Somatic embryogenesis and plantlet regeneration in Pinus elliottiiand its hybrids
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摘要:目的
明确适合湿地松Pinus elliottii及其杂交种的体细胞胚胎发生条件，建立体胚成熟萌发的技术。
方法以2016年6月采集的2个湿地松家系(EE1，EE2)、2个湿地松杂交种家系(EC，EH)的未成熟合子胚(含胚乳)为材料，从诱导、增殖、成熟到萌发配制3个系列培养基，比较外植体采集时间、家系、基本培养基对胚性愈伤组织诱导的影响。用显微镜进行胚性愈伤组织鉴别后，进一步挑选诱导形成的胚性愈伤组织进行增殖、成熟、萌发，最终获得再生植株。
结果湿地松及其杂交种合子胚的发育进程可划分为 8个阶段，阶段Ⅱ、Ⅲ为未成熟胚，适合体胚发生，EC最早出现阶段Ⅲ合子胚。外植体诱导产生具有胚性胚柄团(ESM)结构的胚性愈伤组织，可进一步增殖。诱导培养基对愈伤组织形成及胚性愈伤组织占比具有较大影响，3种诱导培养基(T1、T2和T3)产生愈伤组织效率最高的为T1培养基(49.0%)，胚性愈伤组织所占愈伤组织的比例最高为T2培养基(22.4%)。培养基配方的诱导率存在基因型间的差异，T1培养基整体诱导率低；T2培养基对家系EE1诱导率最高，为5.82%；T3培养基对参试材料均能诱导成功，且平均诱导率最高，为3.75%。随采样时间延后，家系EE1、EH的体胚诱导率逐渐增加，家系EE2、EC的体胚诱导率则随采样时间延后逐渐降低。体胚诱导率与合子发育阶段结果基本一致，阶段Ⅲ合子胚均出现较晚，前期诱导率低。胚性愈伤组织继代24次以后，胚活性逐渐降低。成熟培养基T1S和T3S可完成胚的成熟，平均每克成熟培养基分别成熟23.3和15.9个子叶胚，萌发率为32.1%，移栽保存率为47.8%。
结论诱导培养基T3对参试家系均能诱导成功，T3具有较广泛适用性，各家系在阶段Ⅲ合子胚出现时表现出较大的诱导率，阶段Ⅲ合子胚可能为体细胞胚发生的最佳诱导阶段。建立了湿地松及其杂种体细胞胚发生方法并形成了再生植株。
Abstract:ObjectiveTo define the conditions suitable for somatic embryogenesis of Pinus elliottii and its hybrids, and develop a mature protocol for somatic embryogenesis maturation and germination.
MethodThe immature zygotic embryos (with endosperm) of two P. elliottii families (EE1, EE2) and two P. elliottii hybrid (EC, EH) were sampled in June 2016. Three series of medium from induction, proliferation maturation to germination were set to compare the effects of explant collection date, family and basal medium on embryogenic callus induction. The embryogenic callus were identified through microscopic observation, and choosen for proliferation, maturation and germination culture. Finally the regenerated plantlets were developed.
ResultThe zygotic embryo development processes of P. elliottii and hybrids were divided into eight stages. The immature embryos at stage Ⅱ and Ⅲ were suitable for somatic embryogenesis. The embryos at stage Ⅲ first appeared in EC. The embryogenic callus with embryonal suspensor mass (ESM) could be further proliferated. The induction mediums (T1, T2 and T3) played vital roles in callus formation and embryogenic callus proportion. T1 medium had the highest callus induction efficiency (49.0%), and T2 medium had the highest embryogenic callus proportion (22.4%).The mediums had genotype-specific induction rates. The overall induction rate of T1 medium was low, and T2 medium had the highest induction rate (5.82%) for EE1. T3 medium was suitable for all tested materials, with the highest mean induction rate of 3.75%. The somatic embryo induction rates of EE1 and EH gradually increased with the sampling time extension, while the somatic embryo induction rates of EE2 and EC gradually decreased. The results of somatic embryo induction rate were basically corresponded with zygote development stages. The stage Ⅲ zygotic embryo appeared late, and its induction rate was low in early stage. After the embryogenic callus were subcultured for 24 times, the embryo activities progressively decreased. The maturation medium T1S and T3S could accomplish embryo maturation. Their average ripening efficiencies in per gram of mature medium were 23.3 and 15.9 cotyledons, respectively. The germination rate was 32.1% and the transplanting rate was 47.8%.
