蒙古黄芪的组织培养
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摘要
黄芪(Radix Astragali)是豆科(Leguminosae)蝶形花亚科(Papilionoideae)多年生草本植物,具有很高的药用价值和经济价值。但由于近年来的过度采挖,致使野生黄芪资源几近枯竭,不能满足市场的需要,所以离体培养已成为增加黄芪产量的主要手段之一。本文以购自甘肃省陇西县的优质蒙古黄芪种子为材料,通过组织培养的方法建立其快速繁殖体系。
蒙古黄芪快速繁殖体系的建立,主要是针对种子的消毒和萌发、外植体的筛选、愈伤组织初代诱导培养基、继代增殖培养基和生根培养基的选择,以及褐化现象的抑制进行的。
种子的消毒和萌发率的高低对于无菌条件下获得外植体是很重要的。将蒙古黄芪种子浸泡在适量洗洁精10min中后,再依次进行45s中75%的酒精浸泡和9.5min0.1%的升汞消毒,其萌发率可达100%,且不发生任何污染现象;另外,在低温中存放半年和一年后的种子若经过萌发前蒸馏水中10h的浸泡预处理,其萌发率可有大幅度提高,达到100%。
分别以蒙古黄芪的子叶、下胚轴和胚根作为外植体诱导愈伤组织。结果发现,在分别添加了NAA、2,4-D及6-BA的单一植物激素的MS培养基中,子叶均不能诱导出愈伤组织,而下胚轴在NAA为1.0mg/L时诱导率可达70%;在添加了不同生长素和细胞分裂素组合的培养基中,子叶诱导的愈伤组织褐化程度较轻,但诱导率均较低,而胚根的诱导率虽高于子叶,但褐化严重,只有下胚轴的诱导率最高,可达100%,虽仍有褐化现象发生但生长旺盛。所以,下胚轴是诱导蒙古黄芪愈伤组织的最佳外植体。
培养基中的激素种类与浓度配比是影响蒙古黄芪初代培养、继代增殖及生根培养的重要因素。其中,愈伤组织的最佳初代诱导培养基是:MS+0.5或1.0mg/L6-BA+0.15mg/L NAA。在此激素中,愈伤组织诱导率可达到100%;而继代中仍可以此浓度来进行增殖培养,这样不仅可使愈伤组织达到较好的生长效果而且还具有较高的增殖量。另外,在继代培养时愈伤组织易发生褐化,当用单一防褐剂对其进行抑制时,活性炭(AC)的作用不明显,而一定浓度的Vc对愈伤组织的褐化则起到了较明显的抑制效果;因而进一步通过正交试验对综合因素抑制褐化的效果做了对比,得出当培养基中添加0.1mg/L Vc,前3天进行暗培养,其余几天进行光培养(10h/d),10天后继代时,其褐化得到了较好的控制。在生根培养中,1/2MS+2.0mg/L NAA为最适生根培养基,其中诱导生根率可达到83.3%,若再向培养基中添加0.3%的活性炭(AC)时,则生根数量更多。
通过对蒙古黄芪愈伤组织总的生长趋势进行测定,发现即使按照所有筛选出的最适条件进行培养,愈伤组织的最佳生长旺盛期(即具有最大的增殖率、最佳的生长状态并无褐化现象的发生)只能稳定在3、4代左右,若继续进行增殖培养,则长势逐渐变缓,且伴有褐化现象的发生。
Radix Astragali is perennial herb of Leguminosae Papilionoideae plants, with high medicinal values and economic values. Recently, the number of wild plant resourses have become reduced, which can not meet the need of the market because of the excessive collection. Therefore, the rapid propagation has become one of the major messures to increase the quantity.
The seeds of Radix Astragali selected from Longxi in Gansu province are used to establish the rapid propagation system in this paper.
The steps of the establishment of rapid propagation system contained the sterilization and germination of seeds, the selection of explants, and the selections of callus medium in first and secondary induction, as well as the darkening inhibition medium and rooting medium.
The sterilization of seeds and the germination rates of seeds are important to obtain sterile explants. The best sterilizing method were as follows, immersion in detergents for 10min, sterilization with 75% ethanol for 45s and 0.1% HgCl2 for 9.5min. And the rates of germination was 100%, without any pollution. In addtion, the germination rates of seeds in half-a-year and one-year low temperature storage, could obviously increased, with the highest germinatin rate of 100%, if pre-treament was done to immerse the seeds in distilled water for 10 hours.
Cotyledon, hypocotyl and radicle of Astragalus mongholicus Bunge were used as explants to induce callus. The results showed that, cotyledon could not induce the callus on MS base medium with any grown regulators, but hypocotyl could induce the callus on MS base medium with 1.0mg/LNAA, that the rate of callus induced by cotyledon was lower with slight darkening, and radicle with higher induction rate but heavier darkening. And the rate of callus induced by cotyledon was the highest, with 70%. Therefore, hypocotyl was the best explant in callus induction.
The kinds and proportions of hormone was the key factors in the experiment. The callus induction reached as high as 100% in the combination of 0.5/1.0mg/L6-BA and 0.15mg/L NAA in MS base medium. And in secondary culture, the same medium uesd as in first induction was still good to multiplication of callus. Moreover, Vc was useful in darkening inhibition, and the most effective condition to darkening inhibition was selected, which was as follows, an addition of 0.1mg/L Vc in medium, dark-culturing in the first 3 days, light culturing 10h/d for the rest of days, and with 10-day-generation. For rooting, the best conditin medium was 1/2MS+2.0 mg/L NAA with the highest rate of 83.3%, and there will be more and more roots if 0.3%AC was added.
