CN116508647A - 一种引进外源种属的甘蔗杂交方法 - Google Patents
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Abstract
本发明涉及农业技术领域,特别涉及一种引进外源种属的甘蔗杂交方法。包括以下步骤:(1)以斑茅为母本、割手密为父本,杂交获得斑割杂种;(2)以从步骤(1)获得的斑割杂种鉴定得到的斑割真杂种株系为父本、甘蔗为母本,杂交获得含斑茅、割手密血缘的甘蔗杂种;(3)从步骤(2)获得的甘蔗杂种经鉴定后获得甘蔗真杂种。通过本发明提供的引进外源种属的甘蔗杂交方法获得的甘蔗品种能够提升甘蔗品种农艺性、抗性、蔗糖分等综合性水平,同时克服杂交不亲和的问题,提高杂交结实率。
Description
技术领域
本发明涉及农业技术领域,特别涉及一种引进外源种属的甘蔗杂交方法。
背景技术
甘蔗种质资源是甘蔗育种的基础,甘蔗种质改良和突破性育种的巨大成就主要依赖于种质资源的发掘利用。甘蔗有性杂交育种的100 多年历史实质上是对甘蔗种质资源开发和利用的过程。育种家利用甘蔗属(Saccharum) 内的2 ~ 5 个种(species) 进行“种间杂交”和“高贵化育种”,创造了甘蔗育种史的前半个世纪的辉煌。但是尽管具有丰富的甘蔗种质资源,而优良种质及其利用研究贫乏,甘蔗有性杂交育种大多数是品种间杂交,这些品种绝大多数只限于热带种、印度种和割手密种三个种血缘范围内,其后代亲缘关系密切,遗传血缘贫乏、狭窄,大大制约着甘蔗育种的遗传进展,致使现有品种在产量、糖分、抗性上没有明显的突破。因此,必须利用甘蔗种质资源创新种质,拓宽栽培品种的遗传基础,选育具有突破性的优良亲本和品种。
甘蔗野生种质具有抗逆性强、适应性广、宿根性强等许多优良特性,因此充分利用甘蔗近缘野生种质拓宽遗传基础,就有可能在甘蔗育种基因库增加新的有益基因,提高育成品种的综合性状水平,使甘蔗育种获得新的突破。其中斑茅与割手密被普遍认为最有育种价值的甘蔗近缘野生种质资源。
斑茅是甘蔗近缘属植物之一,因其具有的抗早耐瘠,耐涝,升势旺盛、宿根性好,适应性广泛,抗病、抗冲特性等优良性状,可作为改良甘蔗品种的亲本加以利用。而割手密为复杂多倍体甘蔗属植物被普通认为是现代栽培甘蔗的抗逆型基因种源。近年来,国内外均有报道利用斑茅与割手密杂交获得斑割复合体,再与甘蔗进行杂交获得农艺性状、蔗糖分、抗性等俱佳的优良育种新材料,提高了甘蔗品种的综合性状水平、使斑茅和割手密优异基因资源的发掘利用获得重要进展。但在杂交过程中,由于亲本花粉质量不高、杂交不亲和等原因从而导致杂交成功率低、杂交结实率低等问题的出现。
发明内容
本发明的目的在于提供一种引进外源种属的甘蔗杂交方法,克服亲本花粉质量不高、杂交不亲和、杂交成功率低、杂交结实率低的缺点。
为实现上述目的,本发明提供了一种引进外源种属的甘蔗杂交方法,包括以下步骤:
(1)以斑茅为母本、割手密为父本,杂交获得斑割杂种;
(2)以从步骤(1)获得的斑割杂种经鉴定得到的斑割真杂种株系为父本、甘蔗为母本,杂交获得含斑茅、割手密血缘的甘蔗杂种;
(3)从步骤(2)获得的甘蔗杂种经鉴定获得甘蔗真杂种;
