为优化软枣猕猴桃种苗快繁技术体系,以其茎段、芽作为外植体,开展不同激素配比组合的MS培养基对愈伤组织诱导及增殖、不定芽分化增殖、无菌苗生根和驯化移栽等实验研究。结果表明,茎段外植体表面消毒时长6~7 min,在MS + 0.2 mg/L 2,4-D + 0.4 mg/L 6-BA + 蔗糖30 g/L + 琼脂粉7.8 g/L培养基上愈伤组织诱导率最高,达93.75%,且愈伤组织生长旺盛,呈白绿色湿润疏松状;在MS + 1.3 mg/L 6-BA + 0.25 mg/L NAA软枣猕猴桃初代培养芽苗诱导率、增殖率最高,达到93.75%,在MS + 2.0 mg/L 6-BA + 1.2 mg/L NAA培养基上增殖效率最高,达到86.6%,增殖系数达到5左右;最佳生根培养基为1/2MS + 1.5 mg/L NAA,生根率93.75%,并且每株有4~10条根,根粗苗壮;根长3.0~5.0 cm的再生植株移栽到草炭: 园土: 珍珠岩 = 2:2:1的混合基质中,成活率达86%以上。 In order to optimize the rapid propagation technology system of soft jujube kiwi fruit seedlings, the stem segment and bud were used as explants to carry out experimental studies on callus induction and proliferation, adventitious bud differentiation and proliferation, roots and acclimation and transplanting of sterile seedlings on MS medium with different hormone ratios. The results showed that the callus induction rate reached 93.75% on MS medium with 0.2 mg/L 2,4-D + 0.4 mg/L 6-BA + sucrose 30 g/L + AGAR powder 7.8 g/L after surface disinfection for 6~7 min, and the callus growth was very strong and showed white green moist loose shape; The induction rate and proliferation rate of kiwi soft jujube in MS + 1.3 mg /L 6-BA + 0.25 mg/L NAA reached 93.75%, and the proliferation rate of kiwi soft jujube in MS + 2.0 mg/L 6-BA + 1.2mg /L NAA reached 86.6%. The multiplication coefficient reached about 5. The optimal rooting medium was 1/2MS + 1.5 mg/L NAA, the rooting rate was 93.75%, and each plant had 4~10 roots, thick and strong roots. The regenerated plants with root length of 3.0~5.0 cm were transplanted into the mixture of peat: garden soil: perlite = 2:2:1, and the survival rate was over 86%.
为优化软枣猕猴桃种苗快繁技术体系,以其茎段、芽作为外植体,开展不同激素配比组合的MS培养基对愈伤组织诱导及增殖、不定芽分化增殖、无菌苗生根和驯化移栽等实验研究。结果表明,茎段外植体表面消毒时长6~7 min,在MS + 0.2 mg/L 2,4-D + 0.4 mg/L 6-BA + 蔗糖30 g/L + 琼脂粉7.8 g/L培养基上愈伤组织诱导率最高,达93.75%,且愈伤组织生长旺盛,呈白绿色湿润疏松状;在MS + 1.3 mg/L 6-BA + 0.25 mg/L NAA软枣猕猴桃初代培养芽苗诱导率、增殖率最高,达到93.75%,在MS + 2.0 mg/L 6-BA + 1.2 mg/L NAA培养基上增殖效率最高,达到86.6%,增殖系数达到5左右;最佳生根培养基为1/2MS + 1.5 mg/L NAA,生根率93.75%,并且每株有4~10条根,根粗苗壮;根长3.0~5.0 cm的再生植株移栽到草炭: 园土: 珍珠岩 = 2:2:1的混合基质中,成活率达86%以上。
软枣猕猴桃,诱导分化增殖,植株再生,移栽
Kangju Zhao, Linbin Shen, Qinhong Yu, Lingmei Yin, Tao Ye, Yanfen Huang*
College of Biological Sciences, Guizhou Normal University, Guiyang Guizhou
Received: Mar. 18th, 2022; accepted: Apr. 19th, 2022; published: Apr. 27th, 2022
In order to optimize the rapid propagation technology system of soft jujube kiwi fruit seedlings, the stem segment and bud were used as explants to carry out experimental studies on callus induction and proliferation, adventitious bud differentiation and proliferation, roots and acclimation and transplanting of sterile seedlings on MS medium with different hormone ratios. The results showed that the callus induction rate reached 93.75% on MS medium with 0.2 mg/L 2,4-D + 0.4 mg/L 6-BA + sucrose 30 g/L + AGAR powder 7.8 g/L after surface disinfection for 6~7 min, and the callus growth was very strong and showed white green moist loose shape; The induction rate and proliferation rate of kiwi soft jujube in MS + 1.3 mg /L 6-BA + 0.25 mg/L NAA reached 93.75%, and the proliferation rate of kiwi soft jujube in MS + 2.0 mg/L 6-BA + 1.2mg /L NAA reached 86.6%. The multiplication coefficient reached about 5. The optimal rooting medium was 1/2MS + 1.5 mg/L NAA, the rooting rate was 93.75%, and each plant had 4~10 roots, thick and strong roots. The regenerated plants with root length of 3.0~5.0 cm were transplanted into the mixture of peat: garden soil: perlite = 2:2:1, and the survival rate was over 86%.
