菊芋高效再生体系的建立
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摘要
菊芋(Helianthus tuberosus L.),俗名洋姜、鬼子姜等,属菊科向日葵属多年生宿根草本植物,因其地上似菊、地下为芋而得名。菊芋植株高大粗壮,有机物积累量大,块茎营养丰富、产量高、用途广,是一种新兴的有潜力、多用途的能源植物和经济作物。菊芋是一种自身具有较强耐逆性的植物,不仅耐盐碱、耐旱、耐寒,还可以耐贫瘠、抵抗风沙等,因此,它对生长环境的要求不高,适应性极强,是一种值得广泛推广种植的植物。
目前,我国高度重视对能源植物的开发与利用,菊芋产业快速发展,短期内需求量较大,同时,菊芋的常规繁殖方式为块茎繁殖,属无性繁殖,能够成为病毒的良好寄主,会导致大量病毒病的积累,最终将导致品种的劣化和产量的逐年降低,工业化生产效率不高,优良品种较少,因此建立和完善高效的菊芋组织培养快繁技术是解决这一问题的重要途径。菊芋在农业饲料的生产、工业酒精的制备、食品果糖的提炼等能源开发方面用途极广,因此,产量的提升具有重要的实际意义,脱毒技术在菊芋的种植和增产上将发挥巨大的作用。
本试验以菊芋带芽点的薯块和带节的幼嫩茎段为外植体,诱导不定芽的产生,研究以丛生芽途径建立高效的植株再生体系。主要从外植体灭菌、不定芽诱导、丛生芽增殖、壮苗培养、生根培养、炼苗移栽、田间种植及收获等环节进行试验,集中研究培养基配方、pH值、光照强度、接种方式、丛植密度、移栽条件等多个方面的问题。
通过试验,获得的主要结果如下：
1、通过对薯块外植体的污染率、褐化率、无菌率及成活率的统计,可知,薯块外植体最优的消毒程序：取材→自来水粗洗→洗衣粉上清液浸10min→自来水冲洗30min→75%乙醇表面消毒20s→0.1%升汞溶液浸泡1min→无菌水振荡清洗4-6次→无菌滤纸吸干→接入MS培养基。昼温为(25±2)℃,夜温为(23±2)℃,光照时间为12-14h/d,光照强度为1500-2000Lux,成活率达86%以上。茎段外植体是从茎尖脱毒培养的脱毒苗上截取获得的,茎尖培养的培养基为MS+0.5mg/L6-BA +0.6mg/LIBA。
2、影响不定芽诱导的主要因素是激素水平和外植体的接种方式。实验表明：茎段为最佳的组织培养快速繁殖的外植体材料,不定芽诱导最适宜培养基配方为MS+1.0mg/L 6-BA+0.2mg/L IBA, pH5.8,采用正接的接种方式,诱导率可达93%。
3、影响丛生苗继代增殖的主要因素是6-BA浓度、丛植密度和不定芽株高。继代增殖最适宜培养基为MS+2.0mg/L 6-BA, pH5.8,株高1.0cmm,每瓶10株为最宜,增值倍数平均值最大可达4倍,pH对菊芋的丛生增殖影响不明显。
4、影响壮苗培养的主要因素是光照强度和6-BA浓度。壮苗培养最佳培养基为MS+0.1mg/L6-BA, pH5.8,光强度3000-4000Lux为宜,周期7-10d即可。
5、影响生根的主要因素是NAA浓度。生根适宜培养基为MS+0.2mg/LNAA,pH5.8,生根率可达100%。
6、影响移栽的主要因素是混合基质、温度和湿度。常温炼苗3d后,进行移栽种植,较适宜的混合基质为：泥炭土+蛭石+珍珠岩(2：1：1),温度为26℃,湿度为50%,移栽成活率达95%,大田种植成活率达95%以上
7、五月中旬开始田间种植,十月中旬收获,同时对植株各阶段的生长状况及最终的收获情况等进行统计调查。菊芋的块茎形状多样,多呈长形、瘤形等,产量较高,亩产约1500kg,脱毒后亩产约1695kg,脱毒植株和未脱毒植株的产量比较看出,产量明显提高,约13%,增产效果较好。
通过上述实验,全面研究菊芋组织培养快速繁殖的各个技术环节,筛选出最优的技术参数组合,建立高效的菊芋再生体系,提高菊芋再生增殖系数。同时,脱毒对菊芋增产具有较明显的效果,为菊芋的专业化育苗提供技术支持,填补了我国北方菊芋未见研究的空白,也可以做为菊芋的良种筛选和基因工程研究的基础性材料。
Jerusalem artichoke(Helianthus tuberosus L.) is a perennial plant with persistent roots, belongs to the family of Helianthus annuus L.(Compositae), its common name is Yang Jiang, etc. It likes Chrysanthemum above-ground and likes Taro underground. Jerusalem artichoke is a tall plant grow sturdily, with sufficient organic matter accumulation, nutrient-rich tubers, high output was used widely. It is an emerging potential, multi-energy plants and economic crops. Jerusalem artichoke with strong stress tolerance itself, not only saline tolerance, drought stress and cold, but also anti-poor, resist blown sand, etc. So, it may be widely cultivated species of plants with less demand on the growing environment and highly adaptable.
