CN115428730B - 一种利用山奈酚提高果树自花结实率的方法 - Google Patents
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Abstract
本发明公开了一种利用山奈酚提高果树自花结实率的方法,该方法为在进行梨属等蔷薇科植物果树自交授粉前先采用含有山奈酚的花粉培养基溶液处理花蕾的柱头。果树自交不亲和植株花蕾进入始花期,在柱头上喷洒山奈酚溶液,能不同程度地提高自交亲和性。田间实验结果表明,喷施山奈酚之后,梨自交不亲和系的坐果率由原来的4%提高到18%,花粉管的长度是原来的2倍。本发明的方法适用于梨属等蔷薇科植物自交不亲和系,能有效地提高梨属等蔷薇科植物自交亲和性。
Description
技术领域
本发明属于育种和栽培技术领域,具体涉及一种利用山奈酚提高果树自花结实率的方法。
背景技术
在自然界中,自交不亲和性是植物防止近亲繁殖、促进异交,从而保证物种多样性的一种重要生殖隔离手段。自然界约有60%的植物表现为自交不亲和性,主要分为孢子体型和配子体型两种类型,其中表现为基于S-RNase配子体型自交不亲和性占已知自交不亲和物种的一半以上,涉及到茄科、车前科、芸香科和包括梨在内的蔷薇科等。控制自交不亲和性的遗传位点是S(sterility)位点,表现为高度多态的特征。梨S位点分别编码决定其自交不亲和性的雌蕊因子S-RNase和花粉因子S-locus F-box(SLF)。S-RNase的发现是自交不亲和研究邻域的重大突破。S-RNase的细胞毒素功能主要涉及到花柱拒绝不亲和花粉的机制。当花柱细胞分泌S-RNase到花柱引导组织里,S-RNase能被生长的花粉管在不依赖S-特异性的方式吸收。随后,亲和S-RNase在被SLF蛋白识别后被26S泛素蛋白酶系统降解。而对于不亲和花粉管,S-RNase则行使它的细胞毒素功能,包括启动PCD,阻碍花粉管精子进入子房来终止受精。
在蔷薇科果树中绝大多数成员均存在自交不亲和的现象,如梨、苹果等均为自交不亲和品种。由于生殖隔离的存在,造成了其自花授粉不能坐果,只有异花授粉才能获得相应的品质和产量,这给果树生产中带来了诸多不便。因为梨等蔷薇科果树存在自交不亲和现象,所以在生产中往往要根据栽培品种的S基因型配置一定比例的授粉树;而且在果树开花期还要投入大量人工进行采粉、授粉工作,不仅增加了生产难度,同时还提高了投入成本。因此,克服自交不亲和性,是梨等果树实现可持续生产需要解决的重要问题,同样也急需寻找到一种更高效的具有广谱性的克服自交不亲和性的方法。
发明内容
为了解决背景技术提出的技术问题,本发明的目的在于提供一种利用山奈酚提高果树自花结实率的方法,克服梨等蔷薇科果树自交不亲和性,有效地降低了生产难度,同时还减少了投入成本,为梨等蔷薇科果树实现可持续生产提供了新的方法。
本发明的目的可以通过以下技术方案实现:
山奈酚在提高果树自花结实率中的应用。作为一种优选技术方案:在进行果树自交授粉前先采用含有山奈酚的花粉培养基溶液处理花蕾的柱头。
一种提高果树自花结实率试剂,该试剂为含有山奈酚的花粉培养基溶液。
一种提高果树自花结实率的方法,该方法为在进行果树自交授粉前先采用含有山奈酚的花粉培养基溶液处理花蕾的柱头。
上述的山奈酚在花粉培养基溶液中的终浓度为1~12μM,优选为1.75~10μM;进一步优选为3.5~7μM;最优选为3.5μM。
上述的果树自交授粉的过程为:(1)果树花朵进入大蕾期时,除去花瓣和雄蕊只露出花柱,将含有山奈酚的花粉培养基溶液,喷洒在花蕾的柱头上;(2)柱头干燥后进行自交授粉,授粉后统计坐果率。
进一步优选的:上述的步骤(1)中,于晴天上午9:00-11:00时间段,除去花瓣和雄蕊只露出花柱;使用喷雾器将含有山奈酚的花粉培养基溶液喷洒在花蕾的柱头上,共喷施一次,一次3-5下,每次喷到柱头上有水珠为止;上述的步骤(2)中自交授粉后套收种袋,一星期后摘袋子,授粉后20天统计坐果率。
本发明所述的果树为蔷薇科果树,包括但不限于梨树。
