Tissue culture regeneration of hybrid Liquidambar styraciflua × L. formosana
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摘要:目的枫香具有适应性强、生长速度快、观赏价值高等特点，既可以作为用材树种又可以作为优良的园林绿化树种。北美枫香能够与中国枫香杂交并可产生有杂种优势的后代，但是杂种枫香的快速繁殖技术和遗传转化体系还有待完善。方法本研究以北美枫香为母本，中国枫香为父本进行杂交，选用子一代杂种枫香的子叶、子叶节以及下胚轴为外植体材料建立高效的组培再生体系，并对3种外植体的分化能力进行比较。结果(1) 子叶和子叶节外植体最佳芽诱导培养基为0.2 mg/L TDZ+0.1 mg/L NAA的WPM培养基，而下胚轴外植体最佳芽诱导培养基为0.1 mg/L TDZ+0.1 mg/L NAA的WPM培养基。(2)3种外植体在0.5 mg/L 6-BA+0.1 mg/L NAA+0.8 mg/L GA不加琼脂的WPM芽伸长液体培养基中获得最高的平均不定芽诱导数。(3)不定芽在2.0 mg/L IBA+0.1 mg/L NAA的WPM培养基中生根，生根率达到100%。(4)不定芽生根培养2个月后长为完整植株，移栽成活率在90%以上。结论(1) 杂种枫香3种外植体的分化能力依次为：子叶节>子叶>下胚轴，其中子叶节的分化能力显著高于子叶和下胚轴。(2)TDZ能够有效促进不定芽的诱导，GA对不定芽的伸长具有显著的影响。(3)杂种枫香外植体在固体培养基中获得了最高的不定芽诱导率，在液体培养基中获得了最高不定芽平均数量，而且液体培养中未出现玻璃化。本研究为杂种枫香新品种的快速繁育以及遗传转化体系的建立奠定了良好的基础。Abstract:ObjectiveLiquidambar sp. has strong adaptability, fast growth, high ornamental values, it can be used as commercial tree species and landscaping tree species. Liquidambar styraciflua can be interfertiled with Liquidambar fomorsana, and their offspring can show robust growth. However, the rapid propagation technology and genetic transformation system of hybrid sweetgum need to be improved.MethodIn this study, using Liquidambar styraciflua as female parent and Liquidambar fomorsana as male parent to obtain hybrid sweetgum, and hybrid sweetgum cotyledon, cotyledon node and hypocotyl explants were used to establish an efficient tissue culture regeneration system, we also compared the differentiation ability of the three explants.Result(1) The best bud induction medium of cotyledon and cotyledon node explants was 0.2 mg/L TDZ+0.1 mg/L NAA basal WPM, but the best bud induction medium of hypocotyl explants was 0.1 mg/L TDZ+0.1 mg/L NAA basal WPM; (2) Shoot elongation liquid medium contained 0.5 mg/L 6-BA+0.1 mg/L NAA+0.8 mg/L GA without agar WPM performed the highest average adventitious bud induction number for three explant types; (3) Adventitious buds were rooted in 2.0 mg/L IBA+0.1 mg/L NAA WPM medium, and its rooting rate reached 100%;(4) After the adventitious buds being cultured in rooting medium for 2 months, the plantlets were transferred into greenhouse, and the transplant survival rate was more than 90%.Conclusion(1) The differentiation abilities of the three explants were as follows: cotyledonary node>cotyledon> hypocotyl, in which the differentiation ability of cotyledonary nodes was significantly higher than cotyledon and hypocotyl; (2) TDZ can effectively promote the induction of adventitious buds, and GA has a significant effect on adventitious shoot elongation; (3) The hybrid sweetgum explants obtained the highest adventitious bud induction rate in the solid medium, the highest number of adventitious buds was obtained in the liquid medium, and no vitrification occurred in the liquid culture. The results of this experiment lay a solid foundation for the rapid propagation of new varieties and the establishment of genetic transformation system of hybrid sweetgum.
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Keywords:
- hybrid sweetgum /
- in vitro regeneration /
- two-step culture
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图 1 不同质量浓度组合的TDZ和NAA培养基中的不定芽诱导率
Figure 1. Adventitious bud induction rate in TDZ and NAA medium with different concentrations
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图 2 不同质量浓度组合的TDZ和NAA培养基中的平均
Figure 2. Average adventitious bud induction number in TDZ and NAA medium with different concentrations
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图 5 杂种枫香子叶、子叶节、下胚轴外植体不定芽诱导
A.子叶外植体在0.2 mg/L TDZ+0.1 mg/L NAA的WPM芽诱导培养基中培养1个月后；B.子叶节外植体在0.2 mg/L TDZ+0.1 mg/L NAA的WPM芽诱导培养基中培养1个月后；C.下胚轴外植体在0.2 mg/L TDZ+0.1 mg/L NAA的WPM芽诱导培养基中培养1个月后；D.子叶外植体在0.5 mg/L BA+0.1 mg/L NAA的WPM芽伸长培养基培养1个月后；E.子叶节外植体在0.5 mg/L BA+0.1 mg/L NAA的WPM芽伸长培养基培养1个月后；F.下胚轴外植体在0.5 mg/L BA+0.1 mg/L NAA的WPM芽伸长培养基培养1个月后；G.子叶外植体在0.8 mg/L GA+0 g琼脂的WPM芽伸长培养基培养1个月后；H.子叶节外植体在0.8 mg/L GA+0 g琼脂的WPM芽伸长培养基培养1个月后；I.下胚轴外植体在0.8 mg/L GA+0 g琼脂的WPM芽伸长培养基培养1个月后。
Figure 5. Adventitious bud induction of cotyledon, cotyledon node, hypocotyl explants of hybrid sweetgum
A, cotyledon explants were cultured in 0.2 mg/L TDZ+0.1 mg/L NAA WPM bud induction medium for one month; B, cotyledon node explants were cultured in 0.2 mg/L TDZ+0.1 mg/L NAA WPM bud induction medium for one month; C, hypocotyl explants were cultured in 0.2 mg/L TDZ+0.1 mg/L NAA WPM bud induction medium for one month; D, cotyledon explants were cultured in 0.5 mg/L BA+0.1 mg/L NAA WPM shoot elongation medium for one month; E, cotyledon node explants were cultured in 0.5 mg/L BA+0.1 mg/L NAA WPM shoot elongation medium for one month; F, hypocotyl explants were cultured in 0.5 mg/L BA+0.1 mg/L NAA WPM shoot elongation medium for one month; G, cotyledon explants were cultured in 0.8 mg/L GA+0 g agar WPM bud elongation medium for one month; H, cotyledon node explants were cultured in 0.8 mg/L GA+0 g agar WPM bud elongation medium for one month; I, hypocotyl explants were cultured in 0.8 mg/L GA+0 g agar WPM bud elongation medium for one month.
