组培微环境对球根花卉试管苗生长影响及移栽技术研究
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摘要
在色彩斑谰的百花园中,球根类花卉因其花朵硕大、色彩艳丽、适应性强、既可作切花又可作盆花等特点深受消费者喜爱。组织培养是最近十几年来兴起的应用在球根花卉繁殖上的主要方法,它可以有效的解决种球繁育中所遇到如种球繁殖系数低,病毒侵染严重等问题,但却难以应用于工厂化生产,主要原因是移栽成活率低。试管苗的移栽驯化是组培程序的最后一个环节,也是最为关键的环节。因此,加深对试管苗移栽驯化过程中形态和生理上的变化,有利于制定更有效的移栽驯化措施,提高存活率。
本试验主要以已建立脱毒快繁体系的葡萄风信子和百合试管苗为材料,通过改变生根阶段的光照强度,糖浓度,研究其对风信子、百合移栽过程中植株生长生理以及成活率的影响,并通过一系列移栽试验研究了不同大小球茎、不同基质、不同组培苗质量对移栽成活率的影响,旨在为提高组培苗移栽成活率提供可靠的理论依据与实践经验。
主要研究结果如下:
1.建立了葡萄风信子“白葡萄”、亚洲百合“精粹”的再生快繁体系以葡萄风信子“白葡萄”田间取材的叶片、鳞茎为外植体,通过不同植物激素种类组合和浓度配比诱导产生愈伤组织并且分化出不定芽。结果表明,叶片是葡萄风信子离体再生较合适的外植体类型。以MS+2,4-D0.6mg/L+6-BA0.1mg/L诱导叶片形成愈伤组织效果较好,诱导频率可达100%;鳞茎诱导愈伤组织的适宜培养基为MS+2,4-D3.0mg/L+6-BA0.3mg/L,诱导频率达91.2%;愈伤组织分化不定芽的最适培养基为MS+6-BA2.0mg/L+NAA0.3mg/L,出芽频率可达97.4%;生根培养基为1/2MS+IBA1.0mg/L+0.1%AC,根长势粗壮。
以亚洲百合“精粹”的鳞茎为外植体,通过不同激素种类组合和浓度配比诱导产生不定芽。再以诱导的小植株叶片和鳞茎为外植体,建立百合的快繁再生体系。经过筛选,鳞片分化最佳培养基为MS+ 2.0mg/l6-BA+0.2mg/lNAA。MS + 6- BA1.0mg/L+NAA0.2mg/L为诱导叶片分化率最高的激素组合。MS+ 6-BA2.0 mg/L+ 2,4-D0.1mg/L为诱导离体小鳞茎分化率最高的激素组合,诱导率达100%。生根培养基为MS+NAA0.2mg/L。
2.组培微环境对球根花卉组培试管苗生长及移栽成活率的影响
未生根的葡萄风信子组培苗在3%蔗糖和无糖、百合组培苗在5%蔗糖和无糖的生根培养基中诱导生根,同时分别给予86μmol·m-2·s-1、20μmol·m-2·s-1、48μmol·m-2·s-1三种光照。分别于生根培养15d,30d后,对两者生长形态指标测定表明:添加蔗糖对试管苗的生物量形成有积极的影响,提高光照强度能进一步促进试管苗的生长。风信子和百合组培苗的整体生长势都以处理Ⅳ较好。添加蔗糖提高了组试管苗的根系活力和酶的活性,有糖和高光均提高了植株叶绿素含量。光照强度对气孔开张影响明显,随着光照强度的增加气孔宽度增加,气孔密度减少。蔗糖对气孔开张也有一定的影响。同样光照条件下,无蔗糖处理的植株叶片气孔长度和宽度均高于有糖处理的,气孔密度要少于有糖处理的。本试验中以有糖高光照处理的植株移栽后具有较高的存活率,长势较好。
3.不同大小球茎、不同基质、不同组培苗质量对移栽成活率的影响结果表明:随着试管鳞茎的增大,其移栽成活率升高,成活后苗期新长出的叶片数和株高也随着增加,鳞茎直径≥1.5cm的试管苗,移栽成活率最高,达95%;几种基质的配比中,移栽在混合基质上的试管苗成活率较高、生长状况良好,葡萄风信子以珍珠岩/泥炭土为1/1基质效果最好,成活率为97%,百合试管鳞茎以珍珠岩/泥炭土为1/3基质效果最好,成活率为98%;试管苗质量方面为,正常试管苗的成活率>有愈伤组织的苗子>畸形根的苗子。
In flowers garden, the tuber flower is favoriate by consumers because of its gigantic flowers, gorgeous colors and strong compatibility .They are not only used cutting flowers but also potted. Tissue culture is the main method which used to reproduce tuber flower. It can solve many questions that bulb breeding encounters, such as the lower breeding indexs, severe virus contaminated, but it is difficult to apply to factory-production, the main reason is the lower survival rate. Transplanting and domestication is the last part of the tissue culture process, and is the most critical aspect.So,deepen the study on the shape and physiological changes during transplanting and domestication is beneficial to the plantlets transplanting, improve the survival rate.