父母本的确定是在斑茅、割手密开花期采集花粉置于载玻片上,加I-KI溶液染色,用玻棒捣碎花药,在显微镜下检测花粉育性,结果发现割手密花粉比斑茅花粉发育好,因此在斑割杂交时选择斑茅为母本,割手密为父本;因斑割复合体花粉可育情况良好,故将其作为父本与甘蔗进行杂交利用,而且也有研究表明起始亲本以甘蔗为母本比较容易获得杂交真实后代杂种。
所述步骤(1)、步骤(2)中母本去雄后第二天在第一次杂交授粉前15-20min向母本柱头喷施处理液;所述处理液中各组分按重量百分比为N,N'-二甲基硫脲0.01-0.05%、复硝酚钠0.15-0.3%、硼钼合剂0.03-0.05%,余量为无菌水。进一步的,所述处理液按重量百分比计由以下组分组成:N,N'-二甲基硫脲0.03%、复硝酚钠0.22%、硼钼合剂0.04%,余量为无菌水。母本去雄是在母本雄蕊开花期用医用镊子将雄蕊去除后继续用隔离布套住,有效预防自交;处理液中的N,N'-二甲基硫脲是自由基清除剂,能够清除父母本杂交后产生的高浓度活性氧,有效预防杂交不亲和;硼钼合剂含有的硼元素、钼元素都利于花粉在母本柱头上萌发、生长,同时钼、硼相互促进,相互协同能够进一步促进花粉管进入柱头并完成受精过程从而促进花粉受精;复硝酚钠是广谱型植物生长调节剂同时也是一种强力细胞赋活剂,复硝酚钠与母本柱头接触后能迅速渗透到子房体内,提高胚珠卵细胞的活力,当花粉管长到胚珠柄并进入株孔时,由于卵细胞的活力提高进一步降低了柱头对花粉特异性的识别,使卵细胞与精子能够顺利结合,完成受精,提高杂交成功率。
进一步的,上述引进外源种属的甘蔗杂交方法中, 所述步骤(1)、步骤(2)中的父母本用隔离布罩住在温室大棚中用培养土进行盆栽培养,所述培养土组分按重量计为松果10-15%、硼酸粉0.1-1%,腐熟的猪厩肥5-10%、海藻粉0.2-1%、微硅粉0.05-0.2%、葡萄糖粉1-5%、吲哚丁酸0.08-0.2%,余量为红土壤。在土壤中添加松果,土壤透气透水性好,减少烂根,同时松果能慢慢释放大量矿物质如钙、铁、磷、钾等,能给机体组织提供丰富的营养成分,促进植物跟叶的生长,但是天然采摘的松果比较大,需要将松果捣烂后再使用;海藻粉中的多糖、昆布、植物激素类物质、微生物和微量元素可促进农作物代谢,定向促进开花并提高花粉质量,并为杂交后的种子成熟提供其所必须的微量元素,提高杂交结实率;培养土中还含有硼元素、硅元素、葡萄糖粉以及促根剂吲哚丁酸,都利于促进植物吸收。
进一步的,上述的引进外源种属的甘蔗杂交方法中,所述温室大棚的环境温度为20-28℃,相对湿度为75-95%。采用温室大棚控制亲本培养的环境温度,更有利于亲本繁殖开花。
进一步的,上述的引进外源种属的甘蔗杂交方法中,所述步骤(2)和(3)中鉴定指进行SSR分子标记鉴定。对杂交后代真实性的鉴定,有植物学法、同工酶法、分子标记法等,其中分子标记最为准确,它可从DNA水平上获得最直接的证据。而SSR标记是基于PCR的分子标记,具有简便、快速、精准的特点,在遗传分析中广泛应用。对经形态学方法初步鉴定具有斑茅、割手密形态特征的材料,采用SSR进行鉴定,筛选出两对父本扩增出现特异带的引物,对杂交后的杂种进行扩增,引物结果相互验证鉴定,经比对可知,扩增条带,或来自母本或来自父本,或亲本后代共有,所以在出现父本的特异条带的材料,就可以确定是斑茅割手密杂交的真实后代。
进一步的,上述的引进外源种属的甘蔗杂交方法中,所述杂交授粉为:在母本开花期,收集父本花粉,用毛笔蘸取父本花粉,轻轻点到母本处理后的柱头上,立刻套上隔离布;父本授粉完成后搬离温室大棚,母本继续在温室大棚中培育。
