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植物胚性感受态为植物细胞或组织脱分化能力或潜力,胚性感受态的高低直接决定了植物体细胞胚诱导的难易程度[1]。研究发现,大多数植物的组织或细胞维持高水平胚性感受态的时间极短[2-3]。胚性感受态水平的高低与基因型、外植体的取材部位、生理学年龄等密切相关[4]。在黑核桃Juglans nigra中,授粉后3~4周的幼胚开始具有体胚发生能力,授粉后6~7周时幼胚体胚发生频率最高,此后,体胚发生能力逐渐下降[5]。在梨Pyrus中,具有胚性感受态的时间极短,只有当梨幼胚发育期为鱼雷胚至子叶胚时,才有少量体胚的诱导与发生[6]。榧树Torreya grandis隶属于裸子植物红豆杉科Taxaceae榧属Torreya常绿乔木。香榧Torreya grandis ‘Merrillii’是实生榧树中优良变异类型或优株经人工嫁接繁殖而成的优良品种,为中国特有的珍稀干果和木本油料植物,具有重要的经济、社会和生态价值[7]。由于传统嫁接繁殖难以满足目前市场的巨大需求,近年来,研究人员开展了香榧植株体胚发生及再生体系的建立等研究[8-11]。但由于缺乏对香榧种子内幼胚发育过程及体胚诱导及发生规律的深入了解,导致其体胚发生及再生体系难以稳定,有效繁殖系数低。本研究以不同发育阶段的香榧幼胚为外植体,开展胚性愈伤组织及体细胞胚诱导研究,通过对不同发育时期来源的幼胚进行诱导率分析,同时辅以幼胚内源激素的变化规律,拟阐明香榧幼胚发育状态与其胚性感受态之间的相关性,为建立香榧稳定、高效体胚发生及植株再生体系提供良好的技术平台,同时也为后期香榧遗传转化体系的建立打下良好的基础。
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观察发现,成熟的香榧种子具假种皮,绿色纤维质(图 1A)。剥去假种皮,可见棕褐色外种皮,坚硬木质化(图 1B,图 1C箭头2所示)。内种皮暗红色,褶皱膜质,内陷成脊状(图 1C,箭头1所示)。胚乳单倍体,乳白色,为主要可食用部位(图 1C,箭头3所示)。香榧幼胚位于种子榧眼端雌配子体腔内,细小不明显(图 1C,椭圆所示,图 1D),幼胚胚体和胚柄系统相连,胚柄系统靠近珠孔端,胚头部远离珠孔端。香榧胚发育经历多胚时期、胚胎选择时期和优势胚完全发育等3个时期。通过胚胎选择,多个香榧幼胚最后仅有1个发育较好,其他幼胚在不同发育阶段终止发育而萎缩。香榧早期幼胚为棒状(图 1E, F),随着幼胚发育成熟,头部逐渐膨大,直至子叶展开,胚柄萎缩脱落(图 1G,H)。
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不同发育时期香榧幼胚的内源IAA及ABA质量分数具显著性差异(P<0.05)。香榧幼胚内源IAA的质量分数在胚珠突破种鳞后第8周处于最大值,达53.90 ng·g-1,第9周稍有下降,但与第8周之间无显著性差异,之后迅速下降至44.14 ng·g-1。随着胚龄的逐渐增加,内源IAA质量分数总体呈小幅波动的下降趋势,第15周最低,为35.60 ng·g-1(图 2)。
图 2 香榧幼胚胚龄对其内源IAA及ABA质量分数的影响
Figure 2. Effect of developmental stages on endogenous IAA and ABA content of immature embryo in Torreya grandis 'Merrillii'
香榧幼胚内源ABA质量分数的测定结果表明:幼胚胚龄对其内源ABA的质量分数同样具有显著影响(P<0.05)。在种鳞突破种皮后的第8至第15周内,随着胚龄的增加,内源ABA质量分数呈先下降后上升的总趋势,其中第8周至第10周,内源ABA质量分数先下降后上升,但变化幅度比较小;第10周到第12周处于快速上升期,从第10周的89.80 ng·g-1上升到第12周的144.67 ng·g-1;第13至15周内源ABA处于变化平缓期,且维持在较高水平,其中第14周和第15周内源ABA质量分数无显著差异,第15周达到最大值,为160.20 ng·g-1(图 2)。
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不同发育阶段香榧幼胚的胚性感受态具显著差异;同时,不同离体培养条件对香榧幼胚的胚性感受态激发也有显著差异(表 1,图 3,图 4)。将香榧幼胚接种于1/2 SH基本培养基中培养,当幼胚胚龄较小时,可产生极少量胚性愈伤组织,但从第10周开始,胚性愈伤组织诱导率均为0,但各胚龄幼胚均未诱导直接产生体胚。将幼胚接种于附加0.1 mg·L-1NAA的1/2 SH基本培养基上培养,随着幼胚胚龄的增加,胚性愈伤组织诱导率先上升后下降,其中第8周幼胚的胚性愈伤组织诱导率为48.1%,第9周时略有升高,达到最高值,为48.3%,随后逐渐下降,第14至第15周胚性愈伤组织诱导率为0;将香榧幼胚置于1/2 SH基本培养基附加0.1 mg·L-1NAA的培养基中培养,随着胚龄的增加,诱导率也呈先上升后下降的趋势,第8周体胚诱导率为0,第9周体胚诱导率上升为5.8%,至第10周体胚诱导率达到最高,为42.7%,后随着胚龄的增加,体胚诱导率逐渐下降,第14周至第15周体胚诱导率为0(图 3)。
