||大丽花(Dahlia pinnata)为菊科大丽花属多年生球根花卉,常作花境、花坛或盆栽观赏,是世界著名的园林绿化材料.建立大丽花组培快繁和高效再生体系,可为优良株系的规模繁殖、种质保存以及遗传转化体系建立奠定基础,对通过基因工程实现大丽花种质资源创新具有重要意义.本试验研究了不同因素对大丽花常规快繁和高效再生的影响,其中,重点研究了离体叶片高效再生技术体系,主要结果如下:一、常规快繁①以大丽花茎尖和带腋芽茎段进行外植体比较试验,结果表明,以带腋芽茎段为外植体材料较好,成活率可达到75%;灭菌方法为:先用70%的酒精消毒30 s,再用0.1%升汞或5%的次氯酸钠(滴加1~2滴吐温20)消毒4 min,之后接种到含2 000 mg/L活性炭的初代培养基上,以获得无菌株系.②适宜的壮苗培养基为:MS+6-BA 0.5 mg/L+NAA0.3 mg/+蔗糖50 g/L+琼脂6.5 g/L+B9 50 mg/L.③最佳的增殖培养基是MS+6-BA4 mg/L +NAA0.1 mg/L,增殖系数可达到6.8.其次为MS+KT 6.0 mg/L+ NAA 0.01 mg/L,增殖系数为5.3.其中,蔗糖为30 g/L,琼脂为6.5 g/L;培养室温度为25℃±2℃,光照培养处理的光强为1 000~2 000 lx,光周期为14 h光照,10 h黑暗.④适宜生根的培养基是1/2MS + NAA 0.1~0.3 mg/L+30 g/L蔗糖+6.5 g/L琼脂粉,生根率可达92.0%,平均生根数为10.9.二、高效再生以大丽花无菌苗叶片、叶柄、茎段为试材,采用正交试验设计,对影响离体再生的外源细胞分裂素(6-BA, KT, TDZ)和生长素(IBA, NAA)进行组合和不同配比设计,并对叶片生理状态及暗培养天数等因素进行了研究.结果表明:①以叶片、叶柄和茎段为试材进行离体再生时,离体叶片的再生效果最好,其不定芽再生率显著高于叶柄和茎段;最佳生理状态为顶部充分展开的25 d叶龄,长度为2 cm左右的幼嫩叶片.②实现了大丽花离体叶片两种途径的高效再生.一是通过愈伤组织途径的再生,培养基为:MS+6-BA3 mg/L +IBA0.2 mg/L,愈伤组织的诱导率为76.4%,再生率高达88.0%,平均再生不定芽个数为7.5;二是不通过愈伤组织的直接再生,培养基为:MS+KT 7.0 mg/L+ NAA 0.05 mg/L,再生率为86.0%,平均再生不定芽个数为5.8.③离体叶片暗培养15 d后转到光下培养,愈伤组织诱导率显著提高到89.0%.所建立的大丽花无菌苗离体叶片高效再生技术体系,再生率可保持在86.0%左右,平均再生不定芽个数可提高到8.1个.④叶片最佳的接种方式是以远轴面向下接触培养基,最佳的切割方式是在靠近叶柄的一端垂直中脉切割,平均再生不定芽个数可提高到8.8个.
||Dahlia pinnata, as an perennial flowering bulbs, belongs to the Compositae family Dahlia genus. It is a world-wide famous landscape greening material and is applied in flower borders, flowerbeds and pot flowers. The establishment of tissue culture rapid micropropagation and highly efficient regeneration system can lay foundations for the scale propagation of superior strains, germplasm conservation and the establishment of genetic transformation system, which is also significative for the germplasm innovation of Dahlia pinnata through the means of genetic engineering. In this study, different factors that effect the micropropagation and highly effective regeneration were studied, of which the establishment of leaf in vitro regeneration system was especially studied.The results were as follows:1, Normally rapid micropropagation①The explants comparison test was carried out using the stem tips and stems with axillary bud of Dahlia pinnata as material. The results showed that the stems with axillary bud were the better explants. The rate of survival was 75%. The method for disinfectant was 70% alcohol (washed for 30 s) and 0.1% HgCl2 or 5% NaClO(washed for 4 min)and then inoculate to the original medium which contained 2000mg/L AC. and finally established the sterile system.②The fitting strengthening medium was MS+6-BA 0.5 mg/L+NAA0.3 mg/+ 50 g/Lsugar+6.5 g/L agar +B950 mg/L.③The optimal multiplication medium was MS+6-BA 4 mg/L +NAA 0.1 mg/L. The multiplication coefficient could be reached to 6.8. The inferior medium was MS+KT 6.0 mg/L+ NAA 0.01 mg/L. The multiplication coefficient was 5.3. In which the sugar and agar was respectly 30 g/L and 6.5 g/L. The culture condition was 14/10 light period, 1 000~2 000 light intensity and 25℃±2℃.④The fitting rooting medium was 1/2MS+NAA0.1~0.3 mg/L+30.0 g/L sugar+6.5 g/L agar, the rooting rate was 92.0%, and the mean number of roots was 10.9.2, Highly effective regeneration The experiment was carried out using leaves, petioles, merithals of the test-tube plantlet as material which was orthogonal designed studing the combine and mixture ratio of the exogenous cytokinin(6-BA, KT, TDZ)and auxin(IBA, NAA). It also studied the physiological condition of leaf , the days for darking culture and so on. The results were as follows:①Contrast to other explants such as petioles and merithals,the leaves was the best explants for in vitro regeneration. The rate of adventitious shoots for leaf was significantly higher than petiole and merithal. The best physiological condition of leaf explant was 25 days of test-tube plantlets, enough expanding and 2 cm long.②This study achieved two ways of highly efficient regeneration, one way was through the path of callus tissue,the optimal medium was MS+6-BA 3 mg/L +IBA 0.2 mg/L,the Callus induction rate was 76.4%, regeneration rate was 88.0%, the number of adventitous sprouts per leaf was 7.5; the other was direct regeneration omitting callus induction, the optimal medium was MS+KT 7.0 mg/L+ NAA 0.05 mg/L, regeneration rate was 86.0%, the number of adventitous sprouts per leaf was 5.8.③Fifteen days dark pretreatment and then transferred to normal ray culture can significantly improve the callus induction rate to 89.0%. The regeneration rate of the highly effective regeneration system established by the in vitro leaf of test-tube plantlet of Dahlia pinnata was to keep at about 86.0% and the number of adventitous sprouts per leaf rised to 8.1.④The best ways on the medium was by the abaxial surface(leaf back)touching medium and the best cutting ways of leaves was Cutting the midrib vertically,the number of adventitous sprouts per leaf rised to 8.8.