ConclusionAll tested materials can induce somatic embryo in T3 medium. T3 medium is widely applicable, and all materials have high induction rates when zygotic embryo appears at stage Ⅲ, illustrating that stage Ⅲ may be the optimal for somatic embryo induction. The experiment establishes the protocol for somatic embryogenesis and plantlet regeneration.
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图 1 湿地松及其杂种雌配子体与相应的胚
A：米粒状雌配子体外观；B：切开的米粒状雌配子体；C：取出的胚；标尺=1 mm
Figure 1. The megagametophytes and corresponding embryos of Pinus elliottii and hybrids
A：Appearance of grain-shape megagametophyte；B：The internal structure of grain-shape megagametophyte；C：Embryo taken out；Bar=1 mm
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图 2 湿地松及其杂种球果种子不同生长阶段的合子胚
A：阶段Ⅰ；B：阶段Ⅱ；C：阶段Ⅲ；D：阶段Ⅳ；E：阶段Ⅴ；F：阶段Ⅵ；G：阶段Ⅶ；H：阶段Ⅷ；标尺=500 μm
Figure 2. The zygotic embryos of Pinus elliottii and hybrids at different development stages
A：Stage Ⅰ；B：Stage Ⅱ；C：Stage Ⅲ； D：Stage Ⅳ；E：Stage Ⅴ；F：Stage Ⅵ；G：Stage Ⅶ；H：Stage Ⅷ；Bar=500 μm
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图 3 不同采球果时间各家系合子胚成熟情况
Figure 3. The maturation conditions of zygotic embryos of each family on different sampling dates
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图 4 湿地松及其杂种的胚性及非胚性愈伤组织
A：胚性胚柄团(ESM)结构；B：非ESM结构
Figure 4. The embryonic and non-embryonic callus of Pinus elliottii and hybrids
A：Structure of embryonal-suspensor mass(ESM) ; B: Structure of non-ESM
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图 5 不同采样时间的湿地松及其杂种的诱导率
各图中, 柱子上方的不同小写字母表示差异显著（P<0.05, Tukey 法）
Figure 5. The somatic embryo induction rates of Pinus elliottii and hybrids on different sampling dates
In each figure, different lowercase letters on the bars indicated significant difference（P<0.05, Tukey test）
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图 6 湿地松及其杂种体胚发生过程
A：外植体在诱导培养基T3上培养6周后；B：胚性组织继代24次后；C：胚性愈伤组织在成熟培养基T3S上培养3周后；D：产生子叶胚；E：体胚苗在萌发培养基上培养6周后；F：体胚苗移栽至黄心土1周后生长情况
Figure 6. The somatic embryogenesis stages of Pinus elliottii and hybrids
A：Megasporophylls after six weeks’ culture on the initiation medium T3; B:Embryogenic tissue after 23 cycles of subculture; C:Embryogenic tissue after three weeks of growth on the maturation medium T3S; D: The cotyledon of somatic embryos came out; E: Somatic embryos after six weeks’ culture on the germination medium; F: Somatic seedlings after one week of growth in the yellow soil
表 1 各培养基上胚性愈伤组织的鉴别情况
Table 1 The identification result of embryogenic callus in each medium
培养基
Medium接种总数 (QI)
Quantity of inoculation愈伤数 (CQ)
Callus quantity胚性愈伤数 (ECQ)
Embryogenic callus quantity比率/% rate CQ/QI ECQ/CQ T1 2 000 979 44 49.0 4.5 T2 1 634 223 50 13.6 22.4 T3 1 833 773 78 42.2 10.1 合计Total 5 467 1 975 172 36.1 8.7 
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表 2 不同培养基配方对湿地松及其杂种诱导率的影响1)
Table 2 The effects of different culture mediums on embryogenic induction rates of Pinus elliottiiand its hybrids
家系
Family诱导率/% Induction rate T1 T2 T3 均值 Mean EE1 2.48±1.17a 5.82±1.68a 4.11±1.44a 4.24 EE2 2.51±1.50a 0b 3.79±1.27a 2.10 EC 2.45±1.56a 3.88±4.75ab 2.14±1.56a 2.82 EH 0b 0b 4.13±2.71a 1.38 均值 Mean 2.28 2.68 3.75 1) 同列数据后的不同小写字母表示差异显著 (P<0.05, Tukey 法)
1) Different lowercase letters in the same column indicated significant difference (P<0.05, Tukey test)
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