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蒙古沙冬青(Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f.),又称大沙冬青,属豆科沙冬青属(Ammopiptanthus Cheng f.)常绿阔叶灌木,在我国主要分布于内蒙古、甘肃、宁夏的荒漠地区,具有极强的抗旱、抗寒、抗盐碱性和耐蚀性[1-2],在防沙固沙、防止水土流失等方面具有重要的生态价值。此外,蒙古沙冬青提取物中被证实含有白藜芦醇、生物碱和黄酮类化合物等多种活性成分,具有抗肿瘤、抗炎、抗自由基、抗病毒等药理作用[3-5]。蒙古沙冬青作为一种重要的生态、药用种质资源其开发应用前景十分广阔,但自身狭小的分布范围和近年人类活动造成的生境破坏,其数量正在不断减少,现已被列为国家二级保护植物[6]。对珍稀濒危种质资源采用组织培养的方式进行快速繁殖是重要的保护手段之一。许多研究学者早在20世纪80年代就开始尝试采用蒙古沙冬青无菌幼苗的子叶、胚轴或茎段进行愈伤组织诱导与芽的分化[7-11]。近年,新的研究也多是选用上述外植体诱导培养再生植株[12-13]。通过体细胞胚胎发生的方式进行再生植株诱导,在培养过程中存在愈伤组织易褐化、难继代,分化率低,玻璃化现象严重等问题。本研究选取蒙古沙冬青子叶节为外植体,进行丛生芽诱导培养再生植株,以器官直接分化途径建立的快速繁殖体系,可以明显降低组培褐化率,缩短培养周期,为蒙古沙冬青的种质资源保护提供技术支撑,为进一步开展遗传转化获得抗性更加优良的蒙古沙冬青奠定基础。
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表 1表明:萌发培养基中6-BA的浓度对蒙古沙冬青幼苗生长和子叶节外植体丛生芽的诱导影响显著,在添加6-BA的MS培养基中,蒙古沙冬青幼苗的胚轴均比不添加的粗大,且获得的子叶节外植体在丛生芽诱导阶段的诱导率和平均芽数均显著高于不添加6-BA的萌发处理。使用5.0 mg·L-16-BA萌发培养基获得的子叶节进行从生芽诱导时丛生芽数最多,但子叶节处芽呈簇聚集生长,形态矮小、细弱,无有效芽。使用3.0 mg·L-16-BA萌发培养基获得的子叶节进行从生芽诱导时,丛生芽诱导率最高,但后期生长部分叶片有畸形。因此,综合考虑蒙古沙冬青幼苗生长和子叶节外植体丛生芽诱导状况,确定萌发培养基中6-BA的最佳浓度为2.0 mg·L-1。
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Treatment6-BA浓度
Concentration /(mg·L-1)诱导率
Induction rate/%平均芽数
Average bud number/个生长状况
Growth status1 0.0 18.21±0.38C 0.40±0.23D 子叶节胚轴细弱,零星有芽。
Hypocotyl of cotyledonary node was thin and buds were in a fewer number.2 1.0 36.08±0.62B 1.48±0.12C 子叶节胚轴细弱,芽数清晰,后期有明显茎叶。
Hypocotyl of cotyledonary node was thin; buds can be clearly identified and there were normal stem and leaves in the latter stages.3 2.0 73.32±0.51A 2.06±0.11B 子叶节胚轴粗壮,芽数清晰,后期有明显茎叶。
Hypocotyl of cotyledonary node was thick; buds can be clearly identified and there were normal stem and leaves in the latter stages.4 3.0 75.04±0.33A 2.26±0.17B 子叶节胚轴粗壮,芽数清晰,后期有明显茎叶, 但部分叶片有畸形。
Hypocotyl of cotyledonary node was thick; buds can be clearly identified and both normal and malformed stem and leaves were available in the latter stages.5 5.0 74.83±0.56A 5.02±0.33A 子叶节胚轴粗壮,芽多细密呈簇状,无有效芽。
Hypocotyl of cotyledonary node was thick; buds were densely clustered and there were no normal buds.注:表中计算结果为平均值±标准误,同列不同大写字母表示在0.01水平差异显著,下同。
Note: The results obtained of every row were means±SE (α<0.01).The same as below.Table 1. Effects of concentration of 6-BA in germination medium on cotyledonary nodes growth and induction of cluster bud from of Ammopiptanthus mongolicus
