鸭梨自交不亲和与亲和变异的生理生化特性及分子机理研究
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摘要
梨是配子体自交不亲和果树,生产中必须配置适宜授粉品种或通过人工授粉才能保证产量。自交亲和品种在梨果生产中具有很大优势,20世纪80年代日本已将其作为果树育种的重要任务,且已从“二十世纪(Nijisseiki,基因型S_2S_4)”中选出自交亲和性栽培种“奥嘎二十世纪(Osa-Nijisseiki,基因型S_2S_4~(SM))”,并对其进行了深入的研究。鸭梨是我国白梨系统代表品种,已从中选出金坠梨、魏县阎庄自花结实鸭梨等自交亲和变异,但对其自交亲和机理的研究还未开展,这极大限制了我国梨自交不亲和性研究和自交亲和品种选育工作的开展,因此急需对其亲和机理进行深入研究。
本研究以鸭梨、金坠梨、魏县阎庄自花结实鸭梨为试材,研究了它们的自交不亲和强度、与自交不亲和相关的生理生化机制、自交亲和变异的分子机理及自交不亲和的调控技术,主要结果如下。
1.通过对17个品种自花授粉试验、授粉花柱半离体培养后观察从花柱基部伸出花粉管的数量和花柱比率,来鉴定品种的自交不亲和强度。确定了自交不亲和强度弱的品种5个,即为金坠梨、魏县阎庄白花结实鸭梨、爱宕梨、绿宝石、新雪;中等的4个,分别为喜水、八月红、秦丰、二十世纪;自交不亲和强度强的8个,分别为幸水、丰水、新兴、博斯克、鸭梨、黄金梨、帕克胜利、雪花梨。
2.在生理生化机制方面,发现POD与自交坐果率呈极显著负相关关系,与花序坐果率和花朵坐果率的相关系数分别达到-0.935和-0.888;SOD、CAT、MDA与自交坐果率无显著相关关系。鸭梨、雪花梨自花和异花授粉后,花柱SOD、POD、CAT活性、MDA含量变化均呈单峰曲线,SOD、POD、MDA在授粉后24h达到高峰,CAT活性在授粉后48h达到高峰;在授粉后同一时期,花柱SOD、CAT活性异花授粉高于自花授粉,而POD活性、MDA含量异花授粉低于自花授粉。
3.激素与自交不亲和性关系方面,鸭梨的自交亲和变异金坠梨授粉前花柱中促进生长的激素IAA、GA_3、ZR含量均极显著高于鸭梨,而ABA含量极显著低于鸭梨。2品种自花授粉后72h内,花柱内IAA、ZR含量呈下降趋势;在授粉后24h时内GA_3含量下降,然后上升;金坠梨花柱ABA含量授粉后24h内上升,然后下降;鸭梨花柱ABA含量在白花授粉24h内处于下降趋势,然后上升;鸭梨异花授粉24h后花柱内IAA、GA_3、ZR含量与金坠梨自花授粉的相近。2个品种自花、异花授粉后IAA氧化酶活性变化与花柱IAA含量变化趋势相反;鸭梨异花授粉后花柱IAA氧化酶活性在同一时间低于白花授粉;金坠梨白花授粉后花柱IAA氧化酶活性一直低于鸭梨,且白花授粉24h以后与鸭梨异花授粉花柱IAA氧化酶活性接近。
4.花柱提取液对各品种花粉萌发和花粉管生长均有一定程度抑制作用;随着培养基中添加花柱提取液浓度的提高,花粉萌芽率降低,花粉萌发受抑制程度提高;花粉管生长受抑制程度加剧。鸭梨花柱提取液对鸭梨、魏县阎庄自花结实鸭梨花粉萌发和花粉管生长的抑制程度大于金坠梨;金坠梨花柱提取液只对魏县阎庄自花结实鸭梨花粉萌发和花粉管生长的抑制作用较大,而对金坠梨、鸭梨花粉萌发的抑制作用较小;培养基中添加魏县自花结实鸭梨花柱提取液,对鸭梨和金坠梨花粉萌发和花粉管生长的抑制作用较大,而对魏县自花结实鸭梨花粉萌发和花粉管生长的抑制作用较小。
Pear belongs to the kind of gametophytic self-incompatibility, so it is necessary to arrange varieties for pollination or perform artificial pollination for stable yield. Self-compatible cultivars have many advantages in pear production, so from 20 century 80th, it became the main task of fruit tree breeding of Japanese, and had selected one self-compatible cultivar Osa- Nijisseiki(genotype S_2S_4SM) from Nijisseiki i(genotype S2S4), and studied it thoroughly. Yali pear is the representative of Pyrus bretschneideri Rehd. of China, and two self-compatible cultivars that Jinzhuili pear and Weixianyanzhuangzihuajieshiyali pear were selected from Yali pear, but their self-compatible mechanisms were unknown, this restricted the researches of self-incompatibility of Chinese pear and breeding self-compatible cultivars, so it is necessary to study the mechanism of self-compatible.In this thesis self-incompatible strength, physiological and biochemical characteristics related to self-incompatibility, molecular mechanism of self-compatible and adjustment methods of self incompatibility of Yali were studied. The results were as follows.1. Self incompatible strength of 17 pear cultivars were identified by self pollination tests and observation of the amount of pollen tubes from style bottoms and percentage of style with pollen tubes through their bottoms in semi vitro culture of self pollination styles. Weak SI cultivars were Jinzhui pear, Weixianyanzhuangzihuahieshiyali pear, Atago, Lvbaoshi and Shinsetsu; intermediate SI cultivars were Kisui, Bayuehong, Qinfeng and Nijisseiki; strong SI cultivars were Kohsui, Housui, Shinko, Beurre Bosc, Yali pear, Whangkeumbae, Pakeshengli and Xuehuali pear.2. Physiological and biochemical characteristics aspects, there exited very significance negative correlation between POD and self pollination fruit set percentage, the correlation coefficients among POD and fruit set percentage of inflorescence and fruit set percentage of flower were -0.935and -0.888; their hadn't significance correlations among SOD、 CAT、 MDA and fruit set percentage. SOD、 POD、 CAT activities and MDA content changes in styles of Yali pear and Xuehuali pear after self- and cross-pollination were