进一步的,上述的引进外源种属的甘蔗杂交方法,所述杂交授粉时间为在母本开花期每天上午7:30-8:30和9:30-10:30进行2次授粉,连续授粉7-10天。
进一步的,上述的引进外源种属的甘蔗杂交方法中,所述母本在授粉结束后7-10天取下隔离布,并加强管理直至种子成熟。
与现有的技术相比,本发明具有如下有益效果:
本发明的引进外源种属的甘蔗杂交方法,在杂交授粉前对母本柱头喷施处理液,处理液中的二甲基硫脲是高效活性氧清除剂,有效预防杂交不亲和;硼钼合剂中含有的钼、硼相互促进,相互协同,能够促进花粉管生长,进入柱头并完成受精;复硝酚钠是广谱型植物生长调节剂同时也是一种强力细胞赋活剂,能够提高胚珠卵细胞的活力,降低了柱头对花粉特异性的识别,使卵细胞与精子能够顺利结合,完成受精,提高杂交成功率。该处理液能够有效解决杂交不亲和、提高杂交成功率,进一步提高杂交结实率。
本发明的引进外源种属的甘蔗杂交方法,父母本用本发明提供的培养土进行温室盆栽培养,能够提高促进父母本快速生长,提高开花率,同时提高花粉质量,进一步提高杂交成功率。
说明书附图
图1为本发明实施例1中的育种流程图。
实施方式
下面对本发明的具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。
实施例1
本实施例提供一种引进外源种属的甘蔗杂交方法,如图1所示,包括以下步骤:
(1)以斑茅GXA87-36为母本、割手密GXS79-9为父本,杂交获得斑割杂种;
(2)以从步骤(1)获得的斑割杂种鉴定得到的斑割真杂种株系为父本、桂糖02-761为母本,杂交获得含斑茅、割手密血缘的甘蔗杂种;
(3)从步骤(2)获得的甘蔗杂种经鉴定后获得甘蔗真杂种;
其中步骤(1)中、步骤(2)的母本(斑茅GXA87-36、桂糖02-761)去雄后第二天在第一次杂交授粉前15-20min向母本柱头喷施处理液;处理液中各组分按重量百分比为N,N'-二甲基硫脲0.03%、复硝酚钠0.22%、硼钼合剂0.04%,余量为无菌水。
其中步骤(1)、步骤(2)中的父母本用隔离布罩住在环境温度为20-28℃,相对湿度为75-95%的温室大棚中用培养土进行盆栽培养,培养土组分按重量计为松果12.5%、硼酸粉0.6%,腐熟的猪厩肥7.5%、海藻粉0.6%、微硅粉0.12%、葡萄糖粉2.5%、吲哚丁酸0.12%,余量为红土壤。
SSR分子标记鉴定方法为:提取步骤(1)制得的杂种基因组DNA作为模板,在BiometraPCR扩增仪上进行反应扩增,反应总体积为25μL,每管含10×PCRbuffer2.5μL、30ng/L模板DNA1L、0.24μmol/LdNTPs、0.6μmol/L引物、2.5mmol/LMg2+、Taq DNA聚合酶1.5U。扩增产物经6%的聚丙烯酰胺凝胶电泳分离检测扩增产物,电游结束后直接在PharmaciaBiotech多功能凝胶成像系统观察杂种特征带,通过与父母本特征带对比,在后代扩增出来的特征带或是来自母本,或是来自父本,且出现父本特异条带的样,则可判定为含双亲血缘的真杂种。
在母本开花期每天上午8:00和10:00进行2次授粉,连续授粉9天。杂交授粉的方法为先收集父本花粉,然后用毛笔蘸取父本花粉,轻轻点到母本处理后的柱头上,立刻套上隔离布;父本授粉完成后搬离温室大棚,母本继续在温室大棚中培育;在母本授粉结束后8天取下母本上的隔离布,并加强管理直至种子成熟。
