<graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="0496-3490-39-2247/img_1.png"/>小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

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陈新民, 王凤菊, 李思敏, 张文祥. 小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性 . 作物学报, 2013, 39(12): 2247-2252[CHEN Xin-Min, WANG Feng-Ju, LI Si-Min, ZHANG Wen-Xiang. Stable Production of Wheat Haploid and Doubled Haploid by Wheat × Maize Cross. Acta Agronomica Sinica, 2013, 39(12): 2247-2252] 复制到剪切板

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小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

陈新民^*, 王凤菊, 李思敏, 张文祥

中国农业科学院作物科学研究所, 北京 100081

^* 通讯作者(Corresponding author): 陈新民, E-mail:chenxinmin@caas.cn

收稿日期:2013-07-26

基金:本研究由国家科技支撑计划项目(2011BAD35B03)和引进国际先进农业科学技术计划(948计划)项目(2011-G3)资助。

摘要

小麦与玉米杂交是诱导小麦单倍体最有效的途径之一, 但单倍体和双单倍体产生频率不稳定影响了该技术的应用。选用13个小麦杂种F₁代单交组合与玉米杂交, 研究了不同小麦生长环境、生长素处理、培养基和壮苗处理对单倍体及双单倍体产生频率的影响。小麦生长在大田, 去雄后割穗培养与玉米杂交平均得胚率为23.9%, 每个杂交穗平均得胚数6.8个, 均是返青后从大田移回冷温室盆栽的3倍以上; 不同小麦杂交组合间胚产生频率存在明显差异。生长素Dicamba蘸穗处理平均得胚率是21.5%, 与2,4-D处理得胚率(21.1%)无显著差异, 但不同杂交组合间差异显著。B₅培养基幼胚萌发率为70.9%~88.3%, 平均82.0%; 1/2 MS培养基胚萌发率为70.0%~86.0%, 平均76.6%; 两种培养基平均胚萌发率无显著差异。试管苗经壮苗培养基壮苗处理与试管苗经移栽壮苗处理后加倍效率分别是67.6%和8.6%。移栽壮苗处理的苗分蘖少, 生长较弱, 加倍处理后存活率低和加倍率低是其单倍体加倍效率低的原因。

关键词: 小麦×; 玉米; 单倍体; 激素处理; 培养基

Stable Production of Wheat Haploid and Doubled Haploid by Wheat × Maize Cross

CHEN Xin-Min^*, WANG Feng-Ju, LI Si-Min, ZHANG Wen-Xiang

Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Abstract

One of the most effective ways to produce wheat haploid is the method of crossing wheat with maize. However, the problem of unstable frequencies in haploid and doubled haploid production affects its application. In this study, 13 wheat F₁ hybrids crossed with maize to explore the effects of wheat growth condition, hormone treatment, culture medium, and strong seedling treatment on production of haploid and doubled haploid. The frequency of embryo formation was 23.9% and the number of embryos per spike was 6.8 for the wheat plants grown in filed and then detached before pollination and hydroponically cultured after pollination. The efficiency of this method was three times higher than that of the method with plants transplanted into pot after green recovering in early spring and then grown in the unheated greenhouse condition. Embryo formation frequencies were obviously different among wheat crosses. There was no significant difference in the frequency of embryo formation between treatments of dipping spike with Dicamba (21.5%) and 2,4-D (21.1%). However, wheat cross had significant effect on embryo formation. The mean frequency of embryo germination on B₅ medium was 82.0%, ranging from 70.9% to 88.3%, whereas averaged frequency of embryo germination was 76.6% on 1/2 MS medium, ranging from 70.0% to 86.0%. There was no significant difference between the two culture media in embryo germination. The efficiency of doubled haploid production for the seedlings treated with culture medium and transplanted into pot was 67.6% and 8.6%, respectively. Most of seedlings transplanted into pot had no tiller and grew weakly; this is the reason for low frequency of survival plant and very low frequency of doubling after colchicine treatment.