single apex curves, SOD, POD, MDA reached the peak after pollination in 24h, and CAT activity reached the peak after pollination in 48h. At the same time after pollination, SOD, CAT activities in styles by cross-pollination were higher than self-pollination, but POD activity and MDA content in styles by cross-pollination were lower than self-pollination.3. About hormone and self-incompatibility, endogenous hormones contents of IAA, GA3, ZR that promoted growth in styles before pollination of Jinzhui pear were significantly higher than that in Yali pear. ABA content was significantly lower than that in Yali pear.In the period of 72h after self-pollination, IAA, ZR contents in styles of the two cultivars tended to decline. During 24h after pollination, GA3 contents in styles declined, then rose. ABA content in styles of Jinzhui pear rose at 24h after pollinated, and then declined. During 24h after pollination, ABA content in styles of Yali pear tended to decline, then rose. Changes of endogenous hormones contents in Yali pear styles after cross-pollination were dissimilar from self-pollination, the contents of IAA, GA3, ZR in styles approached the contents of Jinzhui pear after self-pollination. Changes of IAA oxidase activities in styles of the two cultivars after self and cross-pollination opposed to that of IAA contents; at the same time, IAA oxidase activities after self-pollination of Yali pear were lower than that of cross-pollination; IAA oxidase activities in Jnzhuili pear styles after self-pollination were always lower than that of Yali pear, and 24h after self-pollination, it approached the activity of Jinzhui pear after self-pollination.4. There exited inhibition of style extract on pear pollen germination and pollen tube growth, and with the style extract concentration raising, the pollen germination percentage declining, inhibition degree of pollen germination and pollen tube growth raising. The inhibition degree of Yali pear style extract to pear pollen germination and pollen tube growth of Yali pear and Weixianyanzhuangzihuahieshiyali pear was higher than that of Jinzhui pear. The inhibition of Jinzhui pear style extract to Weixianyanzhuangzihuahieshiyali pear was higher than that of Jinzhui pear and Yali pear. The inhibition of Weixianyanzhuangzihuahieshiyali pear style extract to Yali pear and Jinzhuili pear was higher than that of Weixianyanzhuangzihuahieshiyali pear.5. Physiological and biochemical characteristics related to self-incompatibility of Yali pear were as follows. IOD and POD activities which has IOD activity in styles before pollination were higher, endogenous hormones contents of IAA, GA3, ZR that promoted growth were lower, but ABA content was higher; the inhibitions of self-incompatible style extract on its pollen germination and pollen tube growth were strong; but the changes of styles endogenous hormones contents and protected enzymes of self-compatible cultivars after self pollination were similar to that of Yali pear after cross compatible pollination, and the inhibitions of self-compatible style extract on its pollen germination and pollen tube growth were weak.6. S gene PCR amplification of Yali pear and its self-compatible mutations that were
