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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (02): 194-203.doi: 10.3724/SP.J.1006.2020.94065

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2020-02-12 Published: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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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

Fig. 1

Microspore embryogenesis of Orychophragmus violaceus A: some swelled microspores; B: microsporal equal cell division; C: microsporal unequal cell division; D: multicell cluster; E: proembryo with a suspensor-like structure; F: globular proembryo; G: globular embryo; H: round sphere embryoid with a suspensor-like structure; I: oval sphere embryoid; J: heart embryoid; K: cordate embryoid; L: embryoids after 28 days of culture."

Fig. 2

Morphology of the microspore-derived embryoids of O. violaceus A: dicotyledonous embryoid; B: tricotyledonous embryoid; C: tricotyledonous embryoid with an additional radicle; D: monocotyledonous embryoid; E: twin dicotyledonous embryoids; F: torpedo-shaped embryoids; G: heart-shaped embryoids; H: globular embryoids; I: anomalous embryoids; Bar = 2 mm."

Table 1

Microspore-derived embryoids yield of O. violaceus under different heat-shock duration"

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

Table 2

Microspore-derived embryoids yield of O. violaceus under different active charcoal contents"

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

Fig. 3

Regeneration, morphology, fertility, and ploidy of the microspore-derived plantlets in O. violaceus A: germination of embryoids; B: doubled microspore-derived plantlet; C: haploid plantlet; D: flowers and buds of double haploid and haploid plantlets; E: pollens of double haploid plantlet; F: pollens of haploid plantlet; G: chromosomes in the pollen mother cell of doubled plantlet (2n = 24); H: chromosomes in the pollen mother cell of haploid plantlet(2n = 12); bar = 10 μm."

Table 3

Meiotic chromosome pairing in haploid of O. violaceus"

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 — —

Fig. 4

Some meiotic chromosome (2n = 12) pairing configurations in pollen mother cells of the haploid of O. violaceus (Bar = 10 μm) A: 12I; B: 1II+10I; C: 6II; D: 1III+3II+3I; E: 1IV+4II; F: 1IV+1III+1II+3I; G: 2IV+1III+1I; H: 3IV."

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