Pollen Cytology, Stamen Callus Induction, and Seedling Propagation of Paeonia delavayi
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摘要:
目的 研究滇牡丹花蕾与花粉粒发育间的相关性，建立外观形态与花粉粒主导发育时期间的关系，为后期取材提供参考；筛选出滇牡丹雄蕊愈伤组织、增殖和分化诱导的最适培养基，以期建立滇牡丹雄蕊的再生体系。 方法 以红色滇牡丹硬蕾期和透色期的花蕾为研究材料，通过石蜡切片加光学显微观察的途径确定各阶段花粉粒的主导发育时期；结合MS固体培养基，研究不同植物生长调节剂及其质量浓度对滇牡丹雄蕊愈伤组织诱导、增殖和分化的影响。 结果 （1）红色滇牡丹花蕾直径10～17 mm且尚未透色的Y2−Y4阶段，其花粉粒主导发育时期是单核中期；花蕾直径12～15 mm且透色面积小于30%的T1−T2阶段为单核靠边期，花蕊直径15～19 mm且透色面积大于30%的T3−T4阶段为双核期。（2）滇牡丹雄蕊的最佳消毒方式为花蕾经流水冲洗30 min后，使用1% 次氯酸钠消毒8 min。（3）滇牡丹雄蕊的最佳诱导时期为单核靠边期。（4）雄蕊愈伤组织诱导适宜的培养基为MS+0.2 mg·L−1 NAA+0.5 g·L−1PVP；最适增殖培养基为MS+0.5 mg·L−1NAA+0.25 mg·L−16-BA+0.5 mg·L−1GA3+1 g·L−1PVP。 结论 明确了花蕾大小和外观透色形态与各阶段花粉粒的主导发育时期的关系，筛选出红花滇牡丹雄蕊愈伤组织诱导与增殖适宜的培养基和培养条件，为滇牡丹组织培养技术的深入研究提供基础。 Abstract:Objective Developments of flower buds and pollen grains, optimum medium for stamen callus induction, and proper conditions for seedling propagation and differentiation of Paeonia delavayi were investigated. Method Dominant pollen grain development stages were examined under an optical microscope on the paraffin sections of red P. delavayi hard buds with visible color. On an MS solid medium, various plant growth regulators in different concentrations were added to determine the optimal formulation for the induction, proliferation, and differentiation of stamen callus. Result (1) The prevailing stage of pollen grain development for the redP. delavayi, Y2-Y4, occurred in the mid-mononuclear period when the bud diameter was 10-17 mm but the color had yet to show. At T1-T2, the bud grew to a diameter ranging from 12 mm to 15 mm with less than 30% of the bud showing its color. That was in the single-core sideline period. When the diameter of a stamen reached 15-19 mm with a colored area greater than 30%, the T3-T4 stage was in the binuclear period. (2) The stamens were most effectively disinfected with running water for 30min followed by a rinse with 1% sodium hypochlorite for 8min. (3) The stamen could be optimally induced during the single-core sideline period. (4) The medium for optimal stamen callus induction was formulated with MS + 0.2 mg NAA·L−1 + 0.5 g PVP·L−1, and that for the proliferation MS + 0.5 mg NAA·L−1 + 0.25 mg 6-BA·L−1 + 0.5 mg GA3·L−1 + 1 g PVP·L−1. Conclusion This study clearly revealed the floral bud size, appearance, and dominant pollen grain development stages of the red P. delavayi and determined the optimal medium formulations and culture conditions for inducing, proliferating, and differentiating the stamen callus. -
Key words:
- Paeonia delavayi /
- stamens /
- callus /
- plant propagation
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图 1 滇牡丹花粉粒发育时期的细胞学观察（10×100）
A：单核中期； B：单核靠边期；C：双核期；红色箭头为细胞核位置。
Figure 1. Cytological observations on pollen grains of P. delavayi at development stages (10×100)
A: Mid-mononuclear period; B: single-core sideline period; C: binuclear period; red arrow indicates location of nucleus.
图 2 不同生长阶段滇牡丹花蕾
A：不同生长阶段雄蕊； B：不同生长阶段花蕾； Y1：硬蕾期1； Y2：硬蕾期2； Y3：硬蕾期3； Y4：硬蕾期4；T1：透色期1； T2：透色期2；T3：透色期3；T4：透色期4。
Figure 2. P. delavayi buds at different growth stages
A: Stamens at different growth stages; B: buds at different growth stages; Y1: hard bud stage 1; Y2: hard bud stage 2; Y3: hard bud stage 3; Y4: hard bud stage 4; T1: bud with visible color period 1; T2: bud with visible color period 2; T3: bud with visible color period 3; T4: bud with visible color period 4.
图 3 滇牡丹雄蕊不同消毒时间的消毒效果
同组柱状图上的小写字母表示在 P＜0.05 水平差异显著。
Figure 3. Effect of treatment time on disinfection of P. delavayi stamens
Data with lowercase letters in same group indicate significant differences at P＜0.05.
图 4 5种不同状态的愈伤组织
A：带有点状突起的愈伤组织；B：绿色愈伤；C：黄绿色愈伤；D：黄褐色愈伤；E：黑褐色愈伤。
Figure 4. Calluses of 5 different types
A: Callus with punctate process; B: green callus; C: yellow-green callus; D: yellow-brown callus; E: dark-brown callus.