实施例2
本实施例区别于实施例1之处在于处理液中各组分按重量百分比为N,N'-二甲基硫脲0.01%、复硝酚钠0.15%、硼钼合剂0.05%,余量为无菌水。其余步骤与实施例1相同。
实施例3
本实施例区别于实施例1之处在于处理液中各组分按重量百分比为N,N'-二甲基硫脲0.05%、复硝酚钠0.3%、硼钼合剂0.03%,余量为无菌水。其余步骤与实施例1相同。
通过实验可知实施例1-3中开花率高达98%,花粉活力高,斑割杂交成功率高达79%以上,斑割真杂种与甘蔗杂交成功率也在75%以上,甘蔗真杂种花穗发芽百分达95%。
1、培养土对亲本开花率、花粉质量的影响
对比例1
本对比例与实施例1杂交方式相同、柱头处理液相同,不同在于培养土组分按重量计为膨胀珍珠岩12.5%、复合肥3.94%,腐熟的猪厩肥7.5%,余量为红土壤。
对比例2
本对比例与实施例1杂交方式相同、柱头处理液相同,不同在于培养土组分仅为红土壤。
实施例1、对比例1-2分别培育斑茅、割手密、甘蔗各100株,记录开花株数,其结果见表1。
表1为斑茅、割手密、甘蔗的开花株数
由上表可知,用实施例1的培养土培育的亲本,其开花株率达98%。
采集各组分中开花最好的斑茅、割手密、甘蔗花粉,用氯化三苯基四氮唑(TTC)法测定花粉活力,结果表明用实施例1的培养土培育的亲本,斑茅、割手密、甘蔗的花粉染色是最深的,表明用实施例1培养土培养亲本,亲本花粉的活力最好,说明本发明的培养土能够提高花粉质量。
2、处理液对杂交成功率的影响
对比列3-6中杂交方法与实施例1相同,培养土组分含量与实施例1相同,不同在于处理液组分含量不同。实施例1、对比例3-6中处理液组分含量见表2,实施例1、对比例3-6进行斑茅、割手密杂交获得斑割杂种,斑割杂种按照常规方法进行实生苗播种并取斑割杂种实生苗100株通过实施例1中SSR分子标记鉴定方法鉴定斑割真杂种并记录斑割真杂种株数,取斑割真杂种50株与甘蔗杂交后得到含斑茅、割手密血缘的甘蔗杂种,甘蔗杂种按照常规方法进行实生苗播种取甘蔗杂种实生苗50株通过实施例1中的SSR分子标记鉴定甘蔗真杂种,并记录甘蔗真杂种株数,结果见表3。
表2为各组别中柱头处理液的组分含量
表3为各组别杂交后斑割真杂种、甘蔗真杂种数量
由上表可知,实施例1中斑割杂交成功率达80%,斑割真杂种与甘蔗杂交成功率也达78%,而对比例6中斑割杂交成功率仅为51%,斑割真杂种与甘蔗杂交成功率为42%,由此可知在杂交前通过对母本柱头喷施处理液,大大提高杂交成功率和杂交结实率。从对比例3-5的斑割真杂种、甘蔗真杂种数量可知,处理液中缺少了其中的一种或几种,其杂交成功率和杂交结实率比实施例1的低。由此可知,本发明提供的柱头处理液能够有效解决杂交不亲和,获得真杂种的数量大大增加,提高杂交成功率。
3、处理液对杂交结实率的影响
取实施例1和对比例6培育出的甘蔗真杂种后代10株,待种子成熟后,剪下花穗,记录10株花穗总数,对花穗进行常规培育后记录发芽花穗数,结果见表4。
表4为实施例1、对比例6的花穗总数,发芽花穗数
由上表可知,实施例1的发芽花穗量、发芽百分比远高于对比例6,说明本发明提供的柱头处理液能提高种子结实率。