The growth trend of callus of Astragalus mongholicus Bung was tested, that was, the callus of Astragalus mongholicus Bunge could growth well in the 3rd or 4th generation, and multiplication could not occur in the 8th generation.
蒙古黄芪快速繁殖体系的建立,主要是针对种子的消毒和萌发、外植体的筛选、愈伤组织初代诱导培养基、继代增殖培养基和生根培养基的选择,以及褐化现象的抑制进行的。
种子的消毒和萌发率的高低对于无菌条件下获得外植体是很重要的。将蒙古黄芪种子浸泡在适量洗洁精10min中后,再依次进行45s中75%的酒精浸泡和9.5min0.1%的升汞消毒,其萌发率可达100%,且不发生任何污染现象;另外,在低温中存放半年和一年后的种子若经过萌发前蒸馏水中10h的浸泡预处理,其萌发率可有大幅度提高,达到100%。
分别以蒙古黄芪的子叶、下胚轴和胚根作为外植体诱导愈伤组织。结果发现,在分别添加了NAA、2,4-D及6-BA的单一植物激素的MS培养基中,子叶均不能诱导出愈伤组织,而下胚轴在NAA为1.0mg/L时诱导率可达70%;在添加了不同生长素和细胞分裂素组合的培养基中,子叶诱导的愈伤组织褐化程度较轻,但诱导率均较低,而胚根的诱导率虽高于子叶,但褐化严重,只有下胚轴的诱导率最高,可达100%,虽仍有褐化现象发生但生长旺盛。所以,下胚轴是诱导蒙古黄芪愈伤组织的最佳外植体。
培养基中的激素种类与浓度配比是影响蒙古黄芪初代培养、继代增殖及生根培养的重要因素。其中,愈伤组织的最佳初代诱导培养基是:MS+0.5或1.0mg/L6-BA+0.15mg/L NAA。在此激素中,愈伤组织诱导率可达到100%;而继代中仍可以此浓度来进行增殖培养,这样不仅可使愈伤组织达到较好的生长效果而且还具有较高的增殖量。另外,在继代培养时愈伤组织易发生褐化,当用单一防褐剂对其进行抑制时,活性炭(AC)的作用不明显,而一定浓度的Vc对愈伤组织的褐化则起到了较明显的抑制效果;因而进一步通过正交试验对综合因素抑制褐化的效果做了对比,得出当培养基中添加0.1mg/L Vc,前3天进行暗培养,其余几天进行光培养(10h/d),10天后继代时,其褐化得到了较好的控制。在生根培养中,1/2MS+2.0mg/L NAA为最适生根培养基,其中诱导生根率可达到83.3%,若再向培养基中添加0.3%的活性炭(AC)时,则生根数量更多。
通过对蒙古黄芪愈伤组织总的生长趋势进行测定,发现即使按照所有筛选出的最适条件进行培养,愈伤组织的最佳生长旺盛期(即具有最大的增殖率、最佳的生长状态并无褐化现象的发生)只能稳定在3、4代左右,若继续进行增殖培养,则长势逐渐变缓,且伴有褐化现象的发生。
Radix Astragali is perennial herb of Leguminosae Papilionoideae plants, with high medicinal values and economic values. Recently, the number of wild plant resourses have become reduced, which can not meet the need of the market because of the excessive collection. Therefore, the rapid propagation has become one of the major messures to increase the quantity.
The seeds of Radix Astragali selected from Longxi in Gansu province are used to establish the rapid propagation system in this paper.
The steps of the establishment of rapid propagation system contained the sterilization and germination of seeds, the selection of explants, and the selections of callus medium in first and secondary induction, as well as the darkening inhibition medium and rooting medium.
The sterilization of seeds and the germination rates of seeds are important to obtain sterile explants. The best sterilizing method were as follows, immersion in detergents for 10min, sterilization with 75% ethanol for 45s and 0.1% HgCl2 for 9.5min. And the rates of germination was 100%, without any pollution. In addtion, the germination rates of seeds in half-a-year and one-year low temperature storage, could obviously increased, with the highest germinatin rate of 100%, if pre-treament was done to immerse the seeds in distilled water for 10 hours.
Cotyledon, hypocotyl and radicle of Astragalus mongholicus Bunge were used as explants to induce callus. The results showed that, cotyledon could not induce the callus on MS base medium with any grown regulators, but hypocotyl could induce the callus on MS base medium with 1.0mg/LNAA, that the rate of callus induced by cotyledon was lower with slight darkening, and radicle with higher induction rate but heavier darkening. And the rate of callus induced by cotyledon was the highest, with 70%. Therefore, hypocotyl was the best explant in callus induction.
The kinds and proportions of hormone was the key factors in the experiment. The callus induction reached as high as 100% in the combination of 0.5/1.0mg/L6-BA and 0.15mg/L NAA in MS base medium. And in secondary culture, the same medium uesd as in first induction was still good to multiplication of callus. Moreover, Vc was useful in darkening inhibition, and the most effective condition to darkening inhibition was selected, which was as follows, an addition of 0.1mg/L Vc in medium, dark-culturing in the first 3 days, light culturing 10h/d for the rest of days, and with 10-day-generation. For rooting, the best conditin medium was 1/2MS+2.0 mg/L NAA with the highest rate of 83.3%, and there will be more and more roots if 0.3%AC was added.
The growth trend of callus of Astragalus mongholicus Bung was tested, that was, the callus of Astragalus mongholicus Bunge could growth well in the 3rd or 4th generation, and multiplication could not occur in the 8th generation.
引文
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