所述的花粉培养基为本领域技术人员公知的常规花粉培养基溶液,本发明技术方案所采用的为梨花粉培养基溶液,其配方为:5mmol·L-1MES、440mmol·L-1蔗糖、0.55mmol·L-1Ca(NO3)2、1.60mmol·L-1MgSO4、1.60mmol·L-1H3BO3、1.00mmol·L-1KNO3,溶解于蒸馏水后用NaOH调pH值至6.2。但不限于此,只要是常规配方的花粉培养基溶液均可以实现本发明的目的。
本发明利用山奈酚提高果树自花结实率方法的最优选技术方案具体包含如下步骤:
(1)提前配制浓度为1M的山奈酚母液放置于-20℃低温冰箱避光保存,母液的配制及工作液的稀释过程中需注意避光;
(2)梨花进入大蕾期,于晴天上午9:00-11:00时间段,除去花瓣和雄蕊只露出花柱,将山奈酚加入花粉培养基溶液中,山奈酚的终浓度为1~12μM,使用喷雾器,喷洒在花蕾的柱头上,共喷施一次,一次3-5下,每次喷到柱头上有水珠为止;
(3)柱头干燥后进行自交授粉,套收种袋,一星期后摘袋子,授粉后20天统计坐果率。
该方法是利用雾状喷洒将含有山奈酚的花粉培养基溶液喷洒在刚盛开的柱头上,通过降低自交时花柱S-RNase活性所造成的植物自交亲和来实现本发明提高果树自花结实率的目的。
在研究梨的自交不亲和调控机制的过程中,发现自花授粉使花柱产生高水平的S-RNase,从而抑制自花花粉的萌发。本研究发现山奈酚是一种抑制S-RNase活性的药品,使用山奈酚喷施花期柱头,可以显著降低柱头S-RNase的活性,减少对自花花粉的抑制,促进自花花粉的萌发、生长以及进入柱头并完成受精过程。本发明的方法克服自交不亲和的效果显著,且对植物伤害性小,应用范围广,可以应用到梨等蔷薇科植物,对蔷薇科作物的育种及制种具有重要的价值。
本发明的有益效果:
本发明适用于梨等蔷薇科植物。梨自交不亲和植株花蕾进入始花期,在柱头上喷洒山奈酚溶液,能不同程度地提高自交亲和性。田间实验结果表明,喷施山奈酚之后,梨自交不亲和系的坐果率由原来的4%提高到18%,花粉管的长度是原来的2倍。
附图说明
图1为活体喷施山奈酚显著促进自交不亲和梨“喜水”的花粉管的生长(A)及授粉后20d、30d坐果情况(B)。
图2为山奈酚离体处理显著降低花柱S-RNase的活性(A为“砀山酥梨”,B为“黄花”梨)。
图3为山奈酚离体处理能够促进花粉管生长;
其中,A-E分别为对照,自交亲和处理,山奈酚处理,自交不亲和处理以及山奈酚和自交不亲和共同处理“砀山酥梨”花粉的图片,F为不同处理下花粉管长度统计。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
实施例1:以自交不亲和梨“喜水”为例
(1)提前配制浓度为1M的山奈酚母液放置于-20℃低温冰箱避光保存,母液的配制及工作液的稀释过程中需注意避光;
(2)梨花进入大蕾期时,于晴天上午9:00-11:00时间段,除去花瓣和雄蕊只露出花柱,将不同终浓度的山奈酚加入梨花粉培养基溶液中(以未添加山奈酚的梨花粉培养基溶液为对照),使用喷雾器,喷洒在花蕾的柱头上,共喷施一次,一次3-5下,每次喷到柱头表面上沾有水珠为止;
所述梨花粉培养基溶液的配方为:5mmol·L-1MES、440mmol·L-1蔗糖、0.55mmol·L-1Ca(NO3)2、1.60mmol·L-1MgSO4、1.60mmol·L-1H3BO3、1.00mmol·L-1KNO3,溶解于蒸馏水后用NaOH调pH值至6.2。
(3)待柱头上雾滴全部干燥后自交授上新鲜的花粉,去除多余小蕾期花序,套上硫酸纸袋,防止其他花粉污染。7天后摘袋子进行授粉后的花柱收集并进行苯胺蓝染色,授粉20天后统计坐果率。田间实验结果(表1)表明,喷施山奈酚之后,梨自交不亲和系的坐果率由原来的4%提高到18%,同时发现花粉管的长度是原来的2倍(图1)。
表1活体喷施山奈酚提高自交不亲和梨“喜水”的坐果率统计结果(试验1)
实施例2
收集大蕾期的梨花,取下的花柱保存于-80℃备用。通过CM-Sepharose和SP-Sepharose阳离子交换层析柱从花柱中分离并纯化出S-RNase并储存于-80℃备用。S-RNase活性的测定主要参照前人的方法(张绍铃和刘友良,2001),具体步骤如下:
(1)分别向纯化后的2μg的“砀山酥梨”和“黄花”花柱S-RNase中加入山奈酚(终浓度为6mg/L)处理2h;