表 1 滇牡丹花蕾外观形态与花粉粒发育时期的相关性
Table 1. Correlation between floral bud appearance and pollen grain development of P. delavayi
生长阶段
Growth
stage对应时期
Corresponding
period花蕾形态（直径大小）
Bud shape
（Diameter size，Φ）/mm单核中期花粉粒
Mid-mononuclear period pollen grains单核靠边期花粉粒
Single-core sideline period pollen grains双核期花粉粒
Binuclear period pollen grains数量
Amount/个占比
Proportion/%数量
Amount/个占比
Proportion/%数量
Amount/个占比
Proportion/%Y1 硬蕾期 萼片包裹紧实（Φ6～8） — — — — — — Y2 硬蕾期 萼片包裹紧实（Φ10～12） 96 97.96 2 2.04 0 0.00 Y3 硬蕾期 萼片包裹紧实（Φ13～14） 50 54.35 27 29.35 15 16.30 Y4 硬蕾期 萼片包裹紧实（Φ15～17） 39 41.49 29 30.85 26 27.66 T1 透色期 花瓣透色面积＜10%（Φ12～13） 18 19.78 46 50.55 27 29.67 T2 透色期 花瓣透色面积10%～30%（Φ14～15） 11 11.34 52 53.61 34 35.05 T3 透色期 花瓣透色面积30%～60%（Φ15～16.5） 5 5.49 32 35.16 54 59.34 T4 透色期 花瓣开始松散，透色面积＞60%（Φ17～19） 4 4.71 23 27.06 58 68.24 
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表 2 不同生长阶段雄蕊的诱导结果
Table 2. Stamen induction at growth stages
生长阶段
Growth stage花粉粒主导发育时期
Period of dominant pollen grain development外植体褐化率
Browning rate of explants/%诱导率
Induction rate/%愈伤组织生长状态
Callus growth state褐化程度
Browning degreeY3 单核中期 97.80±1.91a 67.78±1.73c 少量白色致密的愈伤 ＋＋ Y4 单核中期 98.90±1.91a 78.89±1.73b 中等白色致密的愈伤 ＋＋ T1 单核靠边期 100.00±0.00a 91.11±7.21a 大量灰白色愈伤，轻微玻璃化 ＋＋＋ T2 单核靠边期 100.00±0.00a 82.22±5.03ab 中等白色致密的团块状愈伤 ＋＋＋ 表中数据为均值±标准差。+表示较轻; ++表示一般; +++表示较重。同列数据后不同小写字母表示在0.05水平下差异显著，表3～5同。
Data are mean±standard error; +means lighter; ++means general; +++means heavier. Data with different lowercase letters on same column indicate significant differences at P＜0.05. Same for Tables 3-5.
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表 3 滇牡丹雄蕊诱导培养基的筛选
Table 3. Medium selection for P. delavayi stamen induction
处理
Treatment植物生长调剂
Plant growth regulator/
（mg·L−1）诱导率
Induction rate/%生长状态
Growth stateNAA 6-BA 1（CK） — — 4.44±1.93d 白色少量米点状愈伤 2 0.05 — 36.67±3.33b 黄白色少量点状愈伤 3 0.2 — 80.00±5.77a 黄白色中等团块状致密愈伤 4 — 0.05 27.78±5.09c 白色少量点状愈伤 5 — 0.2 41.11±1.93b 白色中等团块状愈伤
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表 4 滇牡丹雄蕊愈伤组织增殖培养基的筛选
Table 4. Medium selection for P. delavayi stamen callus proliferation
培养基
Medium植物生长调节剂
Plant growth regulator/(mg·L−1)生长量
Growing quantity/g绿色愈伤占比率
Percentage of green callus/%绿色愈伤出现时间
Time of green callus愈伤组织状态
Callus stateZ1 0.5 GA3 0.11±0.02b 96.30±6.42a 光培养后第9天 黄绿色与淡绿色致密愈伤 Z2 0.5 TDZ 0.27±0.03a 69.64±6.44b 光培养后第14天 浅黄绿色致密愈伤 Z3 0.5 2,4-D 0.06±0.02c 27.04±1.60c 光培养后第20天 白色与浅黄绿色致密愈伤
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表 5 滇牡丹雄蕊愈伤组织分化培养基的筛选
Table 5. Medium selection for P. delavayi stamen callus differentiation
编号
No.植物生长调剂
Plant growth regulator/（mg·L−1）不定芽分化率
Bud differentiation
rate/%绿色愈伤占比率
Percentage of
green callus/%黄绿色愈伤占比率
Percentage of
yellow-green callus/%黄褐色愈伤占比率
Percentage of
yellow-brown callus/%黑褐色愈伤占比率
Percentage of
dark brown callus/%6-BA GA3 PIC CH KT M1 1 0.2 — — — 0a 83.33±5.77b 0d 0d 0c M2 1 0.5 — — — 0a 96.67±2.89a 0d 0d 0c M3 1 1 — — — 0a 88.33±5.77b 0d 0d 0c M2-1 1 0.5 2 — — 0a 26.67±2.89e 35.00±0.00ab 38.33±2.89a 0c M2-2 1 0.5 4 — — 0a 38.33±2.89d 38.33±2.89a 23.33±2.89b 0c M2-3 1 0.5 — 200 — 0a 53.33±2.89c 35.00±5.00ab 11.67±2.89c 0c M2-4 1 0.5 — 400 — 0a 40.00±5.00d 36.67±2.89a 23.33±2.89b 0c M2-5 1 0.5 — — 0.5 0a 23.33±2.89e 25.00±0.00c 16.67±7.64bc 35.00±5.00a M2-6 1 0.5 — — 1 0a 36.67±2.89d 31.67±2.89b 23.33±2.89b 8.33±2.89b
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