Weixianyanzhuangzihuajieshiyali pear and Jinzhuili pear were detected by 8% PAGE, the results showed that there were two binds at about 370bp of Yali pear and Jinzhuili pear, but there was only one bind of Weixianyanzhuangzihuajieshiyali pear, indicated that there was one S gene mutated of Weixianyanzhuangzihuajieshiyali pear, then resulted in self-compatible; more analysis indicated that Yali pear contained SI gene, and SI gene of Weixianyanzhuangzihuajieshiyali pear was normal, its self-compatible molecular mechanism was another S gene mutated. The self-compatible molecular mechanism of Jinzhuili pear might be the modifying gene controlled S gene expression mutated.7. Effects of polyamine on pollen germination and pollen tube growth of Yali pear varied from the concentration of polyamine, the concentrations of spermine that promoted pollen germination and pollen tube growth of Yali pear were 0.005-0.01 mM; that the concentrations of spermidine that promoted pollen germination of Yali pear were 0.01-0.25 mM, at the tested range of spermidine concentrations, pollen tube length of Yali pear was longer than that of control significantly or very significantly; the concentrations of putrescine that promoted pollen germination and pollen tube growth of Yali pear were 0.01-0.5 mM. When the concentration of MGBG was in 0.05 mM, it promoted pollen germination of Yali pear, and other concentrations inhibited pollen germination; when the concentrations of MGBG below 0.25mM, it promoted pollen tube growth of Yali pear very significantly, when it exceeded 0.25mM, it inhibited pollen tube growth of Yali pear.8. Sprayed polyamine in a concentrations range of polyamine enhanced self pollination fruit set of Yali pear very significantly. Semi vitro culture of pollinated styles indicated that there exited very significance of the amount of pollen tubes from style bottoms and percentage of style with pollen tubes through their bottoms by different concentrations of polyamine, the numbers of pollen tubes from style bottoms and percentages of style with pollen tubes through their bottoms treated by 0.0 lmM Spm, 0.05mM Spd and Put were the best. The result showed that polyamine could break self-incompatibility of Yali pear at some extent.9. In a concentrations range of salicylic acid promoted pollen germination and pollen tubes growth, in the range of tested salicylic acid concentrations, percentages of pollen germination of Yali pear were higher than control significantly or very significantly, the best was 0.005 mM, with the concentrations raising, percentages of pollen germination declined rapidly; the suitable SA concentration that promoted pollen tubes growth was 0.005mM, when exceeded this concentration, the promotion effect of SA on pollen tube growth was weakened sharply. Sprayed 0.005-0.025 mM SA, there had lower fruit set percentage of inflorescence and fruit set percentage of flower; the number of pollen tubes from style bottoms and percentage of style with pollen tubes through their bottoms treated by 0.005mM SA was the best. But the fruit set was very low, and couldn't fill the yield, so considered that SA couldn't break self-incompatibility of Yali pear.10. There exited very significance differences of the number of pollen tubes from