Keyword: Wheat ×; maize cross; Haploid; Hormone treatment; Culture medium

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小麦与玉米杂交, 因玉米染色体与小麦纺锤体的不亲和性, 杂合子在最初的几次细胞分裂中会使玉米染色体全部丢失, 产生仅有小麦21条染色体的单倍体, 经过幼胚培养获得单倍体苗, 再经染色体加倍, 就可获得纯合双单倍体DH (doubled haploid)^{[ 1, 2]}。用这种方法产生单倍体有3个优点: 第一, 由于玉米对小麦可交配 Kr基因的不敏感, 杂交得胚率远高于小麦×球茎大麦^{[ 3, 4]}; 第二, 单倍体的产生对于小麦基因型无严格的选择性, 不同基因型的小麦均能获得单倍体^{[ 2, 5, 6, 7]}, 特别是花药培养不能产生愈伤组织或植株的小麦基因型, 在小麦×玉米中具有较高的胚和植株产生率^{[ 8]}, 这一点优于小麦花药培养; 第三, 无白化苗产生, 无体细胞变异, 较易得到稳定的纯合单倍体, 这对于遗传研究有重要价值。单倍体育种可缩短育种年限, 提高育种效率。小麦与玉米杂交, 在杂交的当年就可获得纯合双单倍体, 再经一个生长季的种子繁殖就可参加产量鉴定试验, 比常规杂交育种可缩短3~4年, 特别对于一年一季的冬小麦尤为重要; DH群体还是遗传研究和QTL定位研究的永久群体。因此, 该技术是产生小麦单倍体最有效的途径。自Laurie等^{[ 9]}首次获得小麦×玉米单倍体植株后, 国内外许多学者均进行了有关研究并应用于育种实践^{[ 10, 11]}。国外利用这种方法已培育许多小麦品种在生产上应用。如在加拿大, 从1997年开始利用DH育种技术, 至2011年已审定小麦品种25个, 其中23个是应用小麦×玉米DH技术培育的, 2009年DH品种种植面积占全国小麦面积的1/3^{[ 12]}。我们从1992年开始小麦×玉米产生小麦单倍体和双单倍体的技术研究, 虽然在国内率先建立了利用小麦与玉米杂交产生小麦双单倍体技术体系, 并利用该技术首先培育了小麦新品种^{[ 13]}, 但是由于实验条件和一些技术方面的问题, 单倍体胚和植株产生频率以及单倍体加倍效率还不稳定, 在育种实际应用中存在一些问题。

本文旨在研究小麦生长环境条件、不同激素处理和不同培养基对小麦×玉米产生单倍体的影响及不同壮苗处理方法对单倍体加倍的效果, 为利用该技术稳定、高频率产生DH提供参考。

1 材料与方法

1.1 植物材料

2012年试验所用冬小麦杂种F₁代13个组合(表1)均由中国农业科学院作物科学研究所小麦品质育种课题组组配, 玉米杂交种CRC1引自加拿大农业与农业食品部谷物研究中心(Cereal Research Centre, Agriculture and Agri- Food Canada)。

表1 不同生长条件下小麦×玉米得胚率 Table 1 Embryo Formation by wheat × maize method under different growing conditions

1.2 去雄授粉

玉米分期种植在温室, 小麦种植在大田。为了延长杂交时间, 在返青后将一部分组合从田间移回冷温室盆栽, 抽穗后选择开花前一天的小麦穗子去雄授粉。大田材料采用割穗培养法, 即于去雄次日9:00至10:00从基部剪穗, 在实验室中(20~23℃)授玉米花粉, 授粉后插入1/2 MS (含大量和微量元素)+8 mL L^-1亚硫酸+20 g L^-1蔗糖营养液, 并于16:00至17:00放入生长室, 培养条件为: 温度18~20℃, 相对湿度60%~70%, 光周期16 h光/8 h暗。授粉后1 d, 用100 mg L^-1的2,4-D溶液蘸穗处理杂交穗一次; 授粉2 d后, 第二次蘸穗处理。每隔2 d更换一次培养液。对于盆栽材料, 去雄后第二天上午移至实验室授粉, 16:00至17:00将授粉后的材料放入生长室(生长条件同上)。授粉后1 d用100 mg L^-1的2,4-D溶液蘸穗处理杂交穗一次, 授粉2 d后, 第二次蘸穗处理。

