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作物学报 ›› 2020, Vol. 46 ›› Issue (02): 194-203.doi: 10.3724/SP.J.1006.2020.94065

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

诸葛菜小孢子培养及其单倍体减数分裂染色体配对观察

殷家明1,2,钟荣棋1,2,林呐1,2,唐章林1,2,李加纳1,2   

  1. 1 西南大学农学与生物科技学院, 重庆 400715
    2 重庆市油菜工程技术研究中心, 重庆 400715
  • 收稿日期:2019-04-24 接受日期:2019-08-09 出版日期:2020-02-12 网络出版日期:2019-10-12
  • 作者简介:殷家明, E-mail: 1661096534@qq.com|钟荣棋, 1457139712@qq.com
  • 基金资助:
    本研究由国家重点研发计划项目(2018YFD0100503);国家现代农业产业技术体系建设专项(CARS-12);重庆市社会事业与民生保障科技创新专项(cstc2016shms-ztzx80010);重庆市社会事业与民生保障科技创新专项资助(cstc2017shms-xdny80009)

Microspore culture and observations on meiotic chromosome pairing of the haploid in Orychophragmus violaceus

YIN Jia-Ming1,2,ZHONG Rong-Qi1,2,LIN Na1,2,TANG Zhang-Lin1,2,LI Jia-Na1,2   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2 Chongqing Rapeseed Eengineering and Technology Research Center, Chongqing 400715, China
  • Received:2019-04-24 Accepted:2019-08-09 Published:2020-02-12 Published online:2019-10-12
  • Supported by:
    This study was supported by the National Key Research and Development Plan(2018YFD0100503);the China Agriculture Research System(CARS-12);the Chongqing Science and Technology Innovation Special Project for Social Affairs and People’s Livelihood Insurance(cstc2016shms-ztzx80010);the Chongqing Science and Technology Innovation Special Project for Social Affairs and People’s Livelihood Insurance(cstc2017shms-xdny80009)

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摘要：

诸葛菜是一种极有价值的观赏、蔬菜、饲料和油料作物种质资源。为建立诸葛菜小孢子胚状体诱导再生植株技术, 并为诸葛菜染色体组的起源与进化研究提供相关数据资料, 本研究通过对诸葛菜游离小孢子的培养, 研究了热激培养时间和活性炭浓度对胚状体产量的影响, 并采用常规压片法对诸葛菜单倍体减数分裂染色体配对行为进行了观察。结果表明, 添加活性炭和热激培养对胚状体诱导是必需的。在直径6 cm培养皿中培养4 mL密度为1花蕾花粉 mL -1的小孢子NLN悬液时, 每皿添加1 mg活性炭和32℃热激3 d的培养条件下子叶形胚状体和总胚状体产量最高, 分别为每花蕾0.92±0.18个和1.32±0.25个。子叶形胚状体在1/2 MS培养基上萌发率为27.73%。花粉植株中自然加倍率为25%, 加倍植株染色体数为24, 单倍体植株染色体数为12。诸葛菜单倍体减数分裂染色体的平均配对构型为n = 12 = 6.352I + 2.008II + 0.384III + 0.12IV, 具有二价体及三价体和四价体的细胞比例高达96%, 少量细胞的12条染色体联会形成3个四价体, 说明诸葛菜很可能是起源于染色体基数x = 3的同源八倍体。本试验结果对于诸葛菜新材料新品种选育和基础研究具有重要参考价值。

关键词: 诸葛菜, 小孢子培养, 胚状体, 单倍体, 减数分裂, 染色体配对

Abstract:

Orychophragmus violaceus is extremely valuable as the ornamental, vegetable, forage and oil germplasm resource. In order to develop the technique of microspore-derived embryoid induction and plant regeneration, and provide dada for the origin and evolution of the genome, the effects of the heat-shock incubation duration and the content of additional activated charcoal on embryoid yield were studied through microspore culture, and the meiotic chromosome pairing behavior of the haploid was observed by conventional squashing method in O. violaceus. The activated charcoal addition and heat shock culture were required for embryoid induction. When 4 mL microspore suspension with 1 bud per mL was incubated in a Φ6 cm petri dish at 32℃ of heat shock for three days and supplemented with 1 mg activated charcoal in each dish, the cotyledon-shaped embryoid yield and total embryoid yield were highest, which were 0.92 ± 0.18 and 1.32 ± 0.25 embryoids per bud, respectively. The germination rate of the cotyledon-shaped embryoids in 1/2MS medium was 27.73%. The natural chromosome doubling rate was 25% among the survival microspore-derived plantlets. The chromosome number of the double haploid plants and the haploid plants was 24 and 12, respectively. The meiotic chromosome pairing configuration of the haploid in O. violaceus was averaged as n = 12 = 6.352I + 2.008II + 0.384III + 0.12IV. The percentage of the pollen mother cells with bivalent, trivalent and tetravalent was up to 96%. The 12 chromosomes in 0.8% of pollen mother cells synapsed into three tetravalents. The chromosome pairing behavior strongly suggested that O. violaceus originated from a homologous octoploid with the basic chromosome number of x = 3. The above results provide a reference for breeding new materials and cultivars and for basic research in O. violaceus.