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不同萌发天数的蒙古沙冬青子叶节均能诱导出丛生芽,丛生芽诱导率及芽数随种子萌发天数的增加呈先增加后降低的趋势(表 2)。不同处理间,丛生芽诱导率存在不同程度的差异显著性,其中, 种子萌发1 d的子叶节外植体丛生芽诱导率最低,种子萌发7 d的子叶节丛生芽诱导率最高,但与萌发9、11 d的子叶节丛生芽诱导率差异不显著。种子萌发1~11 d,由子叶节分化产生的丛生芽芽数差异不显著。综合考虑丛生芽诱导率及平均芽数,种子萌发7~11 d后得到的子叶节外植体是诱导子叶节丛生芽分化的最佳材料。
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Treatment种子萌发天数
Seed germination days/d诱导率
Induction rate /%平均芽数
Average bud number /个1 1 18.33±0.42E 1.99±0.06A 2 3 27.35±0.23D 2.01±0.10A 3 5 35.16±0.20C 2.00±0.11A 4 7 74.70±0.56A 2.13±0.06A 5 9 74.53±0.57A 2.10±0.02A 6 11 74.38±0.42A 2.06±0.10A 7 13 51.26±0.42B 1.89±0.04B 8 15 27.37±0.23D 1.56±0.26C Table 2. Effects of seed germination days on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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含有相同浓度6-BA的MS培养基和B5培养基对丛生芽诱导率和丛生芽数的影响差异不显著,但B5培养基抑制褐化的效果显著好于MS培养基(表 3),丛生芽生长30 d时,生长状况良好。故选择B5培养基为丛生芽诱导的基本培养基。
培养基
Medium诱导率
Induction rate /%平均芽数
Average bud number/个生长状况
Growth statusMS 73.35±0.26A 2.06±0.22A 丛生芽生长2030 d时,叶缘、芽尖易褐化,30 d以上易褐变死亡。
Leaf margin and shoot tip were easy to browning when cluster buds were 2030 days old and they were easy to death for browning after 30 d.B5 75.02±0.34A 2.10±0.23A 丛生芽生长30 d时,芽与叶片翠绿,生长健康。
Buds and leaves were green and healthy after 30 d of growth.Table 3. Effects of basal medium on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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蒙古沙冬青子叶节外植体在含不同浓度6-BA的B5培养基中都能产生丛生芽(表 4),随着6-BA浓度的增高,丛生芽诱导率逐渐升高,当6-BA浓度为1.0、1.5、2.5 mg·L-1时,丛生芽诱导率差异不显著;但随着6-BA的浓度的增大,平均芽数增多且差异极显著,当6-BA浓度为1.5、2.5 mg·L-1时,平均芽数虽多但呈簇状,后期生长发育不佳。因此,丛生芽诱导培养基中6-BA的最适浓度为1.0 mg·L-1。
处理
Treatment6-BA浓度
Concentration /(mg·L-1)诱导率
Induction rate/%平均芽数
Average bud number/个生长状况
Growth status1 0.5 73.9±0.17B 1.45±0.19D 芽数较少,生长缓慢。Buds were fewer and slow-growing. 2 1.0 78.1±0.20A 2.26±0.38C 芽数清晰,后期有明显茎叶。
Buds can be clearly identified and there were normal stem and leaves in the latter stages.3 1.5 78.9±0.12A 3.37±0.26B 芽呈簇状,后期有茎叶,但部分为畸形叶。
Buds were clustered and both normal and malformed stem and leaves were available in the latter stages.4 2.5 79.6±0.23A 4.26±0.54A 芽多细密呈簇状,无有效芽。
Buds were densely clustered and there were no normal buds.Table 4. Effects of concentration of 6-BA in induction medium on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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赤霉素能显著促进植物茎叶的伸长与生长,但本研究(表 5)发现:添加不同浓度赤霉素的激素组合,虽然能促进茎叶的生长,但玻璃化现象严重,茎叶均呈不同程度的黄绿色透明状态,生长后期,无有效叶片,易褐化死亡;无赤霉素的激素组合,叶片呈正常绿色,当激素组合为6-BA 1.0 mg·L-1+IAA 0.3 mg·L-1时,茎叶伸长速度加快,与6-BA1.0 mg·L-1+IAA0.5 mg·L-1激素组合差异不显著。因此,选用含有6-BA 1.0 mg·L-1+IAA0.3 mg·L-1激素组合的B5培养基作为蒙古沙冬青丛生芽伸长的最适培养基。