Tuber propagation is the conventional breeding methods for the Jerusalem artichoke. The long-term growth under the ground will lead to the accumulation of large numbers of virus diseases, ultimately deterioration and yield varieties decreased year by year, inefficient industrial production, less Superior species. Establishment of improved artichoke tissue culture technology is an important way to solve this problem. Jerusalem artichoke used widely in the aspect of energy development, such as agricultural forage production, industrial alcohol preparation, food fructose refining, and so on. Therefore, enhance the production has important practical significance, Jerusalem artichoke virus-free technology will play a huge role in planting increase production.
The main results obtained by experiment are as follows:
1. Through the statistical comparisons with the pollution rate and soaked browning rate, sterile rate and survival rate of explant, we can see, two optimum explant disinfection procedures:drawing materials→washing by water→immersing in liquid detergent 10 min→washing by water 30 min→surface sterilizing by 75% ethanol→immersion in 0.1% Mercuric chloride solution→oscillate clean by sterile water 4-6 times→dry by sterile filter paper→access to MS medium. Tuber disinfection with 75% ethanol 20 s and 0.1% Mercuric chloride for 1 min combination treatment. Daytime temperature was (25±2)℃, night temperature was (23±2)℃, illumination time was 12-14 h/d, with 1500-2000 Lux light intensity, survival rate of 86%.
2. The main factors effect shoot induction is the hormone levels and explants inoculation method. The optimal shoot induction medium was MS+1.0 mg/L 6-BA+0.2 mg/L IBA, pH5.8,t positive inoculation (morphology lower down) was the optimal inoculation method. The induction rate is 93%.
3. The main factors effect the shoot subculture proliferation are 6-BA concentration, cluster planting density and shoot height. The optimal subculture medium for the proliferation was MS+6-BA 2.0 mg/L, pH5.8, height 1.0 cm,10 strains every bottle is appropriate, multiple of proliferation could reach 4 times for average.
4. The main factors effect strong seedling are light intensity and 6-BA concentration, the optimal strong seedling medium was MS+0.1 mg/L 6-BA, with the light intensity 1500-2000 Lux.
5. The main factor effect rooting culture is NAA concentration. The optimal rooting medium was MS+0.2 mg/L NAA, H5.8, the rooting rate reached 100%,
6. The main factors effect transplant is mixture, temperature and humidity. After 3 days train seedlings under normal temperature, the plant will be transplant. The optimal mixture was peat soil+ vermiculite+perlite (2:1:1), the temperature is 26℃, humidity is 50%, the survival rate of transplant reach 95%, field planting survival rate of 95%.
7. Planting began in mid-May, harvested in mid-October, while Survey plant growth conditions and the final harvest of the various stages, etc.