Keywords:Soft Jujube Kiwi Fruit, Induced Differentiation and Proliferation, Plant Regeneration, Transplanting
Copyright © 2022 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
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软枣猕猴桃(Actinidia arguta Planch.)属猕猴桃科多年生落叶藤本植物,是我国珍贵的抗寒果树资源,具有较高的营养保健及经济价值 [
带芽的软枣猕猴桃健壮枝条。材料取自贵阳白云黔花香种植养殖专业合作社。
LDZX-50KBS立式压力蒸汽灭菌器,上海向帆仪器有限公司;SW-CJ-2FD型双人单面净化工作台,苏州净化设备有限公司;LT502E电子天平,常熟市天量仪器有限责任公司;PHSJ-3F实验室PH计,上海仪电科学仪器股份有限公司。
6-BA,NAA、IBA、2,4-D、琼脂粉、蔗糖以、MS固体培养基,上海博微生物科技有限公司产品。
取软枣猕猴桃健壮无病虫害的带芽枝条,流水冲洗1 d后用75%的酒精涮洗30 s,再用1%的升汞表面消毒,设置5种表面消毒时间:4 min、5 min、6 min、7 min、8 min;无菌水清洗后沥干水分,接种于MS + 2,4-D 0.2 mg/L + 6-BA 0.4 mg/L蔗糖30 g/L + 琼脂粉7.8 g/L培养基中培养,探寻材料表面消毒时间对初代培养的影响。
将软枣猕猴桃枝条腋芽(侧芽)和顶芽直接切下,茎段切割厚度约为0.2-0.5 cm,芽和茎段接种至(1)-(6)号初代培养基中置于培养室进行培养,筛选最佳初代培养基配方。
1) MS + 1.0 mg/L6-BA + 0.5 mg/L IAA;2) MS + 1.5 mg/L6-BA + 0.5 mg/L IAA;3) MS + 2.0 mg/L6-BA + 0.5 mg/L IAA;4) MS + 0.5 mg/L6-BA + 0.5 mg/L NAA;5) MS + 1.0 mg/L 6-BA + 0.5 mg/L NAA;6) MS + 1.5 mg/L6-BA + 0.5 mg/L NAA。
培养基pH为5.8~6.0,每种培养基接种15瓶,每瓶接种1个外植体。在培养室温度为24℃~26℃;光周期为10 h光照/14h黑暗;相对湿度为45%~60%;光照度为1500~2000 LX培养一周,每2 d/次观察统计褐化率、成活率和污染率、愈伤组织诱导率、芽分化率等。
从初代培养建立的无性系中选出生长势好的壮苗和愈伤组织进行继代增殖培养,培养条件同初代培养,每处理接种15瓶,每2 d/次观察计算增殖周期、增殖倍数等。
筛选质地疏松,颜色鲜绿的愈伤组织进行切割转接,诱导分化培养,经再分化得到再生芽苗。分化培养基参照继代增殖培养基。pH 5.8~6.0,每处理接种15瓶,培养条件同初代培养,每1~2 d观察一次。
挑选继代增殖培养后长势好的再生壮苗作为生根培养的材料,生根培养基采用:1) 1/2 MS + 0.1 mg/L IBA + 2.0 g/L活性炭;2) 1/2 MS + 0.3 mg/L IBA + 2.0 g/L活性炭;3) 1/2 MS + 0.6 mg/L IBA + 2.0 g/L活性炭;4) 1/2 MS + 0.9 mg/L IBA + 2.0 g/L活性炭,培养条件同初代培养。培养3周,观察生长情况,统计生根数,生根率,污染率,成活率等,筛选优化生根培养基配方。
挑选发根多,生长旺盛,苗高度不超过1.5 cm、根长小于0.5 cm的组培苗进行驯化移栽。移栽基质选用体积比为草炭: 园土: 珍珠岩 = 2:2:1的混合基质。移栽后用透明膜覆盖,遮光率不高于60%,空气湿度不低于40%,环境温度保持在15℃~28℃,以达到光照合适、保温、保湿效果。生长几周后,统计成活率、生根率。
