Study On Polyploid And Cross Breeding Of Undaria Pinnatifida And Laminaria Japonica

The induction condition, formation process and its developing characteristics, sex differentiation and required mature time of 2n gametophyte of Undaria pinnatifida and Laminaria japonica were studied. The hybrid from 2n gametophyte and normal gametophyte was conducted and 3n and 4n young sporophytes were produced. 3n and 4n seedlings of U.pinnatifida and L.japonica were gained through temporary sea cultivation. 3n and 4n adult sporophytes of U.pinnatifida were harvested after sea cultivation. Distant crossing breeding between U.pinnatifida and L.japonica was also conducted. Reciprocal hybridization of female and male gametophyte of U.pinnatifida and L.japonica produced high quality hybrids.Polyploid breeding studies showed that optimal conditions for 2n gametophytes of U.pinnatifida and L. japonica induction were juvenile sporophytes in length of 0.1-1.0 cm incubated at 10 g·L^-1 and 20℃, 60 mol·m^-2·s^-1, 12 L:12 D in PESI medium supplied with 100 mg·L^-1NO₃-N and 20 mg·L^-1 PO4-P which renewed 50% in volumes at 15d intervals. The 2n gametophytes induction rate under optimized conditions was as high as 88.2% for U.pinnatifida and 65.5% for L. japonica in 60 days incubation. The formation process of 2n gametophytes was described as follows. Firstly, few survived cells of the juvenile sporophyte turned round and germinated club-shaped protuberance while most of cells were gradually dead. Then the protuberance developed into filament by cell divisions which eventually differentiated into female or male gametophyte during the induction. Temperatures had no difference in the effect on the sexual differentiation of 2n gametophytes(P<0.05)while the high light intensity and longer lighting hours induced more female 2n gametophytes of U.pinnatifida and L. japonica. The percentages of female and male 2n gametophytes were 28% and 72% for U.pinnatifida and 25% and 75% for L. japonica, respectively, measured after differentiation at 20℃, 100 mol·m^-2·s^-1and 12 L:12 D. There was insignificant difference in fertilization rate which were all over 92% among the self-cross in n or 2n gametophytes and 2n gametophytes crossed with n gametophytes(P<0.05).Protoplasts were isolated from 2n, 3n juvenile sporophytes of U.pinnatifida by enzymes and then were stained by Giemsa. The results showed that chromosome number of sporophytes were 2n=60 and 3n=90 which indicated that the induced gametophytes from the body cells of U.pinnatifida were diploid with a chromosome number of 60. The cells of n, 2n female gametophytes and 2n, 3n juvenile sporophytes of L. japonica were stained by Wittmann's method and observed by squashing method. The results showed that chromosome number of normal and induced gametophytes were n=22 and 2n=44 and that of 2n, 3n, 4n sporophytes were 2n=44, 3n=66 and 4n=88 which indicated that gametophytes induced from the body cells of L. japonica were diploid with a chromosome number of 44.In U.pinnatifida, after 30 days'indoor culture, juvenile sporophytes'lengths of 2n♀×n♂and n♀×2n♂grew up to about 510 m which were both longer than 470 m of 2n and 440 m of 4n. After 40 days'temporary sea cultivation, juvenile sporophytes'lengths of 2n♀×n♂and n♀×2n♂grew up to 1.3 cm and 1.2 cm, respectively, which were both longer than 1.0 cm of 2n and 0.8 cm of 4n. In L.japonica, after 30 days'indoor culture, juvenile sporophytes'lengths of 2n♀×n♂and n♀×2n♂grew up to about 570 m which were both longer than 540 m of 2n and 480 m of 4n. After 40 days'temporary sea cultivation, juvenile sporophytes'lengths of 2n♀×n♂and n♀×2n♂grew up to 2.5 cm and 2.3 cm, respectively, which were both longer than 1.5 cm of 2n and 1.1 cm of 4n.In U.pinnatifida, there was very significant difference in length and weight of sporophytes between 3n and 2n (P<0.01) while there being insignificant difference in those between 2n♀×n♂and n♀×2n♂during the period of sea cultivation after seed separation. 3n sporophytes kept a rapid growth in the first and second ten days of May as 2n sporophytes entering reproductive period. On 20th May, 2n♀×n♂and n♀×2n♂sporophytes reached 4.3 m and 2.6 kg, 4.2m and 2.5 kg in length and weight, respectively, which were much better than 2.3 m and 1.4 kg of 2n sporophytes. Compared with 2n sporophytes, 4n sporophytes grew slowly (P<0.01). 4n sporophytes reached the maximal length and weight of 1.8 m and 1.2 kg during the middle ten days of March. The hybridization experiments indicated that 3n sporophytes were of sterility and the sporophylls were not formed. 4n sporophytes were of low-fertility with thimbleful spores released from sporophylls, which were flaky and could produce little sporangia, after drying in the shade.Distant cross breeding studies showed that the fertilization rate of F₁ was 81-92% in the hybridized combination of U.pinnatifida♀×L.japonica♂.There was very significant difference in length and weight of sporophytes among F₁, U.pinnatifida and L.japonica (P<0.01) during the period of sea cultivation. The phenotypic character of F₁ sporophyte of U.pinnatifida♀×L.japonica♂was similar to that of the female parent, namely, U.pinnatifida. On 15th April, F₁ sporophytes reached 3.4 m and 2.1 kg which were much better than 2.4 m and 1.5 kg of U.pinnatifida which showed heterosis in length and weight growth. The sporophylls of F₁ sporophytes were gradually developed in reproductive period. In L.japonica♀×U.pinnatifida♂, the fertilization rate of F₁ was 20-33%. F₁ sporophytes reached 2.3 cm which were much longer than 1.6 cm of L. japonica and 1.3 cm of U.pinnatifida which showed heterosis in growth during the temporarily sea cultivaton. The phenotypic character of F₁ juvenile sporophyte of L.japonica♀×U.pinnatifida♂was similar to that of the female parent, namely, L.japonica which generated no rudiments of the pinnate lobations. The chromosome number of parents and F₁ sporophytes were 2n=60 of U.pinnatifida, 2n=44 of L.japonica and 2n=52 of F₁ of both U.pinnatifida♀×L.japonica♂and L.japonica♀×U.pinnatifida♂.