Callus induction and morphological and cytological observation of unpollinated ovules of Camellia oleifera
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摘要:
目的 筛选适合油茶‘华金’Camellia oleifera‘Huajin’未授粉胚珠诱导愈伤组织的最适培养基，并对愈伤组织进行细胞学观察，为油茶单倍体育种提供依据。 方法 以油茶‘华金’为试材，筛选未授粉胚珠诱导愈伤组织的最适培养基配方，研究基础培养基类型MS培养基、木本植物用培养基(WPM)、MT培养基、1/2MS培养基、B5培养基、生长素萘乙酸(NAA)、吲哚乙酸(IAA)、吲哚丁酸(IBA)、二氯苯氧乙酸(2,4-D)和细胞分裂素苄氨基嘌呤(6-BA)、激动素(KT)、噻苯隆(TDZ)组合，以及植物生长调节剂浓度对愈伤组织诱导的影响；选取不同外部形态的愈伤组织进行石蜡切片，并根据流式细胞仪的倍性鉴定结果，研究愈伤组织来源及发育进程。 结果 MS作为基础培养基，添加1.0 mg·L−1 NAA+2.0 mg·L−1 TDZ+40 g·L−1蔗糖的诱导率最高，为92.97%，NAA和TDZ对诱导率影响显著(P＜0.05)，其中TDZ的影响程度大于NAA。胚性愈伤组织呈浅绿色颗粒状，细胞学特征为排列紧密，核大，倍性鉴定为六倍体。 结论 油茶未授粉胚珠诱导胚性愈伤组织的最适培养基配方为：MS+40 g·L−1蔗糖+7 g·L−1琼脂+1.0 mg·L−1 NAA+2.0 mg·L−1 TDZ；愈伤组织由外珠被细胞脱分化形成。本研究得到较为稳定高效的油茶未授粉胚珠胚性愈伤组织诱导技术体系，为进一步建立完整高效的油茶体细胞胚胎发生和单倍体育种体系提供参考。图4表3参33 Abstract:Objective The objective is to screen the optimal medium suitable for callus induction from unpollinated ovules of Camellia oleifera, and explore the origin and developmental process of callus by cytological observation, so as to provide data reference for haploid breeding of C. oleifera. Method C. oleifera ‘huajin’ was used as the test material to screen the optimal medium formula for inducing callus from unpollinated ovule, and to study the basic medium types MS, WPM, MT, 1/2MS, and B5, as well as effects of plant growth regulator concentration such as auxins NAA, IAA, IBA, and 2,4-D, and cytokinins 6-BA, KT, and TDZ on callus induction. Calluses with different external morphology were selected for paraffin section, and their origin and development process were studied according to the ploidy identification results of flow cytometry. Result MS was used as the basic medium, and the highest induction rate was 92.97% when 1.0 mg·L−1NAA+2.0 mg·L−1 TDZ+40 g·L−1 sucrose was added. NAA and TDZ had significant effects on the induction rate(P＜0.05), and the impact of TDZ was greater than that of NAA. The embryogenic callus was light green granular, with cytological characteristics of close arrangement, large nucleus, and ploidy identification of hexaploid. Conclusion The optimal medium for inducing embrygenic callus from unpollinated ovules of C. oleifera is MS+40 g·L−1sucrose +7 g·L−1 AGAR +1.0 mg·L−1 NAA+2.0 mg·L−1 TDZ. The callus is formed by dedifferentiation of outer integument cells. A relatively stable and efficient technical system for inducing embryogenic callus from unpollinated ovules of C. oleifera is obtained, providing a reference for further establishing a complete and efficient system for somatic embryogenesis and haploid breeding of C. oleifera. [Ch, 4 fig. 3 tab. 33 ref.] -
