Rapid Propagation of Double Delight Chinese Rose Using Tissue Culture
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摘要:
目的 筛选出红双喜月季各阶段最佳培养条件，并建立组培快繁技术体系，为工厂化生产提供科学依据及技术指导。 方法 以红双喜月季茎段为试验材料，采用正交设计和随机区组设计等试验方法，开展红双喜月季初代培养、继代增殖、生根培养以及炼苗移栽的研究。 结果 茎段最佳灭菌处理为75% C2H5OH 30 s+0.1% HgCl2 6 min；最适初代诱导培养基为MS+3.00 mg·L-1 6-BA+0.30 mg·L-1 NAA，诱导率高达95.56%；最适增殖培养基为MS+1.00 mg·L-1 6-BA+0.10 mg·L-1 IBA，增殖系数高达6.61；最适生根培养基为1/4MS+0.05 mg·L-1IBA+2.50 g/L活性炭，生根率高达95.56%；最适移栽基质为V细沙:V蛭石:V珍珠岩:V椰糠=1:2:3:1，移栽成活率高达98.33%。 结论 通过茎段直接诱导丛生芽，可以建立红双喜月季高效快繁技术体系。 Abstract:Objective To optimize the tissue culture conditions for an efficient, rapid propagation of Double Delight Chinese roses for large scale operations. Method Stems cut from the rose plants were used for the tissue culture optimization with an orthogonal experiment and completely randomized block design method. The initial generation culture, subsequent generations enrichment, rooting enhancement, and transplant augmentation for the plant propagation were evaluated. Result The optimal method to disinfect the stems for propagation was to dip them in 75% C2H5OH for 30 s followed by soaking in a 0.1% HgCl2 solution for 6 min. The most efficient 1st generation culture was obtained in a medium containing MS+3.00 mg·L-1 6-BA+0.30 mg·L-1 NAA with an induction rate of up to 95.56%. The selected enrichment medium was MS+1.00 mg·L-1 6-BA+0.10 mg·L-1 IBA to yield a multiplication factor of 6.61. For efficient rooting, the best medium was MS+0.05 mg·L-1 IBA+2.50 mg·L-1 activated carbon to result in a 95.56% rate. The mixing ratio of the substrates for seedling transplanting was optimized to be fine sand:vermiculite:perlite:coconut bran=1:2:3:1 that provided a seedling survival rate as high as 98.33%. Conclusion A highly efficient, rapid propagation method using cut stems from Double Delight Chinese rose plants was established for scale-up productions. -
Key words:
- Double Delight Chinese rose /
- culture seedling /
- rapid propagation /
- orthogonal test /
- randomized block
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图 1 红双喜月季组织培养
注：A为增殖苗；B为生根苗；C为移栽苗；D为移栽3个半月后苗。
Figure 1. Tissue culture of Double Delight Chinese rose
Note:A is a proliferating seedling.B is rooting seedling.C is transplanted seedling. D is a seedling cultured for 3 and a half months after transplanting.
表 1 灭菌时间对外植体接种的影响
Table 1. Effect of disinfection time on explant inoculation
处理
Treatment灭菌时间Disinfection time/min 污染率
Contamination rate/%死亡率
Mortality rate/%75%酒精
75% alcohol0.1%升汞
0.1% mercuric chlorideA1 0.25 4 65.56±0.96a 0 A2 0.25 5 58.89±0.91b 0 A3 0.25 6 34.44±0.76e 0 A4 0.25 7 18.89±0.94g 4.44±0.56c A5 0.50 4 57.78±0.84b 0 A6 0.50 5 45.56±0.96c 0 A7 0.50 6 2.22±1.92h 0 A8 0.50 7 0 17.78±0.94b A9 0.75 4 41.11±2.26d 2.22±1.92d A10 0.75 5 24.44±0.50f 3.33±0.38d A11 0.75 6 0 18.89±0.13b A12 0.75 7 0 26.67±1.53a 注：同列数据后不同字母者表示差异达显著水平(P＜0.05)。表 2~8同。
Note:The difference between different letters after the same column data reached a significant level(P＜0.05).The same as table 2-8.