比较了2,4-D和Dicamba两种激素的效果, 其浓度均为100 mg L^-1, 处理方法一致。

1.3 胚培养

授粉后14~16 d, 剥取籽粒, 在超净工作台上先用70%酒精表面灭菌30 s, 再用0.1%升汞灭菌10 min, 经无菌水充分冲洗3~4次后, 于解剖镜下剥出幼胚, 分别用1/2 MS培养基(含蔗糖2%, 琼脂7.5 g L^-1)和B₅培养基(含蔗糖2%, 琼脂10 g L^-1)培养。接种后先在4℃冰箱中(黑暗)处理2~3 d, 然后放在室温黑暗条件下2 d, 最后在20~23℃、光周期16 h光/8 h暗条件下培养。

1.4 壮苗及春化处理

当幼苗生长到3~5 cm时, 将一部分幼苗转入壮苗培养基(1/2 MS+多效唑0.5 mg L^-1+ IAA 0.2 mg L^-1), 在11~15℃, 每日16 h光照条件下培养2周; 另一部分幼苗移栽至小花盆, 在相同温度、光照和60%湿度条件下生长2周, 每周施用适量稀释16倍的营养液(N∶P∶K= 20∶20∶20) 3次。当幼苗产生1~3个分蘖时, 在2~6℃春化处理1个月(光照16 h d^-1), 再于壮苗生长环境下生长3~4 d, 最后进行加倍处理。

1.5 加倍处理

移出幼苗并剪掉根(留1~2 cm), 用0.2%秋水仙素溶液+DMSO (每10 mL加1滴), 在室温、黑暗、通气条件下处理3~4 h, 用自来水冲洗3~4 h, 然后移栽至壮苗生长环境下缓苗2周, 再逐渐升温, 抽穗后维持在18~22℃, 直到收获。抽穗时将所有植株的穗及时套上羊皮纸袋以防异交, 收获时调查结实率, 凡结实者为加倍成功者。

1.6 统计分析

对不同激素处理试验, 随机将每3个杂交穗混合, 为一个重复, 统计得胚率, 共3个重复, 数据经反正弦转化后再行统计分析。对不同培养基试验, 每个组合每种培养基6个重复, 每重复接种6~10个胚(因为不同组合的得胚率有差异), 按照随机区组试验统计分析。加倍处理效率(%)=结实植株数/加倍处理植株数×100。

2 结果与分析

2.1 不同生长条件对得胚率的影响

在返青时将小麦移栽至冷温室花盆, 这样比大田早抽穗7 d左右, 可多一周时间与玉米杂交。但盆栽与割穗培养两种条件下, 与玉米杂交得胚率存在明显差异(表1)。盆栽6个组合得胚率在5.8%~10.4%间, 平均7.8%; 每个杂交穗得胚1.0~2.5个, 平均1.8个。而割穗培养7个组合得胚率为12.5%~29.6%, 平均23.9%; 每个杂交穗得胚3.6~8.5个, 平均6.8个。小麦生长在大田, 去雄后割穗培养与玉米杂交, 平均得胚率和每个杂交穗平均得胚数均是盆栽的3倍以上。不同小麦杂交组合间胚产生频率存在明显差异。

2.2 不同激素处理对得胚率的影响

Dicamba和2,4-D处理对小麦与玉米杂交诱导单倍体胚频率无显著影响(表2)。Dicamba处理4个组合得胚率为16.2%~27.8%, 平均21.5%; 2,4-D处理胚产生率为6.0%~28.3%, 平均21.1%。但是Dicamba处理平均膨大颖果率(82.4%)显著高于2,4-D处理(58.5%)。不同杂交组合间胚产生频率存在显著差异, 以CA0998/CA0816组合最高(28.1%), 与CA0996/中麦175 组合 (24.2%) 差异不显著, 显著高于轮选987/CA0477 (20.5%)和CA0415/CA0391 (12.4%)组合。

表2 不同激素处理效应 Table 2 Effect of different hormone treatments

2.3 不同培养基对幼胚萌发率的影响

B5培养基平均胚萌发率是82.0%, 变幅70.9%~88.3%; 1/2 MS培养基胚萌发率在70.0%~86.0%间, 平均76.6%; 统计分析表明两种培养基平均胚萌发率无显著差异(表3)。不同杂交组合间平均胚萌发率差异显著, CA0998/CA0816组合萌发率最高 (87.2%), 显著高于组合扬麦16/扬麦15 (73.2%)和轮选987/CA0477 (73.3%), 与组合CA0415/CA0391 (79.2%)、CA0996/中麦175 (81.9%)和扬麦16/中麦895 (81.3%)差异不显著。