由表1可以看出,腋芽(侧芽)、顶芽、茎段外植体随灭菌时间的增加,存活率、芽分化率、愈伤组织诱导率也都随着增加,但达到一定时间后便随着灭菌时间的增加而降低。当灭菌时间为6~7 min时,存活率、芽分化率、愈伤组织诱导率均达到最大值。
灭菌时长(min) | 接种外植体数(个) | 外植体存活率(%) | 芽分化率(%) | 愈伤组织诱导率(%) |
---|---|---|---|---|
4 | 44 | 34.09 | 27.28 | 79.55 |
5 | 32 | 37.50 | 31.25 | 81.25 |
6 | 39 | 74.36 | 64.10 | 89.74 |
7 | 32 | 71.19 | 62.50 | 87.50 |
8 | 32 | 21.88 | 15.63 | 78.13 |
表1. 最适外植体表面消毒时间的筛选
由表2可以看出,软枣猕猴桃初代培养与6-BA和NAA都有关系。当6-BA浓度一致时,随着NAA浓度增加软枣猕猴桃初代培养芽苗诱导率和愈伤组织诱导率也跟着增加;当NAA浓度一致时,随着6-BA浓度增加软枣猕猴桃初代培养芽苗诱导率和愈伤组织诱导率反而降低;当6-BA/NAA约为1:2时,软枣猕猴桃初代培养芽苗诱导率最高;表2中最适培养基为A2:MS + 1.3 mg/L 6-BA + 0.25 mg/L NAA和A3:MS + 0.8 mg/L 6-BA + 0.25 mg/L NAA。
培养基编号 | 培养基配方(mg/L) | 接种外植体数(个) | 芽苗诱导率(%) | 愈伤组织诱导率(%) |
---|---|---|---|---|
A1 | MS + 1.36-BA + 0.15 NAA | 32 | 31.25 | 75.00 |
A2 | MS + 1.36-BA + 0.25 NAA | 32 | 62.50 | 93.75 |
A3 | MS + 0.86-BA + 0.15 NAA | 32 | 71.88 | 90.63 |
A4 | MS + 0.86-BA + 0.25 NAA | 32 | 28.13 | 65.63 |
表2. 培养基配方对软枣猕猴桃初代培养的影响
表3显示,芽苗增殖率和愈伤组织增殖率与6-BA、NAA和IBA都有关系。当6-BA浓度一致时,随着NAA浓度升高芽苗增殖系数、愈伤组织增殖率也跟着增加;6-BA和NAA浓度都增加时,芽苗增殖系数、愈伤组织增殖率也跟着增加;当6-BA/NAA和6-BA/IBA比约为2:1时,愈伤组织增殖率和芽苗增殖系数最高;最佳增殖培养基为3:MS + 2.0 mg/L 6-BA + 1.2 mg/L NAA和4:MS + 3.0 mg/L 6-BA + 1.5 mg/L IBA。
培养基编号 | 激素水平 (mg/L) | 接种芽苗数(芽) | 增殖芽苗数(芽) | 接种愈伤组织数(块) | 增殖愈伤组织数(块) | 芽苗增殖系数 | 愈伤组织增殖率(%) |
---|---|---|---|---|---|---|---|
1 | MS + 1.0 6-BA + 0.4 NAA | 15 | 45 | 32 | 18 | 2.97 | 56.25 |
2 | MS + 1.0 6-BA + 0.8 NAA | 15 | 46 | 30 | 22 | 3.05 | 73.33 |
3 | MS + 2.0 6-BA + 1.2 NAA | 15 | 70 | 30 | 26 | 4.65 | 86.67 |
4 | MS + 3.0 6-BA + 1.5 IBA | 15 | 73 | 26 | 21 | 4.85 | 80.77 |
5 | MS + 2.0 6-BA + 0.8 IBA | 15 | 49 | 28 | 19 | 3.25 | 67.86 |
表3. 激素水平对软枣猕猴桃增殖效果的影响