综上可知,本发明提供的引进外源种属的甘蔗杂交方法,通过对杂交亲本在温室大棚中进行盆栽培育,并且对培养土组分加以改进,能够提高亲本开花率,同时提高花粉质量;在杂交授粉前对母本柱头喷施处理液能够有效解决杂交不亲和、提高杂交成功率,进一步提高杂交结实率。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
Claims (9)
1.一种引进外源种属的甘蔗杂交方法,其特征在于,包括以下步骤:
(1)以斑茅为母本、割手密为父本,杂交获得斑割杂种;
(2)以从步骤(1)获得的斑割杂种鉴定得到的斑割真杂种株系为父本、甘蔗为母本,杂交获得含斑茅、割手密血缘的甘蔗杂种;
(3)从步骤(2)获得的甘蔗杂种经鉴定获得甘蔗真杂种;
所述步骤(1)、步骤(2)中在杂交前将母本去雄,母本去雄后在杂交授粉前5-10min向母本柱头喷施处理液;所述处理液按重量百分比计由以下组分组成:N,N'-二甲基硫脲0.01-0.05%、复硝酚钠0.15-0.3%、硼钼合剂0.03-0.05%,余量为无菌水。
2.根据权利要求1所述的引进外源种属的甘蔗杂交方法,其特征在于,所述处理液按重量百分比计由以下组分组成:N,N'-二甲基硫脲0.03%、复硝酚钠0.22%、硼钼合剂0.04%,余量为无菌水。
3.根据权利要求1所述的引进外源种属的甘蔗杂交方法,其特征在于,所述步骤(1)、步骤(2)中的父母本用隔离布罩住在温室大棚中用培养土进行盆栽培养,所述培养土组分按重量百分比计为松果10-15%、硼酸粉0.1-1%,腐熟的猪厩肥5-10%、海藻粉0.2-1%、微硅粉0.05-0.2%、葡萄糖粉1-5%、吲哚丁酸0.08-0.2%,余量为红土壤。
4.根据权利要求3所述的引进外源种属的甘蔗杂交方法,其特征在于,所述温室大棚的环境温度为20-28℃,相对湿度为75-95%。
5.根据权利要求1所述的引进外源种属的甘蔗杂交方法,其特征在于,所述鉴定是采用SSR分子标记鉴定。
6.根据权利要求1所述的引进外源种属的甘蔗杂交方法,其特征在于,所述杂交授粉为:在母本开花期,收集父本花粉,用毛笔蘸取父本花粉,轻轻点到母本处理后的柱头上,立刻套上隔离布;父本授粉完成后搬离温室大棚,母本继续在温室大棚中培育。
7.根据权利要求1所述的引进外源种属的甘蔗杂交方法,其特征在于,所述杂交授粉时间为在母本开花期每天上午7:30-8:30和9:30-10:30进行2次授粉。
8.根据权利要求1所述的引进外源种属的甘蔗杂交方法,其特征在于,所述杂交授粉为连续授粉7-10d。
9.根据权利要求6所述的引进外源种属的甘蔗杂交方法,其特征在于,所述母本在授粉结束后7-10d取下隔离布,并加强管理直至种子成熟。
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CN102792884A (zh) * | 2012-08-21 | 2012-11-28 | 广州甘蔗糖业研究所 | 室内甘蔗杂交授粉方法 |
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CN115280933A (zh) * | 2022-07-05 | 2022-11-04 | 云南省农业科学院甘蔗研究所 | 一种甘蔗单芽种子制作方法 |
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