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Plant Breeding Institute, Faculty of Agriculture, Hokkaido University, Sapporo, 060 Japan
It is known that the transfer of male-sterile cytoplasm is accelerated by the asymmetric cell-fusion procedure called "donor-recipient method" (Aviv et al. 1984).
Protoplasts isolated from gamma irradiated suspension cells of A-58CMS were electrofused with those from Kitaake after metabolic inhibition with iodoacetoamide. R1 plants were regenerated from the fused products following the usual procedures (Kyozuka et al. 1987).
In R1 plants, a considerable variation in self fertility was produced reflecting pollen sterility, as shown in Table 1. The constitution of nuclear genomes was determined from the expression of morphological and physiological markers. All of completely sterile cybrids possessed the four kinds of plasmids (minicircular DNAs) in their mitochondria, while fertile plants showed the presence of all four
Table 1. Variation of self fertilities in the cybrids between Fujiminori
(nucier parent) and A-58 CMS (cytoplasmic donor)
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Seed fertility
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Nuclear genome Highly Partially Sterile Total
fertile fertile (0%) Unknown
(over 70%) (40-70%)
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Fujiminori type 20(2) 8(2) 24(4) 9(3) 61(11)
A-58 CMS type 0 0 2 0 2
Total 20(2) 8(2) 26 9(3) 63(11)
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Plasmids in B1,B2,B3,B4 all B1,B2,
mitochondria or all absent B3,B4
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* Parentheses mean a number of tetraploid-like plants.
Table 2. Fluctuation of self fertilities during the asexual propagation of a
complete sterile R1 plant
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Seed fertility (%) Mean
Clone* ================================================ Total (%)
0 10 20 30 40 50 60 70 80 90 100
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A 1 1 7 1 10 74+/-10
E 11 2 13 0
C 16 1 2 1 1 21 54+/-19
D 8 2 1 11 52+/-17
E 3 3 1 4 1 1 13 52+/-13
F 18 18 0
G 16 16 0
H 17 17 0
Total 89 2 4 5 5 3 9 2 119
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Divided into eight clones from one sterile plant.
Table 3. Fluctuation of self fertilities in the later sterge of R1 plants
and R2 progenies
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Seed fertility(R1) Mean fertility (R2)
Line ======================== ===========================
Early Late* Pollen Seed
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Cy-141 3(%) 10.3(%) 97.0(%) 72.6(%)
Cy-3A 5 14.1 63.6
Cy-3B 5 14.1 75.2
Cy-6B 5 9.2 69.3 78.7
Cy-4H 37 34.1 - 86.0
Cy-4M 25 34.1 - 68.3
Cy-16A 22 35.7 79.3 -
Cy-16B 27 35.7 95.3 75.7
Cy-16C 21 35.7 93.3 73.8
Cy-14L - 78.7 86.0 80.4
Cy-14M 40 78.7 - 69.0
Cy-44H 75 78.6 93.3 78.6
Cy-44L 22 78.6 82.0 88.2
Cy-44M 50 78.6 88.7 85.8
Cy-48 43 75.9 85.3 78.0
Cy-35 53 83.4 87.3 88.8
Cy-102 56 70.6 96.7 71.1
Cy-62 66 76.0 84.7 58.3
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*Surveyed at one year after regeneration.
plasmids or the absence of all fours.
It is noted that a completely sterile plant produced several clones showing segregation of fertile plants during asexual propagation by division (Table 2). It is probable that sorting out of the sterile elements occurred in the heteroplasmic state (Izhar et al. 1983). Further, the seed fertilities slightly increased one year after regeneration, and the sexual propagation of R1 plants contributed to a considerable increase of pollen and seed fertilities (Table 3). In R2 generation, most of the partially fertile R1 plants restored normal fertility. It seems that sexual reproduction is effective for the restoration of a homoplasmic state for fertile elements.
According to our preliminary observation, most of the cybrids possessed a novel constitution of mitochondrial genome, and the genomic constitution of completely sterile plants resembled that of A-58 CMS. Thus the transfer of male sterile cytoplasm due to cell fusion can be effectively used for rice breeding.
References
Aviv, V., P. Arzee-Gonen, S. Bleichman and E. Galun, 1984. Novel alloplasmic Nicotiana plants by "donor-recipient" protoplast fusion: cybrids having N. tabacum or N. sylvestris nuclear genomes and chondriomes from alien species. Mol. Gen. Genet. 196: 244-253.
Izhar, S., M. Schlicter and D. Swartzberg, 1983. Sorting out of cytoplasmic elements in somatic hybrids of petunia and prevalence of the heteroplasmon through several meiotic cycles. Mol. Gen. Genet. 190: 468-474.
Kyozuka, J., Y. E. Hayashi and K. Shimamoto, 1987. High frequency plant regeneration from rice protoplasts by novel nurse culture method. Mol. Gen. Genet. 206: 408-413.
