百合繁殖器官离体培养研究
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摘要
本论文主要通过对东方百合、亚洲百合的繁殖器官如鳞球茎、珠芽、花器官进行离体组织培养试验,从而比较不同百合品种间繁殖器官的离体培养差异,并分别在灭菌、诱导分化、继代增殖、结鳞茎和生根移栽等方面进行了研究,试验结果表明:
     1、百合不同繁殖器官灭菌效果有差异,珠芽和花器官的灭菌效果好于鳞片;亚洲百合珠芽较适宜的灭菌剂组合为:15%NaClO(15′)+0.1%HgCl_2(1.5′),花器官较适宜的灭菌剂组合为:15%NaClO(10′)+0.15%HgCl_2(10′),鳞片较适宜的灭菌剂组合为:0.1%HgCl_2(10′)+0.15%HgCl_2(10′)。
     2、不同百合品种鳞片的诱导分化能力不同,亚洲百合的诱导分化率为67.74%,平均生成的小鳞茎数为4.480;玛妮莎百合的诱导分化率为58.82%,平均生成的小鳞茎数为3.487;星球战士百合的诱导分化率为52.78%,平均生成的小鳞茎数为4.167。适宜的诱导分化培养基为:Ms基本培养基附加6-BA0.5 mg·L~(-1)+KT0.5 mg·L~(-1)+NAA0.3 mg·L~(-1)。
     3、同一品种不同部位的鳞片诱导能力有差异,分别表现为,亚洲百合:中层>内层>外层;玛妮莎百合:外层>中层>内层;星球战士百合:外层>中层>内层。
     4、激素组合KT2.0 mg·L~(-1)+NAA0.5 mg·L~(-1)对亚洲百合珠芽的诱导分化效果好,诱导分化率为33.33%。
     5、不同激素种类与浓度配比对百合花器官的诱导分化结果不同,当2,4-D浓度为1.0 mg·~(-1)时,有利于愈伤组织的诱导;6-BA2.0 mg·L~(-1)+KT0.5mg·L~(-1)+NAA0.1 mg·L~(-1)分化不定芽的效果好。
     6、不同百合花器官的不同部位愈伤组织分化出芽有明显差异,表现为:亚洲百合:花丝>花瓣>花柱>子房;玛妮莎百合:花丝>花柱>子房>花瓣;星球战士百合:花瓣>花柱>花丝>子房。
     7、两激素组合6-BA1.25 mg·L~(-1)+NAA0.20 mg·L~(-1)对亚洲百合芽增殖效果好;6-BA1.0 mg·L~(-1)+NAA0.25 mg·L~(-1)对玛妮莎百合芽增殖效果好;6-BA1.0 mg·L~(-1)+NAA0.30 mg·L~(-1)对星球战士百合芽增殖效果好。
     8、细胞分裂素KT在壮苗方面优于同浓度水平的6-BA。
     9、不同浓度的三种激素组合对百合芽继代增殖效果不同,以6-BA1.0mg·L~(-1)+KT0.6 mg·L~(-1)+NAA0.2 mg·~(-1)的激素配比对百合的芽继代增殖效果最好。
     10、不同营养附加成分对百合芽继代增殖的影响不同,椰子汁浓度在15—20%范围内有利于芽增殖和试管苗的生长,椰子汁对百合继代效果好于马铃薯汁。
     11、亚洲百合在90g·L~(-1)的蔗糖浓度下结鳞茎率为100%;玛妮莎百合在45 g·L~(-1)下达到100%;星球战士在60 g·L~(-1)下结鳞茎率为100%。高浓度(100~120 g·L~(-1))蔗糖对百合试管苗结鳞茎不利。
     12、浓度范围(10~20 mg·L~(-1))的香豆素对百合结鳞茎有一定的促进作用。
     13、单一生长素NAA对百合的生根壮苗效果好于相同浓度水平的IBA、ABT。
     14、较低浓度范围(0.5~1.0 mg·L~(-1))的多效唑与NAA1.0 mg·L~(-1)配合使用对百合生根壮苗有较好的促进作用。
     15、不同种类的基质按一定比例混和对试管苗的生长有利,较适宜的百合试管苗移栽基质为1/4泥炭+1/4河沙+1/2腐殖土。
     16、百合组培移栽苗以1/6MS培养液作叶面肥效果好。
This paper mainly studied in vitro culture of Lily, which compareddifferent varieties of Lily in vitro culture through Oriental Lily, AsiaticLily reproductive organs such as scales bulb, bulblet and floral organ. Fivestages of primary research were processed, which included sterilization,induction and differentiation, subculture multiplication, bulblet formation,rooting and transplanting. The results showed as follows:
     1.The effects on different reproductive organs sterilization were differentwith different sterilant combinations: 15%NaClO 15 min+0.1%HgCl_2 15 minwas suitable to sterilited the bulblet of Asiatic Lily; 15%NaClO 10 min+0.15%HgCl_2 10 min was suitable to sterilited the floral organs; 0.1%HgCl_2.10min+0.15%HgCl_2 10 min was suitable to sterilited the squamas.