表 1 胚龄及萘乙酸对香榧幼胚胚性感受态的影响
Table 1. Effect of developmental stages and NAA on embryo genic frequency of Torreya grandis 'Merrillii' immature embryo
处理 ρ萘乙酸/(mg·L-1) 胚珠突破种鳞/周 胚性愈伤组织诱导率/% 体胚直接发生率/% 幼胚萌发率/% 1 0 8 5.1 ± 0.8 e 0f 0 k 2 0.1 43.1 ± 0.3 b 0f 0 k 3 0 9 1.2 ± 0.1 f 0f 0 k 4 0.1 48.3 ± 2.6 a 5.8 ± 0.7 e 0 k 5 0 10 0g 0f 5.4 ± 0.8 j 6 0.1 47.4 ± 1.5 ab 42.7 ± 1.3 a 0k 7 0 11 0g 0f 9.8 ± 2.5 P 8 0.1 30.1 ± 0.9 c 39.3 ± 0.2 b 7.2 ± 0.9 i 9 0 12 0g 0f 21.3 ± 1.8 d 10 0.1 13.5 ± 1.1 d 18.6 ± 2.1 c 15.2 ± 1.3 g 11 0 13 0g 0f 44.2 ± 2.4 c 12 0.1 2.1 ± 0.3 f 7.7 ± 0.1 d 19.7 ± 0.7 f 13 0 14 0g 0f 65.1 ± 1.2 a 14 0.1 0g 0f 20.9 ± 0.5 de 15 0 15 0g 0f 63.7 ± 0.9 b 16 0.1 0g 0f 20.3 ± 2.8 ef 图 3 胚龄对胚性愈伤组织及体胚直接发生的影响
Figure 3. Effect of immature embryo developmental stages on embryogenic callus induction and direct somatic embryogenesis (SH+0.1 mg·L-1 NAA)
图 4 胚龄及培养条件对香榧幼胚萌发的影响
Figure 4. Effect of immature embryo developmental stages and media on in vitro germination
第8周至第15周的幼胚进行离体萌发试验发现,不同外植体发育阶段及培养条件对萌发率具有显著影响(图 4)。将幼胚培养于1/2 SH基本培养基中,萌发率随着胚龄的增加而升高,第8周至第9周幼胚萌发率为0,从第10周开始,萌发率逐渐上升,第14周时达最高值,为65.1%,之后略有下降。而当幼胚培养于附加0.1 mg·L-11/2 SH培养基NAA中进行萌发培养时,香榧幼胚极易产生愈伤组织而不能正常萌发形成有效苗(图 5)。
Immature embryo development and embryogenic frequency in Torreya grandis 'Merrillii'
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摘要: 以突破种鳞第8周至第15周种子的香榧Torreya grandis ‘Merrillii’幼胚为外植体,研究了香榧幼胚胚龄、生长素等对香榧胚性愈伤组织以及体细胞胚直接发生的影响,筛选出高胚性感受态的香榧幼胚胚龄,并根据幼胚内源吲哚乙酸(IAA)和脱落酸(ABA)质量分数检测结果,初步确定了高胚性感受态的香榧幼胚生理特点。结果表明:在SH附加0.1 mg·L-1 NAA培养基中培养,第9周幼胚的胚性愈伤组织发生率最高,为48.3%,第10周幼胚的体胚直接发生率最高,为42.7%;而培养于SH基本培养基中的第14周幼胚直接萌发率最高,为65.1%。香榧不同胚龄幼胚内源ABA及IAA质量分数检测结果表明:不同发育时期香榧幼胚的内源IAA及ABA质量分数具显著差异(P < 0.05)。其中香榧第8周幼胚内源IAA的质量分数最高,为53.90 ng·g-1,随着胚龄的逐渐增加,内源IAA质量分数呈下降趋势,第15周内源IAA质量分数最低,为35.60 ng·g-1。内源ABA质量分数随着胚龄的增加呈先缓慢下降,再快速上升,后下降,其中第15周达到最大值,为160.20 ng·g-1。通过对香榧幼胚离体培养及内源激素的测定,发现香榧第8~12周幼胚具有较高的胚性感受态。该研究结果对维持香榧幼胚高胚性感受态,开展香榧胚性愈伤组织及体胚发生培养条件的优化提供了技术指导。