Micropropagated hyacinth and lily were used to study their growth and physiological responses to sucrose concentration added in the MS medium,PPFD during rooting in vitro.And studied the influence of different exercising days, different bulb diameter, different matrix and different plantlets quality on the survival rate.The results were as following: 1.A regeneration system was initiated from Muscari botryoides“Album”and Asiatic hybrid lily“elite”
Leaves and bulbs of Muscari botryoides“Album”were chosen as explants.Different type and concentration of hormone were studied in order to induce callus and adventitious bud. The results showed that the leaf was more appropriate explants for tissue culture of grape hyacinthus than the bulb. The optimum medium for inducing callus form leaves was MS+2,4-D 0.6 mg·L-1+6-BA 0.1 mg·L-1,the induction frequency may reach 100%;The combination of MS+2,4-D 3.0 mg·L-1+6-BA 0.3 mg·L-1 was the appropriate medium for bulbs forming callus with the induction rate 91.2%.The best medium for inducing adventitious buds from callus was MS+6-BA 2.0 mg·L-1+NAA 0.3mg·L-1,the germination rate was 97.4%;The medium of 1/2 MS+IBA 1.0 mg·L-1+1 g·L-1 AC was suitable for rooting with strong roots.
Bulbs of Asiatic hybrid lily“elite”was chosen as explants. Different type and concentration of hormone were studied in order to induce adventitious bud. Then use leaves and bulbs of the new induced plantlets to establish regeneration system from Asiatic hybrid lily“elite”. The adventitious shoots were induced from scales explants based on MS basal medium supplemented with 2.0 mg·L-16-BA and 0.2 mg·L-1NAA. The medium suitable for the leaves of the plantlets to differentiate adventitious bud was MS + 6- BA1. 0 mg·L-1+ NAA0. 2 mg·L-1;The medium suitable for the scales leaves of the plantlets to differentiate bud was MS+6-BA 2.0 mg·L-1+2,4-D 0.1mg·L-1, the induction frequency may reach 100%.MS+NAA 0.2 mg·L-1+0.2%AC was suitable for rooting.
2.Effects of micro-environment factors in vitro on the growth of the bulb flowers and the survival rate after transplant during rooting stage
No-rooting grape hyacinth cuttings were cultured on solid Murashige- Skoog medium with 3% sucrose or without sucrose, lily cuttings were cultured with 5% sucrose or without sucrose, and in 3 photosynthetic photo flux densities(86、20、48mol·m-2·s-1) for 15,30 days respectivly.The results indicated that sucrose was beneficial to biomass formation of plantlets, higher PPFD was benefit to the growth of plantlets. TreatmentⅣwas the best for the growth potential of hyacinth and lily plantlets. With-sucrose raised the activity of roots and antioxidases, with-sucrose and higher PPFD all increased the content of Chl.. Light intensity has visible influence on the stomaal opening and closing. Along with the light intensity increased, the width of stoma also increased, but the density decreased. Sucrose has a certain degree influence on the stomaal opening and closing. On the same PPFD, the length and width with free-sucrose were all above that with-sucrose, the density was lower. The plantlets with sucrose,higher PPFD had the highest survival rate and better growth expression after transplant.
