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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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蒙古沙冬青种子于2016年9月采自甘肃省武威市民勤县沙生植物园。
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种子萌发基本培养基为MS，黑暗、19~24℃温度下萌发；丛生芽诱导基本培养基为MS、B5，伸长、生根基本培养基为B5；诱导、伸长、生根培养温度19~24℃，光照时间14 h·d-1，光照强度1 200 lux。以上培养基均含6 g·L-1琼脂，30 g·L-1蔗糖，pH值5.8。
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挑选饱满、大小一致的蒙古沙冬青种子，用自来水冲洗干净，在无菌条件下用75%的乙醇浸泡1 min，无菌水冲洗2~3次后用0.1% HgCl2浸泡25 min，用无菌水冲洗4~5次，滤纸吸干表面水份。
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将蒙古沙冬青种子接种到含不同6-BA浓度(0.0、1.0、2.0、3.0、5.0 mg·L-1)的MS培养基中，黑暗培养7 d，取萌发的幼苗，保留子叶近胚轴的分生组织部分约2 mm和上胚轴约3 mm，切去其余部分，然后在2片子叶的胚芽处将胚轴纵向切成均等的二半，剔除腋芽后在子叶与胚轴交接处划开2~3个伤口，得到2个子叶节外植体。将外植体的近轴面朝上，倾斜45℃插入添加1.0 mg·L-1 6-BA的MS培养基中诱导丛生芽，每处理各接种10瓶，每瓶接种8个，培养30 d后统计丛生芽诱导率及出芽数，以确定萌发培养基中6-BA的适宜浓度。
丛生芽诱导率=出芽外植体数/接种外植体数×100%
平均芽数=总芽数/出芽外植体数
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取在萌发培养基中黑暗培养1、3、5、7、9、11、13、15 d的蒙古沙冬青幼苗，切取子叶节外植体接种于含1.0 mg·L-1 6-BA的MS培养基中诱导丛生芽，每处理各接种10瓶，每瓶接种8个，培养30 d后统计丛生芽诱导率及出芽数，以确定最适宜的萌发天数。
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将黑暗培养7 d的蒙古沙冬青幼苗的子叶节外植体分别接种于含1.0 mg·L-16-BA的MS培养基和B5培养基中诱导丛生芽，每处理各接种10瓶，每瓶接种8个，培养30 d后统计丛生芽诱导率及出芽数，确定诱导丛生芽的最适基本培养基。
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将黑暗培养7 d的蒙古沙冬青幼苗的子叶节外植体分别接种于含不同浓度6-BA(0.5、1.0、1.5、2.5 mg·L-1)的B5培养基中诱导丛生芽，每处理各接种10瓶，每瓶接种8个，培养30 d后统计丛生芽诱导率及出芽数，确定丛生芽诱导培养基中最适6-BA浓度。
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子叶节处的丛生芽长约0.3~0.5 cm后，剔除剩余子叶，分别转入含1.0 mg·L-16-BA并附加不同的浓度配比GA3和IAA的B5培养基中进行伸长培养，每处理各接种10瓶，每瓶接种4个，30 d后统计丛生芽伸长率及伸长长度，确定最佳伸长培养基。
丛生芽伸长率=抽茎外植体数/接种外植体数×100%
丛生芽伸长长度(cm)=伸长后的长度-伸长前的长度
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当丛生芽伸长至2~3 cm时，单株切下转接到添加不同浓度生长素和生长素组合的1/2B5培养基中诱导生根，每处理各接种10瓶，每瓶接种4个，30 d时统计生根率和根数，确定适宜生根的生长素及最适浓度。以根长达到0.5 cm作为生根的标准。
生根率=生根的外植体总数/接种外植体总数×100%
平均生根数=生根条数/生根的外植体总数
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利用SPSS17.0软件对数据进行统计分析，确定不同阶段各处理平均值之间在0.05和0.01概率水平上的差异显著性。
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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。
表 1 萌发培养基中6-BA浓度对蒙古沙冬青子叶节生长和子叶节丛生芽诱导的影响
Table 1. Effects of concentration of 6-BA in germination medium on cotyledonary nodes growth and induction of cluster bud from of Ammopiptanthus mongolicus
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 0.0 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. -
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。
表 2 萌发天数对蒙古沙冬青子叶节丛生芽诱导的影响
Table 2. Effects of seed germination days on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
处理
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 -
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。
表 3 基本培养基对蒙古沙冬青子叶节丛生芽诱导的影响
Table 3. Effects of basal medium on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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. -
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。
表 4 丛生芽诱导培养基中6-BA浓度对子叶节丛生芽诱导的影响
Table 4. Effects of concentration of 6-BA in induction medium on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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. -
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。
表 5 不同激素组合对蒙古沙冬青子叶节丛生芽伸长培养的影响
Table 5. Effects of different concentration combinations of exogenous hormones on elongation of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