表3 不同培养基胚培养效果 Table 3 Effect of different culture media on embryo culture

2.4 不同壮苗处理方法对单倍体加倍效率的影响

壮苗培养基培养的幼苗经秋水仙素浸根加倍处理后, 存活率和加倍效率分别是92.0%和67.6%, 而经移栽壮苗处理的幼苗加倍处理后存活率和加倍效率分别是46.2%和8.6%, 前者分别是后者的2.0倍和7.9倍(表4)。试管苗经移栽壮苗处理后分蘖少, 生长较弱, 经加倍处理后不仅存活率低, 而且存活者加倍率低, 这是单倍体加倍效率低的原因。

表4 不同壮苗处理方法加倍效果 Table 4 Effect of different strong seedling treatments on doubling

3 讨论

小麦与玉米杂交诱导小麦单倍体后仅有极少部分胚有胚乳,而且胚乳总是异常并退化, 不能提供胚生长发育所需要的营养物质^{[ 1, 4]}, 如果不用生长素处理,让胚在植株上生长到胚培养时, 几乎所有的胚已退化消失^{[ 9]}。因此, 生长素的处理对于维持单倍体胚生长到可以进行幼胚培养阶段必不可少。一般常用生长素2,4-D, 但也有人用Dicamba^{[ 14, 15]}。Knox等^{[ 16]}在硬粒小麦与玉米杂交中发现Dicamba处理较2,4-D处理得胚率显著提高, 但二者使用了不同浓度。在小麦与玉米杂交试验中未见Dicamba与2,4-D处理效果对比报道。本文对比试验表明, 在浓度均为100 mg L^-1下, Dicamba处理较2,4-D处理显著提高了膨大颖果频率, 但这两种生长素处理单倍体胚产生频率无显著差异, 效果几乎相同。Dicamba 处理虽然膨大颖果率高, 但得胚率无差异, 这会增加剥胚工作量, 同时Dicamba价格是2,4-D的数倍。因此, 建议应用生长素2,4-D, 既经济又高效。

小麦生长条件对提高小麦与玉米杂交得胚率有重要作用。研究表明^{[ 17]}, 小麦在未通暖气的冷温室生长条件下与玉米杂交的平均得胚率(29.9%)显著高于大田生长条件下(3.6%), 其原因是两种环境下授粉时的温度、湿度、光照不同。大田小麦与玉米杂交时的温度、湿度、光照不可控制, 特别是遇到阴天、低温, 得胚率会显著降低。这也是大田不同季节或同一季节不同时间小麦与玉米杂交得胚率不稳定的重要原因。割穗培养比大田生长条件下能显著提高得胚率^{[ 18, 19, 20, 21]}。本研究为了延长杂交时间, 在返青后将一部分小麦组合从大田移回冷温室盆栽, 抽穗后去雄与玉米杂交, 并与生长在大田授粉时割穗培养方法比较。割穗培养平均得胚率是盆栽的3倍以上, 虽然2种方法所用小麦组合多不相同, 但就相同的2个组合轮选987/中麦175和CA0996/中麦175 (正反交)而言, 割穗培养平均得胚率分别是27.2%和24.4%, 而盆栽者为10.4%和7.2%, 割穗培养远高于盆栽。2011年也得到相似结果(资料未列出)。两种方法授粉时和授粉后的温度、湿度、光照相同, 只是植株的生长状态不同。盆栽小麦植株分蘖少, 生长势弱于大田小麦, 可能其生理生化活性如内源激素水平、酶活性等也弱于大田小麦, 授粉受精能力较弱, 胚形成频率低; 而割穗培养植株生长健壮, 生理生化活性强, 授粉受精能力强, 有利于胚形成。因此, 盆栽和大田生长割穗培养两种方法得胚率之差异, 主要由小麦植株的生长状态引起。综上所述, 小麦生长环境条件和生长状态是小麦与玉米杂交稳定获得高频率单倍体胚的重要因素。国外一般将小麦和玉米均种植在人工气候室, 分期播种, 一年四季都可以杂交, 小麦生长环境条件如温度、湿度、光照、施肥等均可以控制且保持一致, 这样可以保证始终稳定获得高的得胚率。