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Treatment激素浓度
Hormone concentration/ (mg·L-1)伸长率Elongation rate /% 伸长长度Elongation length /cm 生长状况Growth status 6BA GA3 IAA 1 1.0 0.1 0.0 20.31±0.29CD 0.55±0.32CD 叶片细长,茎叶呈黄绿略透明,玻璃化。
Stem and narrow and long leaves were yellow-green with somewhat transparent and vitrified.2 1.0 0.3 0.0 16.70±0.73D 0.35±0.11D 叶片细长,茎叶呈黄绿略透明,玻璃化。
Stem and narrow and long leaves were yellow-green with somewhat transparent and vitrified.3 1.0 0.5 0.0 5.02±0.54E 0.20±0.04E 叶片增长增宽,黄绿透明,玻璃化。
Leaves became widen, were elongate, yellow-green with transparent and vitrified.4 1.0 1.0 0.0 - 叶片增长增宽,叶片黄绿透明,玻璃化,茎无伸长。
Leaves became widen, were elongate, yellow-green with transparent and vitrified. Stem had no elongation.5 1.0 1.5 0.0 - 叶片增长增宽,叶片黄绿透明,玻璃化,茎无伸长。
Leaves became widen, were elongate, yellow-green with transparent and vitrified. Stem had no elongation.6 1.0 0.0 0.1 20.72±0.42C 0.65±0.16C 叶片正常绿色,茎伸长缓慢。
Leaves were normal and green. Stem were slowly elongate.7 1.0 0.0 0.2 40.35±0.31B 0.92±0.23BC 叶片正常绿色,茎伸长。
Leaves were normal and green. Stem were elongate.8 1.0 0.0 0.3 60.55±0.20A 1.15±0.12AB 叶片正常绿色,茎伸长快速。
Leaves were normal and green. Stem elongated fast.9 1.0 0.0 0.5 61.06±0.16A 1.05±0.09AB 叶片正常绿色,茎伸长快速。
Leaves were normal and green. Stem elongated fast.Table 5. Effects of different concentration combinations of exogenous hormones on elongation of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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不同浓度单一生长素和不同浓度IAA与NAA组合可以不同程度的促进蒙古沙冬青丛生芽幼苗生根(表 6),当IBA浓度为1.0 mg·L-1时,生根率最大,平均生根数最多。因此,选用1.0 mg·L-1IBA用于诱导蒙古沙冬青幼苗生根。
处理
Treatment激素浓度
Hormone concentration /(mg·L-1)生根率
Rooting rate /%平均生根数
Average root number/条IBA IAA NAA 1 0.0 0.0 0.0 0.00 0.00 2 0.5 0.0 0.0 40.76±0.36D 1.02±0.10D 3 1.0 0.0 0.0 52.83±0.29A 2.33±0.38A 4 2.0 0.0 0.0 46.33±0.43B 2.00±0.29AB 5 0.0 0.5 0.0 20.51±0.52IJ 0.56±0.38E 6 0.0 1.0 0.0 28.90±0.31H 0.98±0.22D 7 0.0 2.0 0.0 30.33±0.19G 1.01±0.19D 8 0.0 0.0 0.5 21.27±0.28I 0.78±0.31DE 9 0.0 0.0 1.0 30.63±0.43G 1.23±0.40CD 10 0.0 0.0 2.0 36.72±0.20F 1.07±0.12D 11 0.0 1.0 0.5 20.41±0.56J 1.33±0.19CD 12 0.0 1.5 0.5 42.65±0.24C 1.87±0.07B 13 0.0 2.0 0.5 38.50±0.24E 1.56±0.17C Table 6. Effects of different auxins and concentration on rooting of Ammopiptanthus mongolicus