处理
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. -
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。
表 6 不同生长素及浓度对蒙古沙冬青生根的影响
Table 6. Effects of different auxins and concentration on rooting of Ammopiptanthus mongolicus
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 -
以子叶节为外植体进行丛生芽诱导建立组培再生体系是不发生愈伤组织的器官型的再生方式，此再生方式具有诱导率高、再生周期短、变异率低、再生植株易成活等优点，目前已广泛应用于大豆植株再生和遗传转化技术体系的研究中[14]。蒙古沙冬青属第三代古地中海沿岸的植物，是豆科中比较原始的种[2]。本研究采用蒙古沙冬青无菌苗的子叶节为外植体，诱导丛生芽获得高频率的再生植株在蒙古沙冬青组织培养研究中尚属首次。
本研究结果表明，在蒙古沙冬青种子萌发初期，培养基中6-BA的添加与否对获得的子叶节诱导丛生芽有显著影响。外源细胞分裂素能使子叶节膨大增粗，说明子叶节处的分生细胞生长活跃，从而有利于诱导丛生芽，这与王伟[15]在研究大豆子叶节丛生芽诱导时的结论一致。6-BA浓度并非越高越有利于丛生芽后期的生长，在添加高浓度的6-BA(3、5 mg·L-1)萌发培养基中，丛生芽生长后期会出现畸形芽或因为数量过多，芽与芽之间产生抑制而无有效芽，从而降低再生频率。
种子萌发天数即种子萌发时的生理状态与丛生芽的诱导率密切相关，种子萌发1 d时，由于萌发时间短，种皮未自然脱落，胚轴未伸出，2片子叶较难分离。种子萌发3~5 d时，子叶黄色，胚轴伸长，但较短且细弱，获取子叶节外植体时容易断裂。种子萌发7~11 d时，子叶变成浅黄绿色，胚轴伸出且增粗(图 1A)，2片子叶较易从中轴线分离；而种子萌发天数超过11 d，丛生芽诱导率和出芽数下降，可能是随着萌发天数的增加，胚芽逐渐伸长，顶端生长逐步占据优势，子叶节处分生组织的分生能力逐渐减弱所致。故种子萌发7~11 d时子叶节组织分裂能力最强，是最适宜的子叶节外植体材料。
图 1 蒙古沙冬青子叶节诱导丛生芽再生体系
Figure 1. Cotyledonary nodes regeneration system of Ammopiptanthus mongolicus
丛生芽诱导阶段，本研究选用B5作为诱导的基本培养基，说明在NH4+/NO3-比值较低，VB1和VB6的倍数增大，无甘氨酸的环境中更有利于蒙古沙冬青子叶节丛生芽的再生，这与安利佳等[16]、邵玲玲[17]、李海峰等[18]对豆科植物组织培养研究中培养基的选择一致。细胞分裂素与生长素的组合能促进芽的分化与增殖[19]，在本研究中，选用不同浓度的6-BA与NAA组合使子叶节处除诱导出丛生芽外还会诱导出根，从而影响丛生芽的分化，因此，选用单一细胞分裂素6-BA进行丛生芽的诱导。
蒙古沙冬青组培苗炼苗移植研究结果有待进一步发表。
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通过筛选种子萌发培养基中6-BA浓度、确定适宜的子叶节萌发天数、确定丛生芽诱导的基本培养基和6-BA浓度及研究不同激素对丛生芽伸长和生根的影响，建立的蒙古沙冬青子叶节丛生芽再生体系为：种子在萌发培养基MS+2.0 mg·L-16-BA中黑暗培养7~11 d后，取子叶节，在诱导培养基B5+1.0 mg·L-16-BA中诱导丛生芽，待子叶节处的丛生芽长约0.3~0.5 cm后，剔除剩余子叶，转入丛生芽伸长培养基B5+ 1.0 mg·L-1 6-BA+0.3 mg·L-1IAA中进行伸长培养，当丛生芽伸长至2~3 cm时，单株切下转入生根培养基1/2B5+1.0 mg·L-1 IBA生根。该体系与愈伤组织培养相比，能够极大地改善褐化严重、玻璃化等问题，对蒙古沙冬青的扩繁及进一步的保护利用具有重要意义和理论价值。
蒙古沙冬青子叶节诱导培养再生植株的研究
Research on Cotyledonary Nodes Regeneration System of Ammopiptanthus mongolicus
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摘要:
目的 建立蒙古沙冬青子叶节丛生芽再生体系。 方法 以蒙古沙冬青子叶节为外植体，研究种子萌发培养基中6-BA浓度、子叶节萌发天数、丛生芽诱导的基本培养基及6-BA浓度对丛生芽诱导的影响和外源激素对丛生芽伸长及生根的影响。 结果 表明：（1）添加6-BA的萌发培养基与不添加的相比能够显著促进子叶节的生长及子叶节丛生芽的诱导，且6-BA浓度在2.0 mg·L-1时，诱导率最高可达73.3%，平均芽数2.26个；（2）种子萌发天数对子叶节丛生芽的诱导有显著影响，萌发7 d的子叶节丛生芽诱导率最高，诱导率为74.7%，但与萌发9、11 d的子叶节丛生芽诱导率差异不显著；（3）MS和B5培养基对丛生芽诱导的影响差异不显著，但B5抑制褐化的效果显著好于MS；（4）采用1.0 mg·L-16-BA+0.3 mg·L-1IAA激素组合有利于丛生芽伸长，伸长率为60.5%；（5）不同浓度生长素组合均能促进丛生芽幼苗生根，1.0 mg·L-1IBA生根率最大，生根数最多。 结论 最佳蒙古沙冬青子叶节丛生芽再生体系为：以在MS+2.0 mg·L-16-BA培养基中黑暗培养7 11 d的幼苗子叶节为外植体，在B5+1.0 mg·L-16-BA培养基中诱导丛生芽，待丛生芽长约0.3 0.5 cm后，转入B5+ 1.0 mg·L-1 6-BA+0.3 mg·L-1IAA培养基中进行伸长培养，当丛生芽伸长至2 3 cm时，单株切下转入1/2 B5+1.0 mg·L-1IBA培养基中生根，该体系与愈伤组织培养相比能缩短培养时间，改善褐化严重、玻璃化等问题，可为蒙古沙冬青的扩繁及进一步开展遗传转化奠定基础。 Abstract:Objective To establish cotyledonary nodes regeneration system of Ammopiptanthus mongolicus. Method The effects of concentrations of 6-BA in seed germination medium, seed germination days, type of basal induction medium and 6-BA concentrations on the induction of cluster bud were studied using cotyledonary nodes of A. mongolicus as explants. The effects of exogenous hormones on elongation and rooting of explants were discussed as well. Result The results showed that:(1) The medium containing 6-BA can significantly promote the cotyledonary node growth and the induction of cluster bud compared to the medium without 6-BA. And when the 6-BA concentration was at 2.0 mg·L-1, the induction rate was up to 73.3% with 2.26 buds in