小麦与玉米杂交幼胚培养一般采用1/2 MS培养基, 也有的用Difco Orchid琼脂培养基和B₅培养基^{[ 2, 22]}。本文比较了B₅培养基与1/2 MS培养基幼胚培养效果, 虽然前者较后者胚培养萌发率提高5.4个百分点, 但二者差异不显著。在硬粒小麦与玉米杂交胚培养时也得到了相似结果^{[ 21]}。因此, 在实际应用时可任选其一。

壮苗是提高小麦与玉米杂交单倍体植株加倍效率的关键^{[ 23]}。此前我们是在6月至7月获得单倍体苗后, 放在4℃生长箱越夏处理3~4个月, 等气温降低后再移栽培育壮苗, 最后进行染色体加倍处理, 第2年夏季获得DH种子。为了当季就获得DH种子, 本文比较了壮苗培养基壮苗与移栽壮苗处理方法的加倍效果, 结果前者加倍处理后的存活率和加倍效率分别是后者的2.0倍和7.9倍。其原因是前者培养的壮苗分蘖多, 生长势强, 忍耐秋水仙素毒害能力强, 而后者壮苗效果差, 多数无分蘖, 生长势弱, 耐毒害能力差, 不仅加倍处理后存活率低, 而且存活的植株(可能不在最适加倍期)多数未能加倍结实。结合人工生长室, 在6月至7月获得单倍体苗后, 立即采用壮苗培养基进行壮苗处理, 再经人工春化处理, 在12月至翌年1月就可获得DH种子, 打破休眠后立即播种大田, 覆盖塑料布, 夏收即可获得DH繁殖的种子。秋播大田进行农艺性状和抗性鉴定, 筛选优异材料升入产量鉴定试验, 较常规育种提早3年。

在本实验室, 每获得1000个冬小麦DH株系, 按照现在平均每穗得胚6.8个、胚培养萌发率82%、加倍效率67%计算, 约需要268个小麦与玉米杂交穗。在北京从杂交到收获DH种子需要7~8个月, 并需人工生长室15 m², 温室20~30 m², 低温春化室15 m²。要实现育种上规模化应用, 可像加拿大农业与农业食品部谷物研究中心(Cereal Research Centre, Agriculture and Agri-Food Canada) DH实验室那样, 3个长期技工, 使用6~7个人工气候室, 一年四季均可杂交, 每年可生产15 000个DH株系。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