3.Effect of different exercising days、different diamester bulb、matrix、different plantlets quality on the survival rate
The results showed: the bulb flower plantlets had a higher survival rate through 5 or 7 exercising days before transplant. Bigger the bulbs were, higher the survival rate; the plantlets transplanted on mixed matrix had the better growth potential, higher survival rate. Among those, the 1/1 Perlite/Peat was the most appropriate for the growth of hyacinth plantlets, the is 96%, the 1/3 Perlite/Peat was the most appropriate for the growth of lily plantlets, the survival rate is 98%;on the aspect of the plantlets quality, the survival rate of normal seedlings is higher than that of few roots seedling, seedling of no normal roots was the lowest.
本试验主要以已建立脱毒快繁体系的葡萄风信子和百合试管苗为材料,通过改变生根阶段的光照强度,糖浓度,研究其对风信子、百合移栽过程中植株生长生理以及成活率的影响,并通过一系列移栽试验研究了不同大小球茎、不同基质、不同组培苗质量对移栽成活率的影响,旨在为提高组培苗移栽成活率提供可靠的理论依据与实践经验。
主要研究结果如下:
1.建立了葡萄风信子“白葡萄”、亚洲百合“精粹”的再生快繁体系以葡萄风信子“白葡萄”田间取材的叶片、鳞茎为外植体,通过不同植物激素种类组合和浓度配比诱导产生愈伤组织并且分化出不定芽。结果表明,叶片是葡萄风信子离体再生较合适的外植体类型。以MS+2,4-D0.6mg/L+6-BA0.1mg/L诱导叶片形成愈伤组织效果较好,诱导频率可达100%;鳞茎诱导愈伤组织的适宜培养基为MS+2,4-D3.0mg/L+6-BA0.3mg/L,诱导频率达91.2%;愈伤组织分化不定芽的最适培养基为MS+6-BA2.0mg/L+NAA0.3mg/L,出芽频率可达97.4%;生根培养基为1/2MS+IBA1.0mg/L+0.1%AC,根长势粗壮。
以亚洲百合“精粹”的鳞茎为外植体,通过不同激素种类组合和浓度配比诱导产生不定芽。再以诱导的小植株叶片和鳞茎为外植体,建立百合的快繁再生体系。经过筛选,鳞片分化最佳培养基为MS+ 2.0mg/l6-BA+0.2mg/lNAA。MS + 6- BA1.0mg/L+NAA0.2mg/L为诱导叶片分化率最高的激素组合。MS+ 6-BA2.0 mg/L+ 2,4-D0.1mg/L为诱导离体小鳞茎分化率最高的激素组合,诱导率达100%。生根培养基为MS+NAA0.2mg/L。
2.组培微环境对球根花卉组培试管苗生长及移栽成活率的影响
未生根的葡萄风信子组培苗在3%蔗糖和无糖、百合组培苗在5%蔗糖和无糖的生根培养基中诱导生根,同时分别给予86μmol·m-2·s-1、20μmol·m-2·s-1、48μmol·m-2·s-1三种光照。分别于生根培养15d,30d后,对两者生长形态指标测定表明:添加蔗糖对试管苗的生物量形成有积极的影响,提高光照强度能进一步促进试管苗的生长。风信子和百合组培苗的整体生长势都以处理Ⅳ较好。添加蔗糖提高了组试管苗的根系活力和酶的活性,有糖和高光均提高了植株叶绿素含量。光照强度对气孔开张影响明显,随着光照强度的增加气孔宽度增加,气孔密度减少。蔗糖对气孔开张也有一定的影响。同样光照条件下,无蔗糖处理的植株叶片气孔长度和宽度均高于有糖处理的,气孔密度要少于有糖处理的。本试验中以有糖高光照处理的植株移栽后具有较高的存活率,长势较好。
3.不同大小球茎、不同基质、不同组培苗质量对移栽成活率的影响结果表明:随着试管鳞茎的增大,其移栽成活率升高,成活后苗期新长出的叶片数和株高也随着增加,鳞茎直径≥1.5cm的试管苗,移栽成活率最高,达95%;几种基质的配比中,移栽在混合基质上的试管苗成活率较高、生长状况良好,葡萄风信子以珍珠岩/泥炭土为1/1基质效果最好,成活率为97%,百合试管鳞茎以珍珠岩/泥炭土为1/3基质效果最好,成活率为98%;试管苗质量方面为,正常试管苗的成活率>有愈伤组织的苗子>畸形根的苗子。
In flowers garden, the tuber flower is favoriate by consumers because of its gigantic flowers, gorgeous colors and strong compatibility .They are not only used cutting flowers but also potted. Tissue culture is the main method which used to reproduce tuber flower. It can solve many questions that bulb breeding encounters, such as the lower breeding indexs, severe virus contaminated, but it is difficult to apply to factory-production, the main reason is the lower survival rate. Transplanting and domestication is the last part of the tissue culture process, and is the most critical aspect.So,deepen the study on the shape and physiological changes during transplanting and domestication is beneficial to the plantlets transplanting, improve the survival rate.
