【作者】 王兴翠；

【导师】 徐坤；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2011， 博士

【摘要】 生姜为无性繁殖作物,难以利用常规杂交育种手段进行种质创新及新品种培育。为此,本文以莱芜大姜和山农1号大姜为试材,研究了不同外植体愈伤组织的诱导及生姜植株再生体系的建立,探讨了秋水仙素诱导生姜试管苗多倍体技术及光质对生姜再生苗及根状茎诱导的关系,以期为提高生姜组培快繁效率和创新生姜种质提供参考。主要结果如下：1.生姜离体器官(根、叶鞘、叶)愈伤组织诱导,以叶片为最佳,但培养条件显著影响其诱导效果。生姜叶片愈伤组织诱导以N6培养基较好,MS、MN次之,1/2MS较差;适宜的2,4-D浓度有利于叶片愈伤组织的诱导,且2,4-D与6-BA搭配的诱导效果优于2,4-D与KT搭配；暗培养时间也显著影响叶片愈伤组织诱导率。适于莱芜大姜叶片愈伤组织诱导的最佳培养基为N6+1.0 mg·L-12,4-D+0.5 mg·L-16-BA,在暗培养30 d时,愈伤组织诱导率达77.78%；山农1号大姜叶片愈伤组织诱导培养基为：N6培养基附加1.0 mg·L-1 2,4-D、1.0 mg·L-16-BA,经30 d左右的暗培养效果较好,诱导率达78.89%。2.通过对生姜叶片愈伤组织增殖影响因素的研究得出,愈伤组织在MS和N6培养基上置于白光下培养,均经历缓慢生长、快速生长和生长平缓三个阶段,呈现“S”型曲线,且MS培养基较N6有利于愈伤组织的增殖。提高培养基中的蔗糖浓度,有利于愈伤组织体细胞胚发生。适于生姜胚性愈伤组织增殖的培养基为：MS+0.2 mg·L-1 NAA+5.0mg·L-16-BA+30 g·L-1蔗糖+6 g·L-1琼脂：而适于胚性愈伤组织向体细胞胚发生的培养基则为：MS+0.2 mg·L-1 NAA+5.0 mg·L-1 6-BA+50 g·L-1蔗糖+6 g·L-1琼脂。3.较低浓度的NAA和较高浓度的6-BA有利于叶片愈伤组织分化与植株再生,且6-BA促进器官分化的效果优于KT和TDZ。莱芜大姜愈伤组织分化成苗培养基为MS+0.2 mg·L-1 NAA+10.0 mg·L-16-BA,愈伤组织不定芽分化指数达34.5,分化成苗指数8.5；山农1号大姜愈伤组织不定芽分化指数虽然能达到34.1,但其分化率较莱芜大姜低11.42%,且成苗指数仅5.9。添加活性炭对愈伤组织分化率的促进作用不明显,而且0.5%的活性炭容易导致生姜再生苗玻璃化,不利于其生长。4.不同秋水仙素浓度及处理时间,可显著影响生姜试管苗不定芽再生植株的成活率、变异率及加倍率,其中以秋水仙素浓度、处理时间分别为0.034%～0.187%、4.91～15.62d时,生姜试管苗成活率可望取得达到80%以上；秋水仙素浓度、处理时间分别为0.116%～0.197%、10.63 d～16.29 d时,生姜试管苗变异率可望取得达到5%以上;秋水仙素浓度、处理时间分别为0.118%～0.215%、9.04 d-14.48 d时,生姜试管苗加倍率可望取得达到2%以上。生姜试管苗四倍体植株的叶片下表皮气孔密度减小,气孔增大。愈伤组织经秋水仙素处理后获得的生姜再生植株未发现变异植株。5.通过对不同光质对生姜再生苗继代的影响研究结果表明,蓝光处理能得到健壮的生姜试管苗。与白光处理相比,绿光、红光均显著增加了生姜试管苗的株高,但绿光显著降低了其繁殖系数,而蓝光则刚好相反；黄光对试管苗生长的影响较小。绿光、蓝光、红光均可诱导试管苗较早形成微型姜,而黄光则延迟了微型姜的形成；微型姜鲜重以绿光处理较高,较白光处理增加60%以上,其它处理间则无显著差异；但微型姜干物质、淀粉、粗纤维含量均以绿光处理较低,蓝光处理较高;可溶性糖含量以红光、黄光处理较高,绿光、蓝光处理较低,而可溶性蛋白含量基本呈相反的趋势。生姜试管苗叶片Gs及蒸腾速率则以蓝光、黄光处理较高,红光、绿光处理较低。更多还原

【Abstract】 Ginger is difficult to use conventional means of breeding new varieties of germplasm and cultivation for which is the vegetative propagation of crops. Therefore, in this paper,we studied different explants of "Laiwu" ginger and "Shannong No.1" ginger plantlets as materials for callus induction and plant regeneration, discussed the effect of different concentrations of colchicine on polyploid induction, and the relationship between light quality and the regenerated plants growth as well as the rhizomes induced of ginger, in order to improve an efficiency ginger tissue culture and new germplasm, the main results are as follows:1. Young leaves/sheaths/adventitious roots were as explants for callus induction, and laves were cultured as the best explants. The effect on leaf’s callus inducation of different basic media, hormone levels, time of dark culture and the relation between hormone combination and callus differentiation and plant regeneration were studied in the expeiment. The results showed that leaf-derived callus induction on N6 was better than MS, MN and 1/2 MS. Appropriate concentrations of 2,4-D were beneficial to caluss inducation. The effect of 2,4-D and 6-BA on callus induction was better than 2,4-D and KT, and the time of dark culture was an important factor. The best medium for "Laiwu" ginger leaf-derived callus induction was N6 supplemented with 1.0 mg/L 2,4-D+0.5 mg/L 6-BA+3% sucrose+0.6% agar, and the rate of callus formation was 77.78% when leaf explants were cultureed 30 days in the dark; the best medium for "Shannong No.1" ginger leaf-derived callus induction was N6 supplemented with 1.0 mg/L 2,4-D+1.0 mg/L 6-BA+3% sucrose+0.6% agar, and the rate of callus formation was 78.89% cultureed 30 days in the dark.2. We obtained suitable·culture conditions for callus proliferation and somatic embryogenesis by selecting factors of callus for proliferation. The results showed that calluses cultured on MS and N6 media under white light are experiencing three stages of slow growth, rapid growth and the growth of smooth, showing a "S" curve, and the MS medium for callus proliferation was better than N6 medium. Calluses growed slowly after 30 days, then calluses needed to replace the same composition of medium for continuing to grow. Sucrose concentration changes the physiology of leaf callus and directly affect the maturation of somatic embryos, the study found that concentrations of sucrose increased in the medium is conducive to callus to the transformation of somatic embryogenesis. Therefore, the culture medium for the proliferation of embryogenic callus was:MS+0.2 mg/L NAA+5.0 mg/L 6-BA+30 mg/L sucrose+mg/L agar; and the culture medium for embryogenic callus into somatic embryogenesis was:MS+0.2 mg/L NAA+5.0 mg/L 6-BA+5.0% sugar+0.6% agar.3. Lower concertrations of NAA and higher concertrations of 6-BA was conductive to callus and plant regeneration, and 6-BA which promoted organ differentiation was better than KT. Mediun for "Laiwu" ginger callus and plant regeneration was MS supplemented with 6-BA(10 mg/L) and NAA(0.2 mg/L), and the index of plantlet regeneration was 8.5; Callus differentiation index of "Shannong No.1" ginger could although to achieve 34.1, but the differentiation rate was lower 11.42% than "Laiwu" ginger, and the index of plantlet regeneration was 5.9. The effect of activated carbon added to promote callus differentiation was not obvious, and which added 0.5% activated carbon easily lead to a galass vitro regeneration ginger plants, detrimental to their growth.4. Different colchicine concentration and treatment time, can significantly influence the ginger plantlet survival rate, mutation rate and the doubling rate of shoot regeneration. We calculated that the doubling rate of ginger plantlets was expected to reach more than 2% by regression analysis, when the colchicine concentration and treatment time were 0.118% 0.215% and 9.04 d～14.48 d, and when colchicine concentration and treatment time were 0.034%～0.187%,4.91～15.62 d, the survival rate of ginger plantlets could achieve more than 80%; and colchicine concentration and treatment time were 0.116%～0.197%,10.63 d～16.29 d, the mutation rate could expected to get more than 5%. The tetraploid plants exibited some morphological, including larger stomata and fewer stomata per unit area. There were no plantlets from callues after colchicine concentration treated by morphological trait and flow cytometry techniques.5. In order to improve the efficiency of micropropagation of ginger, the effects of different light quality on the growth and micro-rhizome induction of in vitro cultured ginger were studied. The results showed that compared with white light, green and red. lights increased plant height of in vitro ginger, and green light also reduced its propagation coefficients, significantly, but that of the blue light just was opposite, and yellow light had a little effect on the growth of in vitro ginger. The time of micro-rhizome formation of of in vitro ginger cultured under green, blue and red lights was earlier than that of white light, but later under yellow light. Moreover, the micro-rhizome fresh weight of in vitro ginger under green light was higher, which increased by over 60% compared with that of white light, but there was no significant difference among the other light treatments. Otherwise the dry mass content, starch and crude cellulose of micro-rhizome formated under green light were lowest, but highest under blue light. Soluble sugar content was higher under red and yellow light, while lower under green and blue light, but contrary result appeared with soluble protein content. The Gs and transpiration rate were higher under blue and yellow light, but lower under red and green light.更多还原