参考文献

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[2]	Laurie A D, Reymondie S. High frequencies of fertilization and haploid seeding production in crosses between commercial hexaploid wheat varieties and maize. Plant Breed, 1991, 106: 182-189 [本文引用:3] [JCR: 1.596]
[3]	Inagaki M, Tahir M. Comparison of haploid production frequency in wheat varieties crossed with Hordeum bulbosum L. and maize. Jpn J Breed, 1990, 40: 209-216 [本文引用:1]
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[5]	Chen X-M(陈新民), Lai G-X(赖贵贤), Chen X(陈孝), Zhou J-F(周俊芳), Liu J-X(刘俊秀), Sun F-H(孙芳华). Difference of haploid production in crosses between different F₁ and maize. Acta Agron Sin(作物学报), 1996, 22(4): 437-441 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.8267]
[6]	Mujeeb-Kazi A, Cortes A, Riera-Lizarazu O, Faridi N I, Delgado R. Polyhaploidy in the Triticeae madited by crosses of Triticum species with Zea mays and Trip sacumdatyloides. In: 8th Int Wheat Genet Symp. Beijing: China Agricultural Science and Technology Press, 1995. pp1119-1123 [本文引用:1]
[7]	Suenaga K, Nakajima K. Efficient production of haploid wheat (Triticum aestivum) through crosses between Japanese wheat and maize (Zea mays). Plant Cell Rep, 1989, 8: 263-266 [本文引用:1] [JCR: 2.274]
[8]	Kisan N S, Nkongolo K K, Quick J S, Johnson D L. Production of doubled haploids by anther culture and wheat × maize method in a wheat breeding programme. Plant Breed, 1993, 110: 96-102 [本文引用:1] [JCR: 1.596]
[9]	Laurie A D, Bennett M D. The production of haploid wheat plants from wheat × maize crosses. Theor Appl Genet, 1988, 76: 393-397 [本文引用:2] [JCR: 3.297]
[10]	Chen X-M(陈新民), Chen X(陈孝). Progress on haploid and doubled haploid production by wheat × maize method. Tritical Crops(麦类作物), 1998, 18(3): 1-4 (in Chinese) [本文引用:1]
[11]	Cai H(蔡华), Ma C-X(马传喜), Si H-Q(司红起), Qiao Y-Q(乔玉强), Lu W-Z(陆维忠). Advance on producing double haploid of wheat by wide hybridization between wheat and maize. J Triticeae Crops(麦类作物学报), 2006, 26(4): 154-157 (in Chinese with English abstract) [本文引用:1]
[12]	DePauw R M, Knox R E, Humphreys D G. New breeding tools impact Canadian commercial farmer fields. Czech J Genet Plant Breed, 2011, 47: S28-S34 [本文引用:1] [JCR: 0.532]
[13]	Chen X-M(陈新民), He Z-H(何中虎), Liu C-L(刘春来), Wang D-S(王德森), Zhang Y(张勇). New wheat variety Zhongmai 533 developed by wheat × maize cross. J Triticeae Crops(麦类作物学报), 2011, 31(3): 427-429 (in Chinese with English abstract) [本文引用:1]
[14]	Matzk F, Mahn A. Improved techniques for haploid production in wheat using chromosome elimination. Plant Breed, 1994, 113: 125-129 [本文引用:1] [JCR: 1.596]
[15]	Campbell A W, Grin W B, Conner A J, Rowarth J S, Burritt D J. The effects of temperature and light intensity on embryo numbers in wheat doubled haploid production through wheat × maize crosses. Ann Bot, 1998, 82: 29-33 [本文引用:1] [JCR: 1.014]
[16]	Knox R E, Clarke J M, DePauw R M. Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize. Plant Breed, 2000, 119: 289-298 [本文引用:1] [JCR: 1.596]
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[18]	Gu J(顾坚), Liu K(刘琨), Li S-X(李绍祥), Tian Y-X(田玉仙), Yang H-X(杨和仙), Yang M-J(杨木军). Study on the in vitro culture of cut plant in wheat haploid embryo induction by wheat × maize cross. J Triticeae Crops(麦类作物学报), 2008, 28(1) : 1-5 (in Chinese with English abstract) [本文引用:1]
[19]	Cai H(蔡华), Ma C-X(马传喜), Si H-Q(司红起), Qiao Y-Q(乔玉强), Lu W-Z(陆维忠). Study on increasing frequency of haploid embryo production through wheat × maize. J Triticeae Crops(麦类作物学报), 2005, 25(6): 20-24 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.8529]
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[21]	Cherkaoui S, Lamsaouri O, Chlyah A, Chlya H H. Durum wheat × maize crosses for haploid wheat production: influence of parental genotypes and various experimental factors. Plant Breed, 2000, 119: 31-36 [本文引用:2] [JCR: 1.596]
[22]	Lefebre D, Devaux P. Doubled haploids of wheat from wheat × maize crosses: genotype influence, fertility and inheritance of the 1BL/1RS chromosome. Theor Appl Genet, 1996, 93: 1267-1273 [本文引用:1] [JCR: 3.297]
[23]	Chen X M, Zhang W X, Cui S L, Chen X. Study on chromosome doubling for haploid produced by wheat × maize crossing. Agric Sci China, 2002, 1: 486-490 [本文引用:1] [JCR: 0.449] [CJCR: 0.4431]

1986

0.0

. 1986, 28:313-316

Wheat × maize hybridization

Laurie A D , Bennett M D

Cytogenetic evidence is presented that the cross between hexaploid bread wheat (Triticum aestivum, 2n = 42) and maize (Zea mays, 2n = 20) results in a hