【作者】 姜新超；

【导师】 刘春；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2010， 硕士

【摘要】 为了建立良好的百合悬浮细胞培养体系,本试验首先研究了其愈伤组织诱导与再生体系,进而建立了3个基因型的5个悬浮细胞系。试验具体研究方法及取得的结果和结论如下:(一)愈伤组织诱导与再生体系研究(1)愈伤组织发生能力的测试:为了选择适宜的基因型和外植体类型,试验选用多个百合基因型的多种外植体(试管鳞茎、田间鳞茎、花器官或种子)为试材,利用激素picloram进行愈伤组织诱导。试验结果表明,愈伤组织发生能力为岷江百合、麝香百合>OT系百合、东方百合、亚洲百合>兰州百合,诱导效果为鳞片>花丝>种子>花柱>花药和子房。但具体到某一基因型或同一基因型的不同外植体其愈伤组织发生能力仍然存在差异。(2)愈伤组织诱导与再生的研究:选用愈伤组织发生能力较强的岷江百合和麝香百合‘白狐’的试管鳞茎为试材。在愈伤组织诱导过程中,岷江百合研究了picloram浓度的影响,‘白狐’研究了光照、起始材料状态及激素种类(picloram和2,4-D)和浓度的影响;在愈伤组织再生过程中,两个基因型均研究了MS中大量元素含量和蔗糖浓度的影响。试验结果表明,岷江百合在picloram 2.0 mg?L-1条件下诱导效果最佳,诱导率约为80%;‘白狐’在picloram 1.0 mg?L-1条件下诱导效果最佳,诱导率约为100%,光照和起始材料状态对愈伤组织诱导的影响不显著;2个品种的最佳再生培养基均为?MS +蔗糖15 g?L-1。(3)愈伤组织的再生:将生长良好的愈伤组织接种到培养基?MS+蔗糖15 g?L-1上进行植株再生,试验结果表明,基因型和愈伤组织类型在愈伤组织再生中起着重要作用。在基因型相同的条件下,愈伤组织的再生能力主要与愈伤组织类型有关。胚性愈伤组织的再生能力强,再生率基本为100%,非胚性愈伤组织的再生能力差,再生率偏低。(二)悬浮细胞系建立与植株再生的研究在(一)中选用生长良好且可成功再生的愈伤组织接种到培养液MS(NH4NO3含量减半)+2.0mg?L-1 picloram+30g?L-1蔗糖,置于25℃恒温摇床上,100 r·min-1、黑暗条件下进行悬浮培养。悬浮细胞在再生培养基?MS+15 g?L-1蔗糖上进行植株再生。试验结果表明,各悬浮细胞系的状态和再生情况与其起始愈伤组织的状态和再生情况相对应。结构松散的愈伤组织其悬浮细胞系的建立时间短,分散度、均一度和生长速率高,但由于为非胚性愈伤组织,其悬浮细胞系的再生率也偏低;颗粒状、结构紧实的愈伤组织其悬浮细胞系的建立时间长,分散度、均一度和生长速率均低于前者,但由于为胚性愈伤组织,其悬浮细胞系的再生能力强,再生率为100%。更多还原

【Abstract】 To establish cell suspension cultures of Lilium spp., this research studied the callus induction and regeneration system firstly. Cell suspension cultures were established with the callus which was in good condition and could regenerate successfully. Five cell suspension cultures of three genotypes were established and different cell suspension cultures had different characters and regeneration ability. Specific research methods and the results and conclusions are as follows:(一) Callus induction and regeneration system(1) Callus formation examination: Callus formation were examined in 8 Lilium genotypes,Lilium regale wils., Lilium longiflorum ’White fox’ and ’Snow Queen’ , Lilium Oriental hybrids’ Siberia’ and ’Bernini’, Lilium Oriental×Trumpet ’Manissa’, Lilium davidii var. unicolor, Lilium Asiatic Hybrids ’Vermeer’. Seeds, bulb scales, and flower organs were placed on a medium containing 2.0 mg?L-1 picloram. The results showed that callus formation ability is Lilium regale wils., Lilium longiflorum> Lilium Oriental×Trumpet, Lilium Oriental hybrids, Lilium Asiatic Hybrids > Lilium davidii var. unicolor, respectively. Bulb scales and filament explants showed great ability to form calluses, whereas seeds had poor ability.(2) Callus induction and regeneration: Bulb scales of Lilium regale wils. and Lilium longiflorum ’White fox’ were used as explants to study callus induction and regeneration. In the study of callus induction, the effect of picloram concentrations on Lilium regale wils. and the effect of light, initial materials status and hormone types (picloram and 2,4-D) and oncentrations on Lilium longiflorum ’White fox’ were analysed . During the regenerating, the effects of sucrose concentrations and mineral element content in MS basic medium on both genotypes were studied. The results showed that the optimum concentration for callus induction of Lilium regale wils. was picloram 2.0mg?L-1, and the induction rate was about 80%; the optimum concentration for callus induction of Lilium longiflorum ’White fox’ was picloram 1.0mg?L-1, and the induction rate was about 100%; the optimum regenerating medium of both genotypes was 1/2MS supplemented with sucrose 15 g?L-1.(3) Callus regeneration: Callus in good condition which were obtained from (1) were inoculated on solid 1/2MS medium with 15 g?L-1 sucrose. Genotypes and types of callus had a significant effect on callus regeneration. The genotypes mainly affected the patterns of regeneration and the types of callus had an effect on regeneration capacity. The regenerating rate of embryogenic callus with high regeneration ability was 100%. The non-embryonic callus with low regeneration ability showed lower regenerating rate.(二) Establishment of cell suspension culture and plantlet regeneration: Callus in good condition obtained from (1) and regenerated in (3) were cultured in modified liquid MS medium (NH4NO3 with half-strength) supplemented with picloram 2.0mg?L-1 and sucrose 30g?L-1 , and shook at 100 rpm under dark condition at 25℃. Culture density were 2g(f.w.) per 40mL liquid medium, and cell suspension cultures were subcultured every 1~2w. Suspension cells regenerated plantlets on solid 1/2MS medium with 15 g?L-1 sucrose. The results showed that the state and regeneration ability of cell suspension cultures were related with the initial callus. Stably growing cell suspension cultures were established with loose callus in 10 to 20 days, which had high single-cells ,but low regenerating rate. The cell suspension cultures composed of cell groups,diameter 1~5mm and high regenerating rate,were established by the granular callus.更多还原