本研究以鸭梨、金坠梨、魏县阎庄自花结实鸭梨为试材,研究了它们的自交不亲和强度、与自交不亲和相关的生理生化机制、自交亲和变异的分子机理及自交不亲和的调控技术,主要结果如下。
1.通过对17个品种自花授粉试验、授粉花柱半离体培养后观察从花柱基部伸出花粉管的数量和花柱比率,来鉴定品种的自交不亲和强度。确定了自交不亲和强度弱的品种5个,即为金坠梨、魏县阎庄白花结实鸭梨、爱宕梨、绿宝石、新雪;中等的4个,分别为喜水、八月红、秦丰、二十世纪;自交不亲和强度强的8个,分别为幸水、丰水、新兴、博斯克、鸭梨、黄金梨、帕克胜利、雪花梨。
2.在生理生化机制方面,发现POD与自交坐果率呈极显著负相关关系,与花序坐果率和花朵坐果率的相关系数分别达到-0.935和-0.888;SOD、CAT、MDA与自交坐果率无显著相关关系。鸭梨、雪花梨自花和异花授粉后,花柱SOD、POD、CAT活性、MDA含量变化均呈单峰曲线,SOD、POD、MDA在授粉后24h达到高峰,CAT活性在授粉后48h达到高峰;在授粉后同一时期,花柱SOD、CAT活性异花授粉高于自花授粉,而POD活性、MDA含量异花授粉低于自花授粉。
3.激素与自交不亲和性关系方面,鸭梨的自交亲和变异金坠梨授粉前花柱中促进生长的激素IAA、GA_3、ZR含量均极显著高于鸭梨,而ABA含量极显著低于鸭梨。2品种自花授粉后72h内,花柱内IAA、ZR含量呈下降趋势;在授粉后24h时内GA_3含量下降,然后上升;金坠梨花柱ABA含量授粉后24h内上升,然后下降;鸭梨花柱ABA含量在白花授粉24h内处于下降趋势,然后上升;鸭梨异花授粉24h后花柱内IAA、GA_3、ZR含量与金坠梨自花授粉的相近。2个品种自花、异花授粉后IAA氧化酶活性变化与花柱IAA含量变化趋势相反;鸭梨异花授粉后花柱IAA氧化酶活性在同一时间低于白花授粉;金坠梨白花授粉后花柱IAA氧化酶活性一直低于鸭梨,且白花授粉24h以后与鸭梨异花授粉花柱IAA氧化酶活性接近。
4.花柱提取液对各品种花粉萌发和花粉管生长均有一定程度抑制作用;随着培养基中添加花柱提取液浓度的提高,花粉萌芽率降低,花粉萌发受抑制程度提高;花粉管生长受抑制程度加剧。鸭梨花柱提取液对鸭梨、魏县阎庄自花结实鸭梨花粉萌发和花粉管生长的抑制程度大于金坠梨;金坠梨花柱提取液只对魏县阎庄自花结实鸭梨花粉萌发和花粉管生长的抑制作用较大,而对金坠梨、鸭梨花粉萌发的抑制作用较小;培养基中添加魏县自花结实鸭梨花柱提取液,对鸭梨和金坠梨花粉萌发和花粉管生长的抑制作用较大,而对魏县自花结实鸭梨花粉萌发和花粉管生长的抑制作用较小。
Pear belongs to the kind of gametophytic self-incompatibility, so it is necessary to arrange varieties for pollination or perform artificial pollination for stable yield. Self-compatible cultivars have many advantages in pear production, so from 20 century 80th, it became the main task of fruit tree breeding of Japanese, and had selected one self-compatible cultivar Osa- Nijisseiki(genotype S_2S_4SM) from Nijisseiki i(genotype S2S4), and studied it thoroughly. Yali pear is the representative of Pyrus bretschneideri Rehd. of China, and two self-compatible cultivars that Jinzhuili pear and Weixianyanzhuangzihuajieshiyali pear were selected from Yali pear, but their self-compatible mechanisms were unknown, this restricted the researches of self-incompatibility of Chinese pear and breeding self-compatible cultivars, so it is necessary to study the mechanism of self-compatible.In this thesis self-incompatible strength, physiological and biochemical characteristics related to self-incompatibility, molecular mechanism of self-compatible and adjustment methods of self incompatibility of Yali were studied. The results were as follows.1. Self incompatible strength of 17 pear cultivars were identified by self pollination tests and observation of the amount of pollen tubes from style bottoms and percentage of style with pollen tubes through their bottoms in semi vitro culture of self pollination styles. Weak SI cultivars were Jinzhui pear, Weixianyanzhuangzihuahieshiyali pear, Atago, Lvbaoshi and Shinsetsu; intermediate SI cultivars were Kisui, Bayuehong, Qinfeng and Nijisseiki; strong SI cultivars were Kohsui, Housui, Shinko, Beurre Bosc, Yali pear, Whangkeumbae, Pakeshengli and Xuehuali pear.2. Physiological and biochemical characteristics aspects, there exited very significance negative correlation between POD and self pollination fruit set percentage, the correlation coefficients among POD and fruit set percentage of inflorescence and fruit set percentage of flower were -0.935and -0.888; their hadn't significance correlations among SOD、 CAT、 MDA and fruit set percentage. SOD、 POD、 CAT activities and MDA content changes in styles of Yali pear and Xuehuali pear after self- and cross-pollination were