Through this experiment, it studied comprehensive on Jerusalem artichoke tissue culture propagation in various technical aspects, and selected the best technical parameters, then established an efficient regeneration system of Helianthus tuberosus, increased bud proliferation coefficient. Virus-free yield on the Jerusalem artichoke has obvious effect; it provided the professional technical support to Jerusalem artichoke nursery, also can be the basic material for breed select and gene engineering research.
目前,我国高度重视对能源植物的开发与利用,菊芋产业快速发展,短期内需求量较大,同时,菊芋的常规繁殖方式为块茎繁殖,属无性繁殖,能够成为病毒的良好寄主,会导致大量病毒病的积累,最终将导致品种的劣化和产量的逐年降低,工业化生产效率不高,优良品种较少,因此建立和完善高效的菊芋组织培养快繁技术是解决这一问题的重要途径。菊芋在农业饲料的生产、工业酒精的制备、食品果糖的提炼等能源开发方面用途极广,因此,产量的提升具有重要的实际意义,脱毒技术在菊芋的种植和增产上将发挥巨大的作用。
本试验以菊芋带芽点的薯块和带节的幼嫩茎段为外植体,诱导不定芽的产生,研究以丛生芽途径建立高效的植株再生体系。主要从外植体灭菌、不定芽诱导、丛生芽增殖、壮苗培养、生根培养、炼苗移栽、田间种植及收获等环节进行试验,集中研究培养基配方、pH值、光照强度、接种方式、丛植密度、移栽条件等多个方面的问题。
通过试验,获得的主要结果如下：
1、通过对薯块外植体的污染率、褐化率、无菌率及成活率的统计,可知,薯块外植体最优的消毒程序：取材→自来水粗洗→洗衣粉上清液浸10min→自来水冲洗30min→75%乙醇表面消毒20s→0.1%升汞溶液浸泡1min→无菌水振荡清洗4-6次→无菌滤纸吸干→接入MS培养基。昼温为(25±2)℃,夜温为(23±2)℃,光照时间为12-14h/d,光照强度为1500-2000Lux,成活率达86%以上。茎段外植体是从茎尖脱毒培养的脱毒苗上截取获得的,茎尖培养的培养基为MS+0.5mg/L6-BA +0.6mg/LIBA。
2、影响不定芽诱导的主要因素是激素水平和外植体的接种方式。实验表明：茎段为最佳的组织培养快速繁殖的外植体材料,不定芽诱导最适宜培养基配方为MS+1.0mg/L 6-BA+0.2mg/L IBA, pH5.8,采用正接的接种方式,诱导率可达93%。
3、影响丛生苗继代增殖的主要因素是6-BA浓度、丛植密度和不定芽株高。继代增殖最适宜培养基为MS+2.0mg/L 6-BA, pH5.8,株高1.0cmm,每瓶10株为最宜,增值倍数平均值最大可达4倍,pH对菊芋的丛生增殖影响不明显。
4、影响壮苗培养的主要因素是光照强度和6-BA浓度。壮苗培养最佳培养基为MS+0.1mg/L6-BA, pH5.8,光强度3000-4000Lux为宜,周期7-10d即可。
5、影响生根的主要因素是NAA浓度。生根适宜培养基为MS+0.2mg/LNAA,pH5.8,生根率可达100%。
6、影响移栽的主要因素是混合基质、温度和湿度。常温炼苗3d后,进行移栽种植,较适宜的混合基质为：泥炭土+蛭石+珍珠岩(2：1：1),温度为26℃,湿度为50%,移栽成活率达95%,大田种植成活率达95%以上
7、五月中旬开始田间种植,十月中旬收获,同时对植株各阶段的生长状况及最终的收获情况等进行统计调查。菊芋的块茎形状多样,多呈长形、瘤形等,产量较高,亩产约1500kg,脱毒后亩产约1695kg,脱毒植株和未脱毒植株的产量比较看出,产量明显提高,约13%,增产效果较好。
通过上述实验,全面研究菊芋组织培养快速繁殖的各个技术环节,筛选出最优的技术参数组合,建立高效的菊芋再生体系,提高菊芋再生增殖系数。同时,脱毒对菊芋增产具有较明显的效果,为菊芋的专业化育苗提供技术支持,填补了我国北方菊芋未见研究的空白,也可以做为菊芋的良种筛选和基因工程研究的基础性材料。
Jerusalem artichoke(Helianthus tuberosus L.) is a perennial plant with persistent roots, belongs to the family of Helianthus annuus L.(Compositae), its common name is Yang Jiang, etc. It likes Chrysanthemum above-ground and likes Taro underground. Jerusalem artichoke is a tall plant grow sturdily, with sufficient organic matter accumulation, nutrient-rich tubers, high output was used widely. It is an emerging potential, multi-energy plants and economic crops. Jerusalem artichoke with strong stress tolerance itself, not only saline tolerance, drought stress and cold, but also anti-poor, resist blown sand, etc. So, it may be widely cultivated species of plants with less demand on the growing environment and highly adaptable.