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图 3 不同质量浓度组合的GA和琼脂培养基中的不定芽诱导率
Figure 3. Adventitious bud induction rates in GA and agar medium with different concentrations
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图 4 不同质量浓度组合的GA和琼脂培养基中的平均不定芽诱导数
Figure 4. Average adventitious bud induction number in GA and agar medium with different concentrations
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图 6 杂种枫香各阶段生长情况
A.不定芽在2.0 mg/L IBA+0.1 mg/L NAA的WPM培养基中生长2个月后；B.移栽1个月后；C.移栽3个月后；D.移栽6个月后。
Figure 6. Morphology condition of hybrid sweetgum at different stages
A, adventitious buds grown in 2.0 mg/L IBA + 0.1 mg/L NAA WPM medium for two months; B, one month after transplanting; C, three months after transplanting; D, six months after transplanting.
表 1 不同质量浓度组合的TDZ和NAA对不定芽诱导的效果
Table 1 Effects of different concentrations TDZ and NAA on adventitious bud induction
编号
No.培养基组成
Composition of medium(WPM)子叶
Cotyledon子叶节
Cotyledon node下胚轴
Hypocotyl平均不定芽诱导数
Average adventitious bud induction number不定芽诱导率
Adventitious bud induction rate/%平均不定芽诱导数
Average adventitious bud induction number不定芽诱导率
Adventitious bud induction rate/%平均不定芽诱导数
Average adventitious bud induction number不定芽诱导率
Adventitious bud induction rate/%1 0.2 mg/L TDZ+0.1 mg/L NAA 5.40±0.26a 93.33±4.41a 6.0±0.38a 100 4.35±0.22ab 75±4.04a 2 0.1 mg/L TDZ+0.1 mg/L NAA 4.50±0.18ab 65±5.77bc 5.20±0.28ab 98.33±1.67 4.50±0.21a 83.33±3.84a 3 0.05 mg/L TDZ+0.1 mg/L NAA 2.65±0.16ab 60±3.46bc 1.84±0.13c 100 1.97±0.37b 56.67±2.37abc 4 0.2 mg/L TDZ+0 mg/L NAA 2.45±0.46b 61.67±6.01bc 3.30±0.12abc 90±5.77 2.65±0.17ab 63.33±6.84ab 5 0.1 mg/L TDZ+0 mg/L NAA 2.78±0.29b 73.33±4.37ab 2.85±0.17bc 93.33±3.33 2.22±0.17b 45±1.73bc 6 0.05 mg/L TDZ+0 mg/L NAA 2.47±0.42b 48.33±6.67c 2.04±0.22c 93.33±6.67 1.90±0.24b 35±2.89c 注:平均不定芽诱导数、不定芽诱导率的数据为平均数±标准差。同一列中不同字母表示差异显著(P < 0.05)。下同。Notes: data of the average induction number of adventitious shoots, the induction percentage of adventitious buds are mean ±SD. Different letters in a same column mean significant difference at P < 0.05 level. The same below. 
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表 2 不同质量浓度组合的GA和琼脂对不定芽伸长的效果
Table 2 Effects of different concentrations of GA and agar on adventitious bud elongation
编号
No.培养基组成
Composition of medium (WPM)子叶
Cotyledon子叶节
Cotyledon node下胚轴
Hypocotyl平均不定芽诱导数
Average adventitious bud induction number不定芽诱导率
Adventitious bud induction rate/%平均不定芽诱导数
Average adventitious bud induction number不定芽诱导率
Adventitious bud induction rate/%平均不定芽诱导数
Average adventitious bud induction number不定芽诱导率
Adventitious bud induction rate/%1 0.4 mg/L GA+4 g/L倍力凝+2 g/L琼脂0.4 mg/L GA+4 g/L polygel+2 g/L agar 5.78±0.38b 90±5.77a 6.00±0.61b 100 5.30±0.48b 71.67±2.31b 2 0.4 mg/L GA+0 g/L琼脂0.4 mg/L GA+0 g/L agar 7.70±0.42a 62.67±4.04b 8.18±0.42ab 95±2.89 6.21±0.45ab 63.33±7.26c 3 0.8 mg/L GA+4 g/L倍力凝+ 2 g/L琼脂0.8 mg/L GA+4 g/L polygel+ 2 g/L agar 6.24±0.66b 93.33±1.67a 7.08±0.23ab 100 5.85±0.22b 83.33±1.67a 4 0.8 mg/L GA+0 g/L琼脂0.8 mg/L GA+0 g/L agar 8.29±0.78a 50±2.89b 9.13±0.67a 100 7.34±0.61a 73.33±1.67b
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