(2)向蛋白溶液中加入67μL含有KCl浓度为0.01mol/L的琥珀酸铵缓冲液(pH=6.5)和67μL圆酵母RNA(4mg/mL);
(3)55℃水浴锅内水浴30min,然后加入13μL的0.75%醋酸铀(0.1g/mL)/25%高氯酸(12.2mol/L)溶液;
(4)于4℃下12,000rpm离心10min;
(5)取100μL上清液加入3mL蒸馏水,与OD260 nm下测定吸光值,并换算为比活性。
通过体外山奈酚处理“砀山酥梨”和“黄花”的花柱S-RNase,结果发现花柱S-RNase的活性被山奈酚处理后显著降低(图2)。
实施例3
花粉采自开花前2d大蕾期的梨花,收集花药,室温自然散粉后放于硫酸纸袋-20℃保存备用。使用时将-20℃的花粉置于4℃中2h,25℃继续放置2h后即可进行花粉培养,液体培养基配方为:5mmol·L-1MES、440mmol·L-1蔗糖、0.55mmol·L-1Ca(NO3)2、1.60mmol·L- 1MgSO4、1.60mmol·L-1H3BO3、1.00mmol·L-1KNO3,溶解于蒸馏水后用NaOH调pH值至6.2。
为了探究山奈酚对离体花粉生长和自交不亲和的影响,进行了如下处理:
(1)将花粉浸入2mL培养基中并放入25℃摇床(~100rpm)中预培养90min;
(2)将不同的花柱S-RNase、山奈酚混合均匀,之后加入200μL花粉培养基。设置5个处理:对照处理、添加“黄花”的花柱S-RNase的终浓度为0.23mg/ml(自交亲和处理)、添加“砀山酥梨”的花柱S-RNase的终浓度为0.23mg/ml(自交不亲和处理)、添加山奈酚处理的终浓度为2mg/L以及添加山奈酚(2mg/L)和“砀山酥梨”的花柱S-RNase(0.23mg/ml)的共同处理;
(3)将处理后的花粉置于25℃摇床(~100rpm)中继续培养2h;
(4)显微镜下观察花粉管生长长度,使用配备有CCD照相机的DP80(奥林巴斯,日本)的Olympus IX73显微镜拍照,用Image-Pro Plus软件统计花粉管的长度。
结果发现山奈酚处理可以显著促进花粉的生长;而在自交不亲和处理下明显抑制花粉的生长,但山奈酚和自交不亲和共同处理下,山奈酚可以明显地缓解自交不亲和反应所引起的花粉管生长抑制(图3)。
Claims (4)
1.山奈酚在提高蔷薇科果树自花结实率中的应用,其特征在于:在进行果树自交授粉前先采用含有山奈酚的花粉培养基溶液处理花蕾的柱头;所述的山奈酚在花粉培养基溶液中的终浓度为3.5~7µM。
2.根据权利要求1所述的应用,其特征在于:所述果树自交授粉的过程为:(1)果树花朵进入大蕾期时,除去花瓣和雄蕊只露出花柱,将含有山奈酚的花粉培养基溶液,喷洒在花蕾的柱头上;(2)柱头干燥后进行自交授粉,授粉后统计坐果率。
3.一种提高蔷薇科果树自花结实率的方法,其特征在于:该方法为在进行果树自交授粉前先采用含有山奈酚的花粉培养基溶液处理花蕾的柱头;所述的山奈酚在花粉培养基溶液中的终浓度为3.5~7 µM。
4.根据权利要求3所述的方法,其特征在于:所述果树自交授粉的过程为:(1)果树进入始花期,大量盛开时,除去花瓣和雄蕊只露出花柱,将含有山奈酚的花粉培养基溶液喷洒在花蕾的柱头上;(2)柱头干燥后进行自交授粉,授粉后统计坐果率。
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US5432068A (en) * | 1990-06-12 | 1995-07-11 | Pioneer Hi-Bred International, Inc. | Control of male fertility using externally inducible promoter sequences |
US6005167A (en) * | 1991-04-16 | 1999-12-21 | Mogen International N.V. | Male-sterile plants, method for obtaining male-sterile plants and recombinant DNA for use therein |
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