【作者】 徐彬；

【导师】 王广东； 张效平；

【作者基本信息】 南京农业大学 ， 园林植物与观赏园艺， 2005， 硕士

【摘要】 花烛(Anthurium andraeanum),又名红掌、安祖花,为天南星科(Araceae)花烛属植物,是世界著名的热带花卉。目前,大多数花烛品种用组织培养方法繁殖。但是,花烛品种间基因型差异大,很多商品价值较高的品种没有建立离体再生体系;另外,目前使用的愈伤组织器官发生途径容易引起花烛离体系的生活力衰退和变异。因此,系统研究花烛离体再生中的各种影响因子,探讨几种离体再生途径和增殖方式是解决花烛离体快繁体系中存在问题的根本方法。 本研究首先以叶片为外植体,对几个花烛品种的无菌系的建立进行了研究。结果表明,在无菌水中切割外植体能够有效避免外植体褐化现象的发生;外植体从母株取材后,在高浓度的6-BA和2,4-D溶液中浸泡20min能够有效促进外植体进行脱分化;愈伤组织的发生需要在黑暗条件下诱导;各个品种之间的脱分化和再分化能力不同,对不同浓度的生长调节剂组合反应也不相同,诱导各花烛品种脱分化的适宜培养基为:MN200+0.5～lmg/L6-BA和0.1～0.8mg/L2,4-D;不定芽分化的适宜培养基为:MN200+0.25～0.5mg/L6-BA+0.15mg/L KT+0.2mg/L TFIBA+6g/L琼脂。 部分花烛品种(‘B’和‘B6’)能够在适宜的培养条件下(1/2MS+0.25mg/L 6-BA+0.14mg/L KT+0.2mg/L IAA+0.6%Agar+3%Sucrose,pH 5.5),诱导不定芽和体细胞胚的直接发生。品种‘C3’的愈伤组织在培养基(1/2MS+0.25mg/L 6-BA+0.2mg/LTFIBA+0.2%活性炭)中,有体细胞胚胎的间接发生途径存在。通过组织解剖观察发现,体细胞胚内存在呈极性分布含有丰富淀粉粒结构的细胞;由愈伤组织发育来的不定芽有两种起源方式,由愈伤组织表层分化芽原基的外起源和愈伤组织深层部位分化芽原基的内起源;薄壁细胞和分生原基中普遍存在具有结晶体的分泌细胞;愈伤组织深层的结节组织能够特化为成熟的螺纹式导管。 以花烛品种‘B’为材料,研究了不同植物生长调节剂配比、蔗糖浓度、光照、培养方式等对腋芽增殖的影响,并比较了‘AO’、‘B’、‘B6’等三个品种间的腋芽增殖能力。结果表明:在液体振荡培养下,以Nitsch+1.0mg/L KT+0.5mg/L 6-BA+30g/L蔗糖为培养基,在黑暗下振荡培养25d后转置光下进行液体静置培养,对花烛增殖效果较好;品种间腋芽增殖能力存在差异,但都可以在该体系下快速增殖。腋芽增殖后,将无菌苗转接入固体继代培养基中生根壮苗,45d后切分腋芽能够有效节省切分腋芽中的劳动力。与通过愈伤组织获得的无菌苗相比,通过腋芽增殖获得的无菌苗腋芽发更多还原

【Abstract】 Anthurium andraeanum, one of the most popular tropical flowers, belongs to Araceae. Nowadays, most Anthurium cultivars were propagated by ways of tissue culture. Yet, thanks to the chiasm of genotypes’ in vitro regeneration capabilities, many high valued cultivars have no in vitro regeneration system; and the micropropagation method through indirect shoot-genesis via callus formation gives rise to fading vigor of plantlets and off-types. Thus, investigating the factors affecting in vitro regeneration and multiplication ways systematically is the ultimate solvent.Firstly, the in vitro culture systems of several Anthurium cultivars were established using leaf as explant. Sectioning fully expanded young lamina under sterile water could minimize the browning rate of explant; immersing young lamina cut from potted plant in a solution containing high concentration of 6-BA and 2,4-D could promote dediferentiation of explant; callus should be induced under darkness; according to different cultivars, the dedifferentiation and redifferentiation rate varied and different combinations of auxin and cytokinin should be used; generally, the combinations of 0.5~lmg/L 6-BA and 0.1~0.8mg/L 2,4-D added into MN200 (modified Nitsch basal medium) were suitable to most cultivars to be induced into dedifferentiating; the suitable media for redifferentiation were: MN200 + 0.25~0.5mg/L 6-BA + 0.15mg/L KT + 0.2mg/L TFIBA + 6g/L agar.Some cultivars of Anthurium (’B’ and ’B6’ for example) could be induced to regenerate directly through adventitious shoots or somatic embryos under suitable culture condition ( 1/2MS+0.25mg/L 6-BA+0.14 mg/L KT+ 0.2 mg/L IAA+0.6 % Agar+3 % Sucrose, pH 5.5 ) , while some cultivars could not (’A0’ , ’B1’ , ’C3’ for example). The calli of cultivar ’C3’ could regenerate indirect somatic embryo emergence in one medium (l/2MS+0.25mg/L 6-BA+0.2 mg/L TFIBA+0.2%AC). Polar distributed starch-rich cells were found specifically in somatic embryos; the adventitious shoots regenerated from callus could drive from either outer layers or inner layers of callus; excretory cells containing crystal were found universally in parenchyma and meristem tissues; spiral vessels derived from nodules in the inner callus transporting growth substance and nutrients were oberserved indicating that callus might have primary differentiated structures.Taking Anthurium andraeanum Cultivar. ’B’ as testing material, different combinations of plant growth regulators, concentrations of sucrose, illumination and culture types were studied on the influence of propagation rates and the quality of axillary shoots. The results showed that Nitsch basal medium supplemented with 1mg/L KT, 0.5mg/L 6-BA and 30mg/l sucrose was the suitable medium to proliferate axillary shoots in liquid flask-shaking culture for the first 25 days under darkness and then in still liquid culture under illumination for another 25 days. Three cultivars, ’A0’ , ’B’ , ’B6’ having been propagated in the established suitable culture system showed different capacity of axillary buds emergence, but all were well propagated above the rate of 8 in 50 days. The plantlet with multiple axillary shoots could be transferred directly into solid subculture medium and then be cut into separated single shoots after 45 days to save labor cost. Compared with the plantlets regenerated from callus, the axillary emergency rate of the plantlets gained from this propagation system was slightly higher.The culture conditions of in vitro plantlets of Anthurium andraeanum were systematically investigated. The suitable cutiure medium was 1/2MS+0.25mg/L 6-BA+0.2mg/L TFIBA+0.1%AC, solidified by 0.5 ~ 0.55% agar. 3100~4000Lux illumination was suitable for the strengthening of in vitro plantlets. Under this culture condition, three cultivars, AO, B, B6 were successfully subcultured for 2 years without apparent growth vigor fading or off-type.And also, one variegated plantlet resulting from chlorophyl loss of partly parenchyma tissue was gained from the adventitious buds regenerated from callus. Three types of chimeras w更多还原