Tuber propagation is the conventional breeding methods for the Jerusalem artichoke. The long-term growth under the ground will lead to the accumulation of large numbers of virus diseases, ultimately deterioration and yield varieties decreased year by year, inefficient industrial production, less Superior species. Establishment of improved artichoke tissue culture technology is an important way to solve this problem. Jerusalem artichoke used widely in the aspect of energy development, such as agricultural forage production, industrial alcohol preparation, food fructose refining, and so on. Therefore, enhance the production has important practical significance, Jerusalem artichoke virus-free technology will play a huge role in planting increase production.
The main results obtained by experiment are as follows:
1. Through the statistical comparisons with the pollution rate and soaked browning rate, sterile rate and survival rate of explant, we can see, two optimum explant disinfection procedures:drawing materials→washing by water→immersing in liquid detergent 10 min→washing by water 30 min→surface sterilizing by 75% ethanol→immersion in 0.1% Mercuric chloride solution→oscillate clean by sterile water 4-6 times→dry by sterile filter paper→access to MS medium. Tuber disinfection with 75% ethanol 20 s and 0.1% Mercuric chloride for 1 min combination treatment. Daytime temperature was (25±2)℃, night temperature was (23±2)℃, illumination time was 12-14 h/d, with 1500-2000 Lux light intensity, survival rate of 86%.
2. The main factors effect shoot induction is the hormone levels and explants inoculation method. The optimal shoot induction medium was MS+1.0 mg/L 6-BA+0.2 mg/L IBA, pH5.8,t positive inoculation (morphology lower down) was the optimal inoculation method. The induction rate is 93%.
3. The main factors effect the shoot subculture proliferation are 6-BA concentration, cluster planting density and shoot height. The optimal subculture medium for the proliferation was MS+6-BA 2.0 mg/L, pH5.8, height 1.0 cm,10 strains every bottle is appropriate, multiple of proliferation could reach 4 times for average.
4. The main factors effect strong seedling are light intensity and 6-BA concentration, the optimal strong seedling medium was MS+0.1 mg/L 6-BA, with the light intensity 1500-2000 Lux.
5. The main factor effect rooting culture is NAA concentration. The optimal rooting medium was MS+0.2 mg/L NAA, H5.8, the rooting rate reached 100%,
6. The main factors effect transplant is mixture, temperature and humidity. After 3 days train seedlings under normal temperature, the plant will be transplant. The optimal mixture was peat soil+ vermiculite+perlite (2:1:1), the temperature is 26℃, humidity is 50%, the survival rate of transplant reach 95%, field planting survival rate of 95%.
7. Planting began in mid-May, harvested in mid-October, while Survey plant growth conditions and the final harvest of the various stages, etc.
Through this experiment, it studied comprehensive on Jerusalem artichoke tissue culture propagation in various technical aspects, and selected the best technical parameters, then established an efficient regeneration system of Helianthus tuberosus, increased bud proliferation coefficient. Virus-free yield on the Jerusalem artichoke has obvious effect; it provided the professional technical support to Jerusalem artichoke nursery, also can be the basic material for breed select and gene engineering research.
引文
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