     2. There were different frequencies of buds induction and differentiatiationwith squama of different varieties. The frequency of buds differentiation withAsiatic Lily was 67.74%, the average of induced bulblets was 4.48; that ofManissa was 58.82%and 3.487; that of Starfighter was 52.78%and 4.167. Theappropriate medium for induction and differentiation was MS basic medium added6- BA 0.5mg·L~(-1)+KT0.5mg·L~(-1)+NAA 0.3mg·L~(-1).
     3. Different part of a squamas gave different responses to the effect ofinduction on the same varieties. The inducting fequencies of middle scales withAsiatic Lily was the highest, inner scales was the second, and outer scaleswas the lowest. The inducting fequencies of outer scales with Manissa Lily wasthe highest, middle scales was the second, and inner scales was the lowest.But the results of Starfighter Lily showed that the highest was outer scales and the lowest was inner scales.
     4. The optimal hormone Combination for bulblets of Asiatic Lily was 2.0mg·L~(-1)KT and 0.5mg·L~1NAA. The induction frequencies of buds was 33.33%.
     5. There were different hormone categories and concentration combinationson induction and differentiation of floral organ. When the concentration of2, 4-D was 1.0 mg·L~(-1), it was good for callus induction. When the medium combinedwith 2.0mg·L~(-1)6-BA, 0.5mg·L~(-1)KT and 0.1mg·L~(-1)NAA, it was more profit forbuds induction.
     6. There were obvious difference in the bud differentiation of calli thatinduced from different parts of Lily floral organ, in which showed that thebud differentiation rate from filament was the highest, followed by petal, thelowest was from ovary of Asiatic Lily; while in Manissa Lily, the highest buddifferentiation rate was from filament, followed by style, and the lowest wasfrom petal. For starfighter Lily, the highest bud differentiation rate was frompetal, followed by style, and the lowest was from ovary.
     7. The optimal combination of two hormone for buds subculture multiplicationof Asiatic Lily was 1.25mg·L~(-1)6-BA+0.20mg·L~(-1)NAA; that of Manissa was 1.0mg·L~(-1)6-BA+0.25mg·L~(-1) NAA; that of Starfighter was 1.0mg·L~(-1) 6-BA+0.30mg·L~(-1)NAA.
     8. At the same concentration level, the cytomin KT was better than 6-BA insound plantlet aspect.
     9. Different combinations of three hormones gave different responses to theeffect of buds subculture multiplication. The treatment with 6-BA1.0mg·L~(-1)+KT0.6mg·L~1+NAA0.2mg·L~(-1) showed the better effect than the others.
     10. There were different effects on subculture multiplication of Lily withadditional different nutritional components. Concentration in the range ofcoconut milk (15-20%) was favorable to buds multiplication and plantlet growth.The effect of coconut milk on subculture multiplication was better than thatof potato juice.
     11. Under 90g·L~(-1)sucrose concentration, the rate of bulblet formation withAsiatic Lily reached 100%; that of Manissa Lily and Starfighter Lily were 45 g·L~(-1) and 60g·L~(-1). High concentrations(100-120g·L~(-1))sucrose was unfavourableto bulblet formation of Lily plantlet.
     12. Concentration range of cumarin(10-20mg·L~(-1)) could promot bulbletformation.
     13. The rooting and sound plantlet effect of NAA was superior to the sameconcentration level of IBA or ABT.
     14. Low concentration range of MET(0.5-1.0mg·L~(-1)) combined with(1.0mg·L~(-1))NAA, which could accelerate root induction and sound plantlet.
     15. Different types of matrix mixture according to a fixed percentagebenefited Lily plantlet growth. The optimal translation matrix mixture was 1/4peat +1/4 sand +1/2 humus soil.
     16. 1/6MS liquid proved the best concentration in leaf-sprinkle fertilizersfor the plantlets translation of Lily.
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