从表4可以看出,生根率和NAA的浓度有关。当NAA浓度增加时生根率也随着增加,但是达到一定浓度后生根率就会降低,可见NAA浓度对软枣猕猴桃根的诱导具有一定的抑制作用,最适生根培养基为1/2MS + 1.5 mg/L NAA。
培养基编号 | 培养基配方(mg/L) | 接种不定芽数(芽) | 生根不定芽数(个) | 生根率(%) |
---|---|---|---|---|
I | 1/2MS + 1.0 NAA | 16 | 14 | 87.50 |
II | 1/2MS + 2.0 NAA | 16 | 12 | 75.00 |
II | 1/2MS + 1.5 NAA | 16 | 15 | 93.75 |
表4. 生根培养基筛选
由表5看出,软枣猕猴桃驯化移栽成活率与移栽基质配比有关。当草炭:珍珠岩 = 1:1时,随着园土基质增加时,移栽称呼哦了反而降低;当园土: 珍珠岩 = 1:1时,随着草炭基质增加时,移栽成活率跟着增加;当草炭: 园土 = 2:2时,随着珍珠岩基质增加,移栽成活率反而降低。得出最佳驯化移栽基质配比为草炭: 园土: 珍珠岩 = 2:2:1,移栽成活率高达86.6%。
编号 | 基质配比 | 移栽盆数(盆) | 成活盆数(盆) | 成活率(%) |
---|---|---|---|---|
1 | 草炭: 园土: 珍珠岩 = 1:1:1 | 15 | 6 | 40.0 |
2 | 草炭: 园土: 珍珠岩 = 1:2:1 | 15 | 4 | 26.7 |
3 | 草炭: 园土: 珍珠岩 = 2:2:1 | 15 | 13 | 86.6 |
4 | 草炭: 园土: 珍珠岩 = 2:1:1 | 15 | 5 | 33.3 |
5 | 草炭: 园土: 珍珠岩 = 2:2:2 | 15 | 8 | 53.3 |
表5. 驯化移栽基质筛选
实验结果表明,灭菌时长为6~7 min时软枣猕猴桃茎段在存活率、芽苗诱导率、愈伤组织诱导率均高于比其他灭菌时间,为最适灭菌时长;初代培养基MS + 1.3 mg/L 6-BA + 0.25 mg/L NAA和MS + 0.8 mg/L 6-BA + 0.15 mg/L NAA对软枣猕猴桃芽苗和愈伤组织诱导率最高,分别达到93.75%和90.63%;在继代增殖培养基MS + 2.0 mg/L 6-BA + 1.2 mg/L NAA和MS + 3.0 mg/L 6-BA + 1.5 mg/L IBA上增殖效率最高,达到86.67%和80.77%,增殖系数达到5左右,增殖周期为15~16 d;最佳生根培养基为1/2MS + 1.5 mg/L NAA,生根率达93.75%,并且每株有根数可达4~10条,根粗苗壮;根长3.0~5.0 cm的再生苗,移栽到草炭: 园土: 珍珠岩 = 2:2:1的混合基质中,成活率达86%以上。实验中还发现,在切割转接愈伤组织时,如果愈伤组织块底部出现褐变,可在底部留一层薄薄的褐变层,这样更有利于愈伤组织块在新鲜培养基上的扩繁,这一现象的发生机制有待进一步的研究探明。
植物组织培养技术具有生育周期短、不会受到季节限制的特点,利用组织培养技术繁育软枣猕猴桃种苗,既可以提高繁殖速度,又可以增加种苗数量,并且有利于对野生种质资源的保存、驯化及利用,但是愈伤组织和不定芽的诱导是组织培养再生体系建立中最关键的一步,如何在最短的时间、利用最低的成本获得最高的再生效率是组培育苗的重要研究内容 [
教育部大学生创新创业计划项目“软枣猕猴桃产业发展——种苗快速繁殖技术研究”[
赵康举,申林彬,郁琴红,殷领梅,叶 桃,黄燕芬. 软枣猕猴桃种苗离体快繁Soft Jujube Kiwi Fruit Seedlings in Vitro and Rapid Propagation[J]. 农业科学, 2022, 12(04): 294-300. https://doi.org/10.12677/HJAS.2022.124044