Key words:
- Camellia oleifera /
- unpollinated ovule /
- callus /
- cytological characteristics /
- ploidy identification
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图 2 胚珠愈伤组织生长过程中的外部形态变化
Figure 2 External morphological changes during ovule callus growth
图 3 胚珠外珠被细胞产生愈伤组织的发育过程
Figure 3 Development of callus from cells of the outer integument of ovule
表 1 不同基础培养基处理下油茶胚珠愈伤组织的诱导情况
Table 1. Callus induction of C. oleifera ovule under different basal mediums
编号 培养基类型 诱导率/% 褐化率/% 1 MS 72.50±5.15 a 21.65±1.88 d 2 WPM 69.16±1.74 a 37.38±1.02 b 3 MT 26.30±3.44 c 82.17±0.27 a 4 1/2MS 71.18±4.49 a 26.35±5.62 cd 5 B5 50.19±6.64 b 28.87±0.64 c 说明：表中不同的小写字母表示在P＜0.05水平上有显著差异。 
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表 2 不同激素组合处理下油茶胚珠愈伤组织的诱导情况
Table 2. Callus induction of C. oleifera ovary under different hormone combinations
编号 生长素质量浓度/(mg·L−1) 细胞分裂素及质量浓度/(mg·L−1) 诱导率/% 褐化率/% NAA IAA IBA 2,4-D 6-BA KT TDZ 1 1.0 0 0 0 2.0 0 0 26.92±1.92 de 54.33±25.99 bc 2 1.0 0 0 0 0 2.0 0 52.05±11.53 c 34.02±4.24 cd 3 1.0 0 0 0 0 0 2.0 88.89±5.16 a 11.11±2.34 e 4 0 1.0 0 0 2.0 0 0 30.83±7.89 d 91.76±2.14 a 5 0 1.0 0 0 0 2.0 0 30.90±5.90 d 76.83±9.85 a 6 0 1.0 0 0 0 0 2.0 69.72±9.08 b 48.68±14.33 bc 7 0 0 1.0 0 2.0 0 0 18.85±1.55 e 88.45±2.25 a 8 0 0 1.0 0 0 2.0 0 37.70±3.88 d 69.83±10.15 ab 9 0 0 1.0 0 0 0 2.0 85.75±5.17 a 47.70±5.95 c 10 0 0 0 1.0 2.0 0 0 72.50±1.55 c 21.65±5.18 de 11 0 0 0 1.0 0 2.0 0 60.04±1.15 bc 42.59±14.37 cd 12 0 0 0 1.0 0 0 2.0 55.94±4.05 c 39.69±5.16 cd 说明：表中不同的小写字母表示在P＜0.05水平上有显著差异
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表 3 激素和蔗糖组合L9(33)正交试验中油茶胚珠愈伤组织的诱导率情况
Table 3. Induction rate of C. oleifera ovary callus in the L9 (33) orthogonal test of hormone and sucrose combination
编号 激素及质量浓度/(mg·L−1) 蔗糖质量浓度/(g·L−1) 愈伤组织诱导率/% 褐化率/% 愈伤组织形态 NAA TDZ 1 0.5 1.0 40 70.65±7.62 bc 50.09±8.61 ab 质地疏松，黄褐色 2 0.5 2.0 50 71.26±12.06 bc 36.01±13.26 ab 质地疏松，浅黄色 3 0.5 3.0 60 68.90±16.16 bc 54.10±1.47 a 质地疏松，乳白色或黄褐色 4 1.0 1.0 50 85.65±7.65 abc 33.79±11.81 b 质地紧密，黄白色 5 1.0 2.0 60 89.11±2.46 ab 10.81±5.16 c 质地紧密，浅绿色 6 1.0 3.0 40 73.15±15.61 abc 48.26±10.57 ab 质地紧密，乳白色 7 2.0 1.0 60 71.44±11.95 bc 46.30±5.26 ab 质地疏松，黄褐色 8 2.0 2.0 40 92.97±5.97 a 34.26±12.90 b 质地紧密，黄色 9 2.0 3.0 50 64.92±2.42 c 52.43±12.15 ab 质地疏松，浅黄色或黄褐色 $\overline {X}_1$ 70.27 75.91 78.93 $\overline {X}_2$ 82.64 84.45 73.94 $\overline {X}_3$ 76.44 68.99 76.48 R 12.37 15.46 4.99 说明：表中不同的小写字母表示在P＜0.05水平上有显著差异。表中$\overline X_1 $、$\overline X_2 $、$\overline X_3 $代表正交试验的3个水平，R为诱导率的极差
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