single apex curves, SOD, POD, MDA reached the peak after pollination in 24h, and CAT activity reached the peak after pollination in 48h. At the same time after pollination, SOD, CAT activities in styles by cross-pollination were higher than self-pollination, but POD activity and MDA content in styles by cross-pollination were lower than self-pollination.3. About hormone and self-incompatibility, endogenous hormones contents of IAA, GA3, ZR that promoted growth in styles before pollination of Jinzhui pear were significantly higher than that in Yali pear. ABA content was significantly lower than that in Yali pear.In the period of 72h after self-pollination, IAA, ZR contents in styles of the two cultivars tended to decline. During 24h after pollination, GA3 contents in styles declined, then rose. ABA content in styles of Jinzhui pear rose at 24h after pollinated, and then declined. During 24h after pollination, ABA content in styles of Yali pear tended to decline, then rose. Changes of endogenous hormones contents in Yali pear styles after cross-pollination were dissimilar from self-pollination, the contents of IAA, GA3, ZR in styles approached the contents of Jinzhui pear after self-pollination. Changes of IAA oxidase activities in styles of the two cultivars after self and cross-pollination opposed to that of IAA contents; at the same time, IAA oxidase activities after self-pollination of Yali pear were lower than that of cross-pollination; IAA oxidase activities in Jnzhuili pear styles after self-pollination were always lower than that of Yali pear, and 24h after self-pollination, it approached the activity of Jinzhui pear after self-pollination.4. There exited inhibition of style extract on pear pollen germination and pollen tube growth, and with the style extract concentration raising, the pollen germination percentage declining, inhibition degree of pollen germination and pollen tube growth raising. The inhibition degree of Yali pear style extract to pear pollen germination and pollen tube growth of Yali pear and Weixianyanzhuangzihuahieshiyali pear was higher than that of Jinzhui pear. The inhibition of Jinzhui pear style extract to Weixianyanzhuangzihuahieshiyali pear was higher than that of Jinzhui pear and Yali pear. The inhibition of Weixianyanzhuangzihuahieshiyali pear style extract to Yali pear and Jinzhuili pear was higher than that of Weixianyanzhuangzihuahieshiyali pear.5. Physiological and biochemical characteristics related to self-incompatibility of Yali pear were as follows. IOD and POD activities which has IOD activity in styles before pollination were higher, endogenous hormones contents of IAA, GA3, ZR that promoted growth were lower, but ABA content was higher; the inhibitions of self-incompatible style extract on its pollen germination and pollen tube growth were strong; but the changes of styles endogenous hormones contents and protected enzymes of self-compatible cultivars after self pollination were similar to that of Yali pear after cross compatible pollination, and the inhibitions of self-compatible style extract on its pollen germination and pollen tube growth were weak.6. S gene PCR amplification of Yali pear and its self-compatible mutations that were