Micropropagated hyacinth and lily were used to study their growth and physiological responses to sucrose concentration added in the MS medium,PPFD during rooting in vitro.And studied the influence of different exercising days, different bulb diameter, different matrix and different plantlets quality on the survival rate.The results were as following: 1.A regeneration system was initiated from Muscari botryoides“Album”and Asiatic hybrid lily“elite”
Leaves and bulbs of Muscari botryoides“Album”were chosen as explants.Different type and concentration of hormone were studied in order to induce callus and adventitious bud. The results showed that the leaf was more appropriate explants for tissue culture of grape hyacinthus than the bulb. The optimum medium for inducing callus form leaves was MS+2,4-D 0.6 mg·L-1+6-BA 0.1 mg·L-1,the induction frequency may reach 100%;The combination of MS+2,4-D 3.0 mg·L-1+6-BA 0.3 mg·L-1 was the appropriate medium for bulbs forming callus with the induction rate 91.2%.The best medium for inducing adventitious buds from callus was MS+6-BA 2.0 mg·L-1+NAA 0.3mg·L-1,the germination rate was 97.4%;The medium of 1/2 MS+IBA 1.0 mg·L-1+1 g·L-1 AC was suitable for rooting with strong roots.
Bulbs of Asiatic hybrid lily“elite”was chosen as explants. Different type and concentration of hormone were studied in order to induce adventitious bud. Then use leaves and bulbs of the new induced plantlets to establish regeneration system from Asiatic hybrid lily“elite”. The adventitious shoots were induced from scales explants based on MS basal medium supplemented with 2.0 mg·L-16-BA and 0.2 mg·L-1NAA. The medium suitable for the leaves of the plantlets to differentiate adventitious bud was MS + 6- BA1. 0 mg·L-1+ NAA0. 2 mg·L-1;The medium suitable for the scales leaves of the plantlets to differentiate bud was MS+6-BA 2.0 mg·L-1+2,4-D 0.1mg·L-1, the induction frequency may reach 100%.MS+NAA 0.2 mg·L-1+0.2%AC was suitable for rooting.
2.Effects of micro-environment factors in vitro on the growth of the bulb flowers and the survival rate after transplant during rooting stage
No-rooting grape hyacinth cuttings were cultured on solid Murashige- Skoog medium with 3% sucrose or without sucrose, lily cuttings were cultured with 5% sucrose or without sucrose, and in 3 photosynthetic photo flux densities(86、20、48mol·m-2·s-1) for 15,30 days respectivly.The results indicated that sucrose was beneficial to biomass formation of plantlets, higher PPFD was benefit to the growth of plantlets. TreatmentⅣwas the best for the growth potential of hyacinth and lily plantlets. With-sucrose raised the activity of roots and antioxidases, with-sucrose and higher PPFD all increased the content of Chl.. Light intensity has visible influence on the stomaal opening and closing. Along with the light intensity increased, the width of stoma also increased, but the density decreased. Sucrose has a certain degree influence on the stomaal opening and closing. On the same PPFD, the length and width with free-sucrose were all above that with-sucrose, the density was lower. The plantlets with sucrose,higher PPFD had the highest survival rate and better growth expression after transplant.
3.Effect of different exercising days、different diamester bulb、matrix、different plantlets quality on the survival rate
The results showed: the bulb flower plantlets had a higher survival rate through 5 or 7 exercising days before transplant. Bigger the bulbs were, higher the survival rate; the plantlets transplanted on mixed matrix had the better growth potential, higher survival rate. Among those, the 1/1 Perlite/Peat was the most appropriate for the growth of hyacinth plantlets, the is 96%, the 1/3 Perlite/Peat was the most appropriate for the growth of lily plantlets, the survival rate is 98%;on the aspect of the plantlets quality, the survival rate of normal seedlings is higher than that of few roots seedling, seedling of no normal roots was the lowest.
引文
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