花楸不同外植体的茎丛增生和愈伤组织诱导及植株再生
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摘要
花楸(Sorbus pohuashanesis Hedl)是北方未被充分开发利用的优良的绿化树种。我们以幼胚、成熟胚、无菌茎丛的茎段、成年树的嫩叶和茎段等为外植体，首次通过不定芽和腋芽增殖途径建立了两套高效、可靠的组培再生系统。
1、以花楸幼胚为外植体的研究表明：
(1)取材时期和激素浓度配比是影响花楸幼胚丛生芽诱导效果的主要因素。最适采种时期为7月下旬至8月上旬，筛选出最佳的丛生芽诱导培养基为MS+BA0.5mg／L+NAA0.05mg／L+蔗糖15～20g／L+琼脂6g／L，丛生芽诱导率最高可达22.22％；
(2)丛生芽最佳的增殖培养基为WPM+BA0.3～0.5mg／L+蔗糖20g／L+琼脂7g／L，经继代培养一个幼胚可诱导出7～20个小茎丛；带有矮小不定芽的组织块可继代培养多次；
(3)实验从改变培养基种类、降低激素浓度、增加琼脂浓度等方面采取有效的措施消除了茎丛增殖过程中的玻璃化现象；
(4)当茎丛高达2～3cm时，转入生根培养基诱导生根，筛选出最佳的生根培养基为1／2MS+NAA0.3mg／L或IBA0.2mg／L；
(5)移栽前将根系发达的生根苗打开瓶盖驯化3-5天，移入装有珍珠岩的塑料钵中炼苗一周，然后移栽到泥炭土和河沙以2：1混合的基质中，保持25±1℃，湿度＞80％，成活率可达80％以上。
2、以无菌茎丛的茎段为外植体，筛选出最佳的腋芽增殖培养基为：WPM+IBA(或NAA)0.05mg／L+BA1.0mg／L+蔗糖20g／L+琼脂7g／L，腋芽最大增殖倍数可达6。
3、以花楸成熟胚为外植体，在添加BA、TDZ、IBA、2，4-D、NAA组成的不同激素组合的MS培养基上培养。结果表明：
(1)相同条件下，沙藏处理3个月的种子愈伤组织诱导率(100％)明显高于未经处理的种子(55.56％)；
(2)诱导愈伤组织的最佳激素组合和浓度为NAA0.5-1.0mg／L+BA1.0mg／L；
(3)当NAA或2，4-D浓度≤0.1mg／L与BA组合能诱导出不定芽，经过继代可产生5～20个不定芽，经生根培养可成苗。
4、以成年树嫩叶和茎段为外植体，在添加BA、TDZ、IBA、NAA、2，4-D组成的不同激素组合的MS培养基上培养，以诱导愈伤组织的结果表明：
(1)以幼嫩的茎段为外植体时，从愈伤组织的诱导率、体积和生长状态综合考
虑，得出生长素的效应大小为NAA>2，4一D>IBA，最佳激素种类组合为
NAAI.omg/L+BAO.5一3.omg/L、2，4一Dl.omg/L+BAO.5一3.omg/L或TDZO.01~0.5
mg/L;
(2)以幼嫩的叶片为外植体时，虽然诱导率可达100%，但只是在切口处长小米
粒大小的愈伤组织，生长素的诱导效应大小为2，4一D>NAA>1 BA，最佳激素组合为
2，4一D 1 .omg/L+BAO.5~3.omg/L:
(3)无论以嫩叶还是茎段为外植体，将经初代培养诱导的愈伤组织转入增殖培
养基，增殖效果都非常好，4周后愈伤组织长满培养瓶。
Sorbus pohuashanesis Hed1 is a valuable ornamental species in northern urban areas of our country. We took young embryos, mature embryos, stem segments of tissue-cultured shoots, tender leaves and stem segments of adult trees as explants, two high effective in vitro culture systems of Sorbus pohuashanesis plants were established through adventitious shoot and axillary bud regeneration approaches for the first time.
1. When young embryos of Sorbus pohuashanesis were taken as explants, the following results were obtained:
(1) The collection period of young embryos and the concentration and combination of hormones were the principal influencing factors for shoot regeneration from the young embryos. The optimum collection period of young embryos was from the late July to the beginning of August. The optimum induction medium was MS+BA0.5mg/L+NAA0.05mg/L+ sucrose 15~20g/L + agar 6g/L, and the induction rate of adventitious shoots was up to 22.22%.
(2) The best proliferation medium of adventitious shoots was WPM+BA 0.3 -0.5mg/L+ sucrose 20g/L + agar 6g/L. Seven to twenty shoots were able to obtain from one embryo in the first subculture. Some remained organ blocks with short adventitious shoot could be subcultured several times.
