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Research Article

Mitochondrial-targeted Expression of orf456 Causes Male Sterility in Chinese Cabbage (Brassica rapa L.)

Li Liu1, Yeong Deuk Jo1, Won-Hee Kang1, Dosun Kim2, Byoung-Cheorl Kang1,*
Plant Breeding and Biotechnology 2013;1(2):196-204.
Published online: June 30, 2013

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*Corresponding author: Byoung-Cheorl Kang, bk54@snu.ac.kr, Tel: +82-2-880-4563, Fax: +82-2-873-2056
• Received: June 20, 2013   • Revised: June 24, 2013   • Accepted: June 27, 2013

Copyright © 2013 The Korean Society of Breeding Science

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  • Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea
    Caitao Chang, Deling Sun, Kana Hondo, Fumika Kakihara, A.‐M. Chevre
    Plant Breeding.2014; 133(5): 620.     CrossRef

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Mitochondrial-targeted Expression of orf456 Causes Male Sterility in Chinese Cabbage (Brassica rapa L.)
Plant Breed. Biotech.. 2013;1(2):196-204.   Published online June 30, 2013
DOI: https://doi.org/10.9787/PBB.2013.1.2.196
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Mitochondrial-targeted Expression of orf456 Causes Male Sterility in Chinese Cabbage (Brassica rapa L.)
Plant Breed. Biotech.. 2013;1(2):196-204.   Published online June 30, 2013
DOI: https://doi.org/10.9787/PBB.2013.1.2.196
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Mitochondrial-targeted Expression of orf456 Causes Male Sterility in Chinese Cabbage (Brassica rapa L.)
Image Image Image Image
Fig. 1 Orf456 chimeric gene construct and transformation in B.rapa. a) Schematic diagram illustrating the construction and ligation of orf456 and coxIV into plant transformation vector, pMDC32, driven by the 35S promoter. b) Polymerase chain reaction (PCR) analysis of chimeric gene genetically modified situation in T0 individual transgenic B. rapa plants. Actin was used as internal control to probe Chinese cabbage gDNA quality. c) Polymerase chain reaction (PCR) analysis of chimeric gene genetically modified situation in T1 individual transgenic B. rapa plants. HPT was hygromycin B, an antibiotic produced by the bacterium Streptomyces hygroscopicus. d) RNA and RT-PCR analysis of T1 individual transgenic B. rapa lines.
Fig. 2 Phenotypes of wild-type and orf456 transformants. The morphological characters were observed in transgenic plants in which the chimeric orf456 gene was expressed. Results suggested that orf456 expression may have functioned in the various reproductive growth performance of transgenic B. rapa lines. a) Transgenic T1 B. rapa plants, b) buds, c) blossoms, d) siliques, e) pods, f) seeds.
Fig. 3 Pollen viability tests. a) The plasmolysis test was performed to probe the pollen. In hypertonic (25% glycerol) solution, pollen from orf456 transformants B. rapa burst within 10–15 min, while the wild-type pollen grains remained intact even after 24 hr. b) Staining test to determine pollen enzymatic activity and membrane integrity. c) In vitro germination assays determine the actual germination ability of pollen under suitable conditions. After 4 hrs of in vitro germination, pollen from wild-type B. rapa were able to produce pollen tubes which did not occur in transgenic B. rapa.
Fig. 4 Analyses of pollen abortion phenotypes in the CMS lines. a) Three T0 lines (1, 6, and 10) showed semi-sterile phenotype, and higher ratio than other lines. This classification was based on malformed pollen morphology. b) Analysis result of seeds and siliques of wild-type and transgenic T1 B. rapa plants.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Mitochondrial-targeted Expression of orf456 Causes Male Sterility in Chinese Cabbage (Brassica rapa L.)

Analysis of malformed pollens ratio of T1 B. rapa plants.

Pollen
Blossom Normal blossom Malformed blossom
Brassica rapa Normal blossome Malformed blossom Malformed blossom ratio (%) Normal pollen Malformed pollen Malformed pollen ratio (%) Normal pollen Malformed pollen Malformed pollen ratio (%)
WT 25 0 — 824 8 0.96 915 7 0.76
1-5 17 6 26.09 424 49 10.36 417 711 63.03
1-7 22 4 15.38 589 72 10.89 885 199 18.36
1-8 32 1 3.03 394 46 10.45 — — —
1-9 22 7 24.14 533 218 29.03 682 243 26.27
1-11 25 0 — 325 187 36.52 — — —
2-3 25 0 — 814 62 7.08 — — —
3-4 20 4 16.67 731 213 22.56 79 725 90.17
3-11 31 4 11.42 230 349 60.28 6 1291 99.54
3-12 6 1 14.28 229 152 39.89 — — —
3-15 12 4 25 574 78 11.96 328 517 61.18
3-16 79 4 4.82 417 57 12.03 5 500 99
4-1 25 0 — 661 46 6.51 — — —
4-4 25 0 — 703 57 7.5 — — —
4-8 25 0 — 718 49 6.39 — — —
4-9 25 0 — 897 42 4.47 — — —
6-1 25 0 — 719 34 4.52 — — —
6-7 25 0 — 974 29 2.89 — — —
6-8 25 0 — 485 40 7.61 — — —
6-9 12 9 42.86 636 212 25 355 335 48.55
6-10 25 0 — 712 12 1.66 — — —
7-1 12 1 7.69 921 55 5.64 — — —
7-2 13 1 7.14 894 210 19.02 — — —
7-3 25 0 — 702 27 3.7 — — —
7-4 47 13 21.67 528 265 33.42 246 87 26.13
7-6 25 0 — 582 192 24.81 — — —
10-5 25 0 — 592 45 7.06 — — —
10-6 25 0 — 482 72 12.99 — — —
10-7 25 0 — 633 48 7.05 — — —
10-8 9 3 25 732 108 12.86 312 270 46.39
10-9 23 1 4.17 505 23 4.36 — — —
Table 1 Analysis of malformed pollens ratio of T1 B. rapa plants.

Table 1

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