Weixianyanzhuangzihuajieshiyali pear and Jinzhuili pear were detected by 8% PAGE, the results showed that there were two binds at about 370bp of Yali pear and Jinzhuili pear, but there was only one bind of Weixianyanzhuangzihuajieshiyali pear, indicated that there was one S gene mutated of Weixianyanzhuangzihuajieshiyali pear, then resulted in self-compatible; more analysis indicated that Yali pear contained SI gene, and SI gene of Weixianyanzhuangzihuajieshiyali pear was normal, its self-compatible molecular mechanism was another S gene mutated. The self-compatible molecular mechanism of Jinzhuili pear might be the modifying gene controlled S gene expression mutated.7. Effects of polyamine on pollen germination and pollen tube growth of Yali pear varied from the concentration of polyamine, the concentrations of spermine that promoted pollen germination and pollen tube growth of Yali pear were 0.005-0.01 mM; that the concentrations of spermidine that promoted pollen germination of Yali pear were 0.01-0.25 mM, at the tested range of spermidine concentrations, pollen tube length of Yali pear was longer than that of control significantly or very significantly; the concentrations of putrescine that promoted pollen germination and pollen tube growth of Yali pear were 0.01-0.5 mM. When the concentration of MGBG was in 0.05 mM, it promoted pollen germination of Yali pear, and other concentrations inhibited pollen germination; when the concentrations of MGBG below 0.25mM, it promoted pollen tube growth of Yali pear very significantly, when it exceeded 0.25mM, it inhibited pollen tube growth of Yali pear.8. Sprayed polyamine in a concentrations range of polyamine enhanced self pollination fruit set of Yali pear very significantly. Semi vitro culture of pollinated styles indicated that there exited very significance of the amount of pollen tubes from style bottoms and percentage of style with pollen tubes through their bottoms by different concentrations of polyamine, the numbers of pollen tubes from style bottoms and percentages of style with pollen tubes through their bottoms treated by 0.0 lmM Spm, 0.05mM Spd and Put were the best. The result showed that polyamine could break self-incompatibility of Yali pear at some extent.9. In a concentrations range of salicylic acid promoted pollen germination and pollen tubes growth, in the range of tested salicylic acid concentrations, percentages of pollen germination of Yali pear were higher than control significantly or very significantly, the best was 0.005 mM, with the concentrations raising, percentages of pollen germination declined rapidly; the suitable SA concentration that promoted pollen tubes growth was 0.005mM, when exceeded this concentration, the promotion effect of SA on pollen tube growth was weakened sharply. Sprayed 0.005-0.025 mM SA, there had lower fruit set percentage of inflorescence and fruit set percentage of flower; the number of pollen tubes from style bottoms and percentage of style with pollen tubes through their bottoms treated by 0.005mM SA was the best. But the fruit set was very low, and couldn't fill the yield, so considered that SA couldn't break self-incompatibility of Yali pear.10. There exited very significance differences of the number of pollen tubes from
引文
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