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表 2 诱导率正交试验极差分析及多重比较
Table 2. Extreme difference analysis and multiple comparison on induction rates obtained from orthogonal test
处理
Treatment因素Factor 诱导率
Inductivity/%A：基本培养基
Minimal mediumB：6-BA 6-Benzylaminopurine
/(mg·L-1)C：NAA 1-naphthlcetic acid
/(mg·L-1)1 1(MS) 1(1.00) 1(0.10) 83.33±3.34b 2 1 2(2.00) 2(0.20) 47.78±5.09c 3 1 3(3.00) 3(0.30) 95.56±1.93a 4 2(B5) 1 2 20.00±3.33de 5 2 2 3 11.11±3.85f 6 2 3 1 14.44±1.93ef 7 3(WPM) 1 3 16.67±3.34ef 8 3 2 1 23.33±3.34d 9 3 3 2 18.89±3.85de X1 75.56 a 40.00 a 40.37 a X2 15.18 b 27.41 b 28.89 b X3 19.63 b 42.96 a 41.11 a R 60.37 15.56 11.48 最优组合 A1 B3 C3 注：Xv1、X2、X3分别为各列各水平诱导率均值；R为极值。
Note:X1, X2, X3 are the mean values of induction rate at each level in each column, R is the extreme value.
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表 3 6-BA、IBA水平间增殖系数的多重比较
Table 3. Multiple comparison on proliferation coefficients between 6-BA and IBA levels
6-BA
6-Benzylaminopurine/
(mg·L-1)均值
Mean valueIBA
Indole-3-Butytric acid(mg·L-1)均值
Mean value0.50 4.40b 0.10 5.20a 1.00 5.12a 0.20 4.65b 1.50 3.78c 0.30 3.40c 2.00 2.34d 0.40 2.39d
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表 4 不同质量浓度6-BA与IBA配比对增殖的影响
Table 4. Effects of 6-BA and IBA concentrations on seedling proliferation
处理
Treatment6-BA 6-Benzylaminopurine
/(mg·L-1)IBA Indole-3-Butytric acid/(mg·L-1) 增殖系数
Propagation coefficient生长状态
Growing statusB1 0.50 0.10 6.07±0.20b 叶片绿色，株高4 cm左右 B2 0.50 0.20 5.19±0.23c 叶片翠绿色，株高4.5 cm左右 B3 0.50 0.30 4.15±0.15e 部分叶片黄色，株高4.8 cm左右 B4 0.50 0.40 2.20±0.10i 叶片绿色，株高3 cm左右 B5 1.00 0.10 6.61±0.11a 叶片翠绿，较多腋芽，株高6 cm左右 B6 1.00 0.20 5.99±0.19b 腋芽叶片黄绿色，株高7 cm左右 B7 1.00 0.30 4.01±0.15ef 叶绿色个别脱落，株高5.5 cm左右 B8 1.00 0.40 3.85±0.29f 叶片黄绿色，株高4 cm左右 B9 1.50 0.10 4.73±0.06d 叶小绿色，株高3.5 cm左右 B10 1.50 0.20 4.55±0.11d 叶片微卷，株高8 cm左右 B11 1.50 0.30 3.58±0.23g 叶翠绿色较小，株高5 cm左右 B12 1.50 0.40 2.25±0.05i 叶黄脱落，基部有愈伤，株高3 cm左右 B13 2.00 0.10 3.40±0.56g 叶小，部分发白，株高3.5 cm左右 B14 2.00 0.20 2.86±0.02h 叶浅绿色，株高4 cm左右 B15 2.00 0.30 1.84±0.02j 叶绿色，株高3 cm左右 B16 2.00 0.40 1.25±0.04k 叶基部浅黄色，株高2.5 cm左右
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表 5 基本培养基对生根的影响
Table 5. Effect of basal medium on rooting of seedlings
处理
Treatment基本培养基
Minimal mediumIBA
Indole-3-Butytric acid/(mg·L-1)生根率
Rooting rate
/%平均根数
Average rootnumber/条平均根长
Average root length/cm生长状态
Growing statusC1 MS 0.10 76.67±3.34b 3.50 2.00 褐色，细弱 C2 1/2MS 0.10 54.44±1.93c 2.10 4.80 白色，较粗 C3 1/4MS 0.10 84.44±5.09a 5.10 4.30 白色，较粗 C4 1/6MS 0.10 72.22±3.85b 7.60 6.00 黄褐色，较细 注：生根率为均值±标准差；表 6、7同。
Note:Rooting rate is mean±standard deviation, table 6 and 7 identical.
