜学， 2005， 博士

【摘要】 生姜（Zingiber.officinale Rosc.）是姜科、姜属能形成根状茎的一个栽培种。广泛分布于亚洲热带、亚热带地区,在南非、北美和澳大利亚也有栽培,是集调味品、食品加工原料和药用为一体的高效多用途蔬菜。生姜以根状茎作为繁殖材料,繁殖系数低,容易通过种姜传播病害,而且长期无性繁造成种性退化。由于姜很少开花结籽,因此无法通过有性杂交手段进行种质创新和品种改良。本论文以河南鲁山县张良姜、陕西城固黄姜、湖北枣阳生姜、四川竹根姜为试材,建立了生姜胚性愈伤组织诱导和植株再生体系、胚性悬浮系再生体系,获得了原生质体再生植株,分析了离体培养过程中的体细胞变异情况,获得如下结果: 1 以各生姜地方品种的茎尖组织为材料,诱导产生了胚性愈伤组织,建立了高频再生体系。筛选出了适于胚性愈伤诱导及扩增最佳培养基MSN+1.0mg·L^-12,4-D+0.2mg·L^-1KT+3.0%蔗糖+0.7%琼脂;胚性愈伤组织分化培养基MS+0.2mg·L^-12,4-D+5.0mg·L^-1BA+3.0%蔗糖+0.7%琼脂;根状茎形成培养基MS+3.0mg·L^-1BA+0.1mg·L^-1NAA+6.0%蔗糖+0.7%琼脂。植物激素、氮源对生姜茎尖胚性愈伤组织形成和再分化有较大影响。蔗糖比葡萄糖更利于根状茎的诱导,活性炭对根状茎诱导没有明显作用。不同品种间胚性愈伤组织诱导、愈伤组织再分化以及植株再生能力存在一定的差异。 2 以胚性愈伤组织为材料,建立了胚性细胞悬浮系,并获得了再生植株。结果表明悬浮细胞干重和pH之间有一定的相关性。接种量和AgNO₃含量对悬浮细胞的生长都有影响:最佳接种量为1.0%,最适AgNO₃浓度为6.0mg·L^-1。不同品种间胚性细胞悬浮系的生长能力不同。悬浮系愈伤组织的分化能力低于其对应的胚性愈伤组织分化能力。 3 利用生姜胚性细胞悬浮系成功分离得到原生质体,获得SC姜再生植株。酶种类和浓度、渗透压调节剂种类和浓度对分离原生质体有重要影响,最佳酶液组成为4.0%纤维素酶、1.0%离析酶、0.1%果胶酶、11%甘露醇、0.5%CaCl₂和0.1%MES。原生质体产量达到6027×10⁶g^-1,原生质体活力为90.7%。悬浮培养时间对分离原生质的产量和活力有影响。葡萄糖比蔗糖更利于纯化后原生质体分裂和生长。低浓度的AgNO₃(2.0-6.0mgl^-1)可以降低原生质愈伤组织褐化速度。不同品种间在原生质体分裂、愈伤组织分化、植株再生方面有显著差异。 4 对各品种生姜的胚性愈伤组织再生植株、胚性悬浮系再生植株、原生质体再生植株进行形态学、细胞学、分子生物学鉴定。结果表明,再生植株外部形态没有明显变异。再生植株的染色体数目没有变异。RAPD多态性分析表明,再生植株存在较广泛的体细胞变异。对各品种胚性愈伤组织、胚性悬浮系愈伤组织、原生质体再生愈伤组织进行细胞学鉴定和RAPD分析表明,愈伤组织的体细胞变异率远远高于再生植株的体细胞变异率。继代次数对体细胞变异率也有影响,随着继代时间的延长,体细胞变异率有上升趋势。更多还原

【Abstract】 Ginger (Zingiber officinale Rose.) is a cultivar producing rhizomes of the family Zingiberaceae. It is mostly distributed in the Asian tropical and subtropics region. And South Africa, North America and Australia also plant gingers. Gingers are rich in volatile oils and widely used as a condiments, dyes, beverage, medicine and vegetable. Gingers annually propagate themselves by rhizomes with low proliferation rate. They are heavily attacked by soil-born pathogens. And the quality of rhizomes degenerates for the long-term agamic propagation. Gingers seldom flowers and have seeds, so it is difficult to cultivate new variety through crossbreeding. In this paper, in vitro tissue culture techniques were carried out including somatic and embyogenic callus induction, embryogenic susppension culture and protoplast culture.1 Somatic embryogenic calli of 4 ginger genotypes were produced. Somatic embryogenic callus was induced from ginger shoot tips on MS medium supplemented with 1.0 mg/ L 2,4-D and 0.2mg·L-1 KT. The callus (about 1.5cm in diameter) were transformed into the MS+ 0.2mg·L-12,4-D+5.0mg·L-1 BA +3%sucrose+0.7%agar, and shoots and roots were formed. And shoots developed into complete plantlets with rhizomes on solid MS medium supplemented with 3.0mg·L-1 BA and 0.1mg·L-1NAA and 6% sucrose. The effect of phytohormones on embryogenic callus formation and redifferenciation was discussed. The effect of sucrose on rhizome formation was researched.2 Somatic embryogenic suspension cultures of 4 ginger genotypes were established. From which, regenerated plantlets were obtained. The relationship between the dry weight (DW) of suspension cultures and pH changes in medium was also discussed. The effects of inoculation and AgNO3on the growth of embryogenic suspensions were also discussed. The optimum concentration of inoculation and level of AgNO3 were 1.0% and 6.0 mg · L-1, respectively.3 Protoplasts were isolated from embryogenic suspensions incubating in enzyme solution containing 4.0% cellulose Onozuka R10, 1.0% maceroyme R10,0.1%PectolyaseY-23,0.5% CaCl2, 0.1% MES and 11% mannitol. The enzyme mixture were kept in a stationary position for 6 h and shaken (60 rpm) for 6 h at 27℃:. The yield and viability were 6.27 × 106g-1 and 90.7%, respectively. Purifying protoplasts were cultured initially in shallow liquid layers.4 The chromosome numbers of embryogenic callus-derived plants, embyogenic suspension-derived plants and protoplast-derived plants were 22, equal to their parent plants. There were differences of morphological characters between in vitro plants and parent plants such as leaf thick, leaf color and leaf index. The RAPD analysis of regenerated plants indicated relatively somatic variation. The chromosome number determination and RAPD analysis of embyogenic callus and embryoenic suspension-derived and protoplast-derived callus showed greater somatic variation than those of regenerated plants. Somatic variation frequency of in vitro plants was increased with the increasing subculture time.更多还原