average. (2) The days of seed germination also had a significant effect on the induction rate of cluster bud. The induction rate of the 7-day-old cotyledonary node reached the maximum of 74.7%, but had no statistically significant difference with the induction rate of the 9-, and 11-day-old cotyledonary nodes. (3) B5 or MS as the basal medium for induction of cluster bud had no difference, but B5 worked better in inhibiting browning. (4) For elongation culture, the optimal condition was 1.0 mg·L-1 6-BA+0.3 mg·L-1IAA with an elongation of 60.5%. (5) Although the root can be induced from the combination of different concentrations of auxins, the best rooting rate and the maximum number of root occurred when IBA was 1.0 mg·L-1. Conclusion The optimal cotyledonary nodes regeneration system of A. mongolicus includes the following steps:the cotyledonary nodes, cultivated on the MS medium containing 2.0 mg·L-16-BA in the dark after 7 to 11 days, are used in B5 medium containing 1.0 mg·L-16-BA to regenerate cluster bud. When the length of buds is about 0.3-0.5 cm, they are transferred to B5 medium with 1.0 mg·L-16-BA and 0.3 mg·L-1IAA for elongation culture. When the clumps stretch to 2-3 cm, the single plants are cut into 1/2B5 +1.0 mg·L-1IBA medium to take root. Compared with the callus tissue culture, this process can shorten the culture time, improve the serious brown and vitrification, and will lay the foundation of propagation and further genetic transformation for A. mongolicus. -
Key words:
- Ammopiptanthus mongolicus
- / cotyledonary nodes
- / cluster bud
- / regeneration plant
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图 1 蒙古沙冬青子叶节诱导丛生芽再生体系
Figure 1. Cotyledonary nodes regeneration system of Ammopiptanthus mongolicus
表 1 萌发培养基中6-BA浓度对蒙古沙冬青子叶节生长和子叶节丛生芽诱导的影响
Table 1. Effects of concentration of 6-BA in germination medium on cotyledonary nodes growth and induction of cluster bud from of Ammopiptanthus mongolicus
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 0.0 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.
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表 2 萌发天数对蒙古沙冬青子叶节丛生芽诱导的影响
Table 2. Effects of seed germination days on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
处理
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
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表 3 基本培养基对蒙古沙冬青子叶节丛生芽诱导的影响
Table 3. Effects of basal medium on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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.
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表 4 丛生芽诱导培养基中6-BA浓度对子叶节丛生芽诱导的影响
Table 4. Effects of concentration of 6-BA in induction medium on induction of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
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.
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表 5 不同激素组合对蒙古沙冬青子叶节丛生芽伸长培养的影响
Table 5. Effects of different concentration combinations of exogenous hormones on elongation of cluster bud from cotyledonary nodes of Ammopiptanthus mongolicus
处理
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.
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表 6 不同生长素及浓度对蒙古沙冬青生根的影响
Table 6. Effects of different auxins and concentration on rooting of Ammopiptanthus mongolicus
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
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