(3) Changing the kind of medium, decreasing the concentration of hormone and increasing the concentration of agar were proved to be effective steps to eliminate the serious vitrification during the proliferation of adventitious shoot.
(4) When clustered shoots grew up to 2-3cm, they were transferred onto rooting medium. The best rooting medium was 1/2MS medium supplemented 0.3mg/L NAA or 0.2mg/L IBA.
(5) Before transplanting, the plantlets were domesticated by removing the tube cover for 3-5 days, transplanted into container with perlite for a week, then transplanted to mixed media (peat/sand=2:1) and keeping environment temperature at 25 C + 1 C and moisture over 80%. The survive rate could be more than 80%.
2. The stem segments from in vitro cultured shoots derived from young embryos were cultured onto the axillary bud proliferation medium, of which the optimum one
was WPM+IBA(or NAA)0.05mg/L+BA1.0mg/L+sucrose 20g/L+agar 7g/L. Six shoots could obtained at most from one axillary bud.
3. Mature embryos of Sorbus pohuashanensis were cultured on MS medium supplemented with different hormone combinations including BA, TDZ, IBA, 2,4-D and NAA. The results showed that:
(1) The induction rate of callus from seeds by stratification for 3 months (100%) was higher than that of seeds without treatment ( 55.56%) in the same condition.
(2) The best hormone combination for callus induction was NAA0.5-1.0 mg/L+BA1.0mg/L.
(3) The adventitious shoots could be induced on medium supplemented with BA and NAA (less than 0.1 mg/L) or 2,4-D (less than 0.1 mg/L). The induced shoots could further generate 5-20 shoots in subculture. Shoots rooted and plantlets established.
4. Tender leaves and stem segment of adult trees were cultured for callus induction on MS medium supplemented with different hormone combinations including BA, TDZ, IBA, NAA and 2,4-D. The results showed that:
(1) When tender stem segments of adult trees was taken as explants the effect sequence of auxin was NAA> 2,4-D > IBA and the optimum hormone combination was 1.0mg/L NAA+0.5~3.0mg/L BA, 1.0mg/L 2,4-D+0.5~3.0mg/L BA or 0.01 -0.5mg/L TDZ, considering from the callus induction rate, volume and growth status.
(2) When tender leaves of adult trees was taken as explants, the induction rate could reach 100%, but the callus was very small and can only regenerated from the cut parts of explants. The effect sequence of auxin was 2,4-D > NAA > IBA and the optimum hormone combination was 2,4-D1.0mg/L+0.5~BA3.0mg/L.
(3) When the callus from both tender leaf and stem segments of adult trees in first culture was transferred onto proliferation medium, they would grow full of bottle after four weeks culture.
1、以花楸幼胚为外植体的研究表明：
(1)取材时期和激素浓度配比是影响花楸幼胚丛生芽诱导效果的主要因素。最适采种时期为7月下旬至8月上旬，筛选出最佳的丛生芽诱导培养基为MS+BA0.5mg／L+NAA0.05mg／L+蔗糖15～20g／L+琼脂6g／L，丛生芽诱导率最高可达22.22％；
(2)丛生芽最佳的增殖培养基为WPM+BA0.3～0.5mg／L+蔗糖20g／L+琼脂7g／L，经继代培养一个幼胚可诱导出7～20个小茎丛；带有矮小不定芽的组织块可继代培养多次；
(3)实验从改变培养基种类、降低激素浓度、增加琼脂浓度等方面采取有效的措施消除了茎丛增殖过程中的玻璃化现象；
(4)当茎丛高达2～3cm时，转入生根培养基诱导生根，筛选出最佳的生根培养基为1／2MS+NAA0.3mg／L或IBA0.2mg／L；
(5)移栽前将根系发达的生根苗打开瓶盖驯化3-5天，移入装有珍珠岩的塑料钵中炼苗一周，然后移栽到泥炭土和河沙以2：1混合的基质中，保持25±1℃，湿度＞80％，成活率可达80％以上。
2、以无菌茎丛的茎段为外植体，筛选出最佳的腋芽增殖培养基为：WPM+IBA(或NAA)0.05mg／L+BA1.0mg／L+蔗糖20g／L+琼脂7g／L，腋芽最大增殖倍数可达6。
3、以花楸成熟胚为外植体，在添加BA、TDZ、IBA、2，4-D、NAA组成的不同激素组合的MS培养基上培养。结果表明：
(1)相同条件下，沙藏处理3个月的种子愈伤组织诱导率(100％)明显高于未经处理的种子(55.56％)；
(2)诱导愈伤组织的最佳激素组合和浓度为NAA0.5-1.0mg／L+BA1.0mg／L；
(3)当NAA或2，4-D浓度≤0.1mg／L与BA组合能诱导出不定芽，经过继代可产生5～20个不定芽，经生根培养可成苗。
4、以成年树嫩叶和茎段为外植体，在添加BA、TDZ、IBA、NAA、2，4-D组成的不同激素组合的MS培养基上培养，以诱导愈伤组织的结果表明：
(1)以幼嫩的茎段为外植体时，从愈伤组织的诱导率、体积和生长状态综合考
虑，得出生长素的效应大小为NAA>2，4一D>IBA，最佳激素种类组合为
NAAI.omg/L+BAO.5一3.omg/L、2，4一Dl.omg/L+BAO.5一3.omg/L或TDZO.01~0.5
mg/L;
(2)以幼嫩的叶片为外植体时，虽然诱导率可达100%，但只是在切口处长小米
粒大小的愈伤组织，生长素的诱导效应大小为2，4一D>NAA>1 BA，最佳激素组合为
2，4一D 1 .omg/L+BAO.5~3.omg/L:
(3)无论以嫩叶还是茎段为外植体，将经初代培养诱导的愈伤组织转入增殖培
养基，增殖效果都非常好，4周后愈伤组织长满培养瓶。
Sorbus pohuashanesis Hed1 is a valuable ornamental species in northern urban areas of our country. We took young embryos, mature embryos, stem segments of tissue-cultured shoots, tender leaves and stem segments of adult trees as explants, two high effective in vitro culture systems of Sorbus pohuashanesis plants were established through adventitious shoot and axillary bud regeneration approaches for the first time.