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表 6 不同浓度IBA、NAA对生根的影响
Table 6. Effect of IBA and NAA concentrations on rooting of seedlings
处理
Treatment培养基
MediumIBA
Indole-3-Butytric acid/(mg·L-1)NAA
Naphthlcetic acid/(mg·L-1)生根率
Rooting rate/%平均根数
Average root number/条平均根长
Average root length/cm生长状态
Growing statusD1 1/4MS 0.05 - 91.11±6.94a 5.40 4.90 白色，粗 D2 1/4MS 0.10 - 84.44±5.09a 5.10 4.30 白色，较粗 D3 1/4MS 0.15 - 71.11±5.09b 5.60 2.10 黑色，细弱 D4 1/4MS - 0.05 60.00±3.33cd 1.00 2.50 黄褐色，细弱 D5 1/4MS - 0.10 67.78±6.94bc 2.30 3.20 黄白色，较粗壮 D6 1/4MS - 0.15 54.44±5.09d 3.50 4.70 褐色，较细
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表 7 不同活性炭浓度对生根的影响
Table 7. Effect of activated carbon concentration on rooting of seedlings
处理
Treatment活性炭浓度
Activated carbon concentration/(g·L-1)生根率
Rooting rate
/%平均根数
Average root number/条平均根长
Average root length/cm生长状态
Growing statusE1 0 91.11±5.09 ab 4.20 4.50 根较粗，白色 E2 1.50 76.67±3.34 d 3.50 3.20 根细，白色 E3 2.00 80.00±3.34 cd 3.00 6.50 根细，白色 E4 2.50 95.56±1.93 a 4.60 5.30 根较细，白色 E5 3.00 85.56±5.09 bc 5.20 5.60 根细，白色
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表 8 不同基质正交试验结果极差分析及多重比较
Table 8. Range analysis and multiple comparisons on culture media tested with orthogonal matrix
处理
Treatment因素Factor 成活率
Survival rate/%A(细沙)Fine sand B(蛭石)Vermiculite C(珍珠岩)Perlite D(椰糠)Coconut bran F1 0 1 1 0 63.33±5.77 de F2 0 2 2 1 76.67±2.89 c F3 0 3 3 2 70.00±5.00 cd F4 1 1 2 2 55.00±5.00 f F5 1 2 3 0 96.67±2.89 a F6 1 3 1 1 86.67±5.77 b F7 2 1 3 1 66.67±2.89 de F8 2 2 1 2 75.00±5.00 c F9 2 3 2 0 61.67±2.89 ef X1 70.00 b 61.67 c 75.00 a 74.44 a X2 80.00 a 83.33 a 64.67 b 76.67 a X3 67.78 b 72.78 b 78.33 a 66.67 b R 12.22 21.67 13.87 10.00 最优组合 A2 B2 C3 D2 注：X1、X2、X3分别为各列各水平成活率均值，R为极值。
Note:X1、X2、X3 are the mean values of survival rate at each level in each column, R is the extreme value.
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