Key words: Orychophragmus violaceus, microspore culture, embryoid, haploid, meiosis, chromosome pairing

图1

诸葛菜小孢子胚胎发生过程 A: 部分小孢子膨大; B: 小孢子近均等分裂; C: 小孢子不均等分裂; D: 多细胞团; E: 具胚柄状结构的原胚; F: 球形原胚; G: 球形胚; H: 具胚柄状结构的圆球形胚状体; I: 卵球形胚状体; J: 桃心形胚状体; K: 心脏形胚状体; L: 培养28 d后的胚状体。"

图2

诸葛菜小孢子胚状体形态 A: 双子叶胚状体; B: 三子叶胚状体; C: 具1胚根状结构三子叶胚状体; D: 单子叶胚状体; E: 合生双胚; F: 鱼雷形胚状体; G: 心形胚状体; H: 球形胚状体; I: 不规则形胚状体; 标尺 = 2 mm。"

表1

不同热激时间下诸葛菜小孢子胚状体产量"

热激时间
Heat-shock duration (d)		 子叶形胚状体
Cotyledonous embryoids (No. per bud)		 总胚状体
All embryoids (No. per bud)
0 0 0
1 0.13±0.13 c 0.57±0.13 c
3 0.92±0.18 a 1.32±0.25 a
5 0.48±0.10 b 0.95±0.09 b
7 0.15±0.09 c 0.60±0.13 c

表2

不同活性炭浓度下诸葛菜小孢子胚状体产量"

活性炭浓度
Active charcoal content (mg dish-1)		 子叶形胚状体
Cotyledonous embryoids (No. per bud)		 总胚状体
All embryoids (No. per bud)
0 0 0
1 0.92±0.18 a 1.32±0.25 a
2 0.85±0.15 a 1.25±0.10 ab
3 0.37±0.10 b 0.80±0.17 b

图3

诸葛菜小孢子植株的再生、形态、育性和倍性 A: 胚状体萌发; B: 加倍的小孢子植株; C: 单倍体植株; D: 加倍和单倍体植株的花和花蕾; E: 加倍植株花粉; F: 单倍体植株花粉; G: 加倍植株花粉母细胞染色体(2n = 24); H: 单倍体植株花粉母细胞染色体(2n = 12); 标尺 = 10 μm。"

表3

诸葛菜单倍体减数分裂染色体配对构型"

序号
No.		 染色体构型 Chromosome configuration 细胞数
No. of cells		 百分比
Percentage (%)
IV III II I
1 0 0 0 12 5 4.0
2 0 0 1 10 16 12.8
3 0 0 2 8 21 16.8
4 0 0 3 6 22 17.6
5 0 0 4 4 6 4.8
6 0 0 5 2 3 2.4
7 0 0 6 0 3 2.4
8 0 1 0 9 3 2.4
9 0 1 1 7 9 7.2
10 0 1 2 5 15 12.0
11 0 1 3 3 2 1.6
12 0 2 0 6 2 1.6
13 0 2 1 4 3 2.4
14 0 2 2 2 2 1.6
15 0 2 3 0 1 0.8
16 1 0 0 8 1 0.8
17 1 0 1 6 3 2.4
18 1 0 2 4 2 1.6
19 1 0 3 2 1 0.8
20 1 0 4 0 1 0.8
21 1 1 0 5 1 0.8
22 1 1 1 3 1 0.8
23 2 1 0 1 1 0.8
24 3 0 0 0 1 0.8
合计Total 15 48 251 794 125 100.0
平均Average 0.12 0.384 2.008 6.352 — —

图4

部分诸葛菜单倍体(2n = 12)花粉母细胞减数分裂染色体配对构型(标尺 = 10 μm)"

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