1. When young embryos of Sorbus pohuashanesis were taken as explants, the following results were obtained:
(1) The collection period of young embryos and the concentration and combination of hormones were the principal influencing factors for shoot regeneration from the young embryos. The optimum collection period of young embryos was from the late July to the beginning of August. The optimum induction medium was MS+BA0.5mg/L+NAA0.05mg/L+ sucrose 15~20g/L + agar 6g/L, and the induction rate of adventitious shoots was up to 22.22%.
(2) The best proliferation medium of adventitious shoots was WPM+BA 0.3 -0.5mg/L+ sucrose 20g/L + agar 6g/L. Seven to twenty shoots were able to obtain from one embryo in the first subculture. Some remained organ blocks with short adventitious shoot could be subcultured several times.
(3) Changing the kind of medium, decreasing the concentration of hormone and increasing the concentration of agar were proved to be effective steps to eliminate the serious vitrification during the proliferation of adventitious shoot.
(4) When clustered shoots grew up to 2-3cm, they were transferred onto rooting medium. The best rooting medium was 1/2MS medium supplemented 0.3mg/L NAA or 0.2mg/L IBA.
(5) Before transplanting, the plantlets were domesticated by removing the tube cover for 3-5 days, transplanted into container with perlite for a week, then transplanted to mixed media (peat/sand=2:1) and keeping environment temperature at 25 C + 1 C and moisture over 80%. The survive rate could be more than 80%.
2. The stem segments from in vitro cultured shoots derived from young embryos were cultured onto the axillary bud proliferation medium, of which the optimum one
was WPM+IBA(or NAA)0.05mg/L+BA1.0mg/L+sucrose 20g/L+agar 7g/L. Six shoots could obtained at most from one axillary bud.
3. Mature embryos of Sorbus pohuashanensis were cultured on MS medium supplemented with different hormone combinations including BA, TDZ, IBA, 2,4-D and NAA. The results showed that:
(1) The induction rate of callus from seeds by stratification for 3 months (100%) was higher than that of seeds without treatment ( 55.56%) in the same condition.
(2) The best hormone combination for callus induction was NAA0.5-1.0 mg/L+BA1.0mg/L.
(3) The adventitious shoots could be induced on medium supplemented with BA and NAA (less than 0.1 mg/L) or 2,4-D (less than 0.1 mg/L). The induced shoots could further generate 5-20 shoots in subculture. Shoots rooted and plantlets established.
4. Tender leaves and stem segment of adult trees were cultured for callus induction on MS medium supplemented with different hormone combinations including BA, TDZ, IBA, NAA and 2,4-D. The results showed that:
(1) When tender stem segments of adult trees was taken as explants the effect sequence of auxin was NAA> 2,4-D > IBA and the optimum hormone combination was 1.0mg/L NAA+0.5~3.0mg/L BA, 1.0mg/L 2,4-D+0.5~3.0mg/L BA or 0.01 -0.5mg/L TDZ, considering from the callus induction rate, volume and growth status.
(2) When tender leaves of adult trees was taken as explants, the induction rate could reach 100%, but the callus was very small and can only regenerated from the cut parts of explants. The effect sequence of auxin was 2,4-D > NAA > IBA and the optimum hormone combination was 2,4-D1.0mg/L+0.5~BA3.0mg/L.
(3) When the callus from both tender leaf and stem segments of adult trees in first culture was transferred onto proliferation medium, they would grow full of bottle after four weeks culture.
引文
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