Abstract: To obtain high embryogenic frequency in the immature embryo developmental stage, immature embryos of Torreya grandis 'Merrillii' from seeds (8 to 15 weeks after ovule breakthrough from the scales) were sampled as explants to optimize the best developmental stage for somatic embryogenesis. Endogenous Indole-3-acetic Acid (IAA) and Abscisic Acid (ABA) of immature embryos were also analyzed using enzyme-linked immunosorbent assay (ELISA) method. Results showed that the embryogenic callus frequency (up to 48.3%) and direct somatic embryogenesis frequency (up to 42.7%) were highest with immature embryos from seeds at 9 weeks after scale breakthrough on Schenk and Hilderbrandt (SH) basal medium supplemented with 0.1 mg·L-1 1-Naphthaleneacetic acid (NAA). In vitro germination frequency was highest (up to 65.1%) with immature embryos from seeds at 14 weeks after scale breakthrough. Significant differences (P < 0.05) among development stages of different immature embryos were found. With the development of immature embryos, endogenous IAA decreased gradually being highest with immature embryos from seeds at 8 weeks after scale breakthrough[up to 53.90 ng·g-1 fresh weight (FW)]. Meanwhile, endogenous ABA initially decreased followed by a rapid increase with the highest content being 160.20 ng·g-1 FW for immature embryos from seeds at 15 weeks after scale breakthrough. This study revealed that with immature embryos of seeds from 8 to 12 weeks, embryogenic frequency was high which could help optimize embryogenic callus induction and somatic embryo genesis in Torreya grandis 'Merrillii'.
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表 1 胚龄及萘乙酸对香榧幼胚胚性感受态的影响
Table 1. Effect of developmental stages and NAA on embryo genic frequency of Torreya grandis 'Merrillii' immature embryo
处理 ρ萘乙酸/(mg·L-1) 胚珠突破种鳞/周 胚性愈伤组织诱导率/% 体胚直接发生率/% 幼胚萌发率/% 1 0 8 5.1 ± 0.8 e 0f 0 k 2 0.1 43.1 ± 0.3 b 0f 0 k 3 0 9 1.2 ± 0.1 f 0f 0 k 4 0.1 48.3 ± 2.6 a 5.8 ± 0.7 e 0 k 5 0 10 0g 0f 5.4 ± 0.8 j 6 0.1 47.4 ± 1.5 ab 42.7 ± 1.3 a 0k 7 0 11 0g 0f 9.8 ± 2.5 P 8 0.1 30.1 ± 0.9 c 39.3 ± 0.2 b 7.2 ± 0.9 i 9 0 12 0g 0f 21.3 ± 1.8 d 10 0.1 13.5 ± 1.1 d 18.6 ± 2.1 c 15.2 ± 1.3 g 11 0 13 0g 0f 44.2 ± 2.4 c 12 0.1 2.1 ± 0.3 f 7.7 ± 0.1 d 19.7 ± 0.7 f 13 0 14 0g 0f 65.1 ± 1.2 a 14 0.1 0g 0f 20.9 ± 0.5 de 15 0 15 0g 0f 63.7 ± 0.9 b 16 0.1 0g 0f 20.3 ± 2.8 ef -
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2018.05.010