【作者】 李晓丽；

【导师】 董双林； 张泽宇；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2008， 博士

【摘要】 本文主要研究了裙带菜、海带2n配子体的诱导条件、产生过程、及其发育特点、性分化、成熟时间等,并进行了2n配子体间及与正常配子体的杂交,培育出3n、4n幼孢子体,通过海区暂养,得到裙带菜、海带3n、4n幼苗;通过海区栽培,得到裙带菜3n、4n成熟藻体。并且进行了裙带菜、海带远缘杂交育种的研究,以裙带菜、海带为亲本,进行了正反杂交,选育出具有杂种优势的远缘杂交组合。多倍体试验结果表明:藻体长度为0.1-1.0 cm的裙带菜、海带幼孢子体切除固着器后在添加了100 mg·L^-1NO₃-N、20 mg·L^-1 PO4-P的PESI溶液中,在20℃、60 mol·m^-2·s^-1、12 L:12 D,培养密度为10 g·L^-1,每15d更换50%培养液的条件下,大部分营养细胞逐渐死亡,部分存活细胞变圆在细胞的一端或两端产生棒状突起,经细胞分裂后形成丝状体,丝状体进一步分化成雌雄配子体,培养60d后裙带菜2n配子体的诱导率达到88.2%,海带2n配子体的诱导率达到65.5%。温度对裙带菜、海带2n配子体性分化影响不显著（P<0.05）;光照强度、光照时数的增加有利于2n雌配子体的分化;在20℃、100 mol·m^-2·s^-1、12 L:12 D下,裙带菜2n配子体雌雄比例分别为28%和72%,海带的为25%和75%。裙带菜、海带2n雌雄配子体自交及与单倍n雌雄配子体杂交的受精率都达到92%以上,与单倍n雌雄配子体自交差异不明显（P<0.05）。裙带菜2n、3n幼苗经酶解为原生质团后,通过Giemsa染色观察到的染色体数分别为2n=60,3n=90,表明体细胞诱导产生的裙带菜配子体为2n配子体,染色体数为2n=60;海带n雌配子体、2n雌配子体及2n、3n、4n海带幼孢子体经Wittmann法染色,压片法观察到的染色体数分别为n=22,2n=44及2n=44,3n=66,4n=88,说明体细胞诱导产生的海带配子体为2n配子体,染色体数为2n=44。经30d室内培养后,裙带菜2n♀×n♂、n♀×2n♂两组合3n幼孢子体长度为510 m左右,大于n♀×n♂组合2n幼孢子体的470 m和2n♀×2n♂组合4n幼孢子体的440 m;经40d海区暂养后,3n幼孢子体的两个组合藻体长度分别达到1.3 cm和1.2 cm,已经明显地大于2n幼孢子体的1.0 cm,4n幼孢子体的藻体长度也增长至0.8 cm。经30d室内培养后,海带2n♀×n♂、n♀×2n♂两组合3n幼孢子体长度达到570 m左右,大于n♀×n♂组合2n幼孢子体的540 m和2n♀×2n♂组合4n幼孢子体的480 m;经40d海区暂养后,3n幼孢子体的两个组合藻体长度分别达到2.5 cm和2.3 cm,已经明显地大于2n幼孢子体的1.5 cm,4n幼孢子体的藻体长度也增长至1.1 cm。分苗后海区栽培期间,在藻体的长度和重量方面,裙带菜2n♀×n♂、n♀×2n♂两个3n孢子体组合间差异不显著（P>0.05）,与对照组合2n孢子体之间的差异极显著（P<0.01）。在裙带菜进入繁殖期的5月上中旬,3n孢子体仍保持较快的生长速度,在5月20日测定两个组合的藻体长度和重量分别达到4.3 m、2.6kg和4.2 m、2.5kg,而对照组合2n孢子体仅达到2.3 m和1.4 kg。4n孢子体生长明显地慢于对照的2n孢子体（P<0.01）,藻体生长最大值的时间为3月中旬,最大藻体长度和重量仅为1.8 m和1.2 kg。裙带菜3n孢子体不发育,在繁殖期内无孢子叶形成,表现出不育的特性;4n孢子体形成孢子叶,但孢子叶很薄,孢子囊形成的数量少,发育水平也很低,经阴干刺激仅有极少量的孢子放出,显示出低育的特性。远缘杂交试验研究结果表明:裙带菜♀×海带♂杂交组合的受精率为81-92%,F₁孢子体表现出母本裙带菜性状,在海区栽培期间,在藻体长度和重量方面,与对照组合裙带菜、海带孢子体差异极显著（P<0.01）。在4月15日测定,F₁孢子体的长度和重量达到3.4 m、2.1 kg,大于裙带菜的2.4 m、1.5 kg（P<0.01）,表现出生长优势;进入繁殖期后,F₁孢子体正常形成孢子叶。海带♀×裙带菜♂杂交组合的受精率为20-33%,在暂养期间F₁幼孢子体达到2.2 cm,大于海带、裙带菜幼孢子体的1.8 cm和1.1 cm,表现出生长优势;F₁幼孢子体表现出母本海带的性状,无裂叶缺刻雏形的形成。对各杂交组合的亲本及子代的染色体鉴定得出:裙带菜2n=60,海带2n=44,二者正反杂交F₁子代都为2n=52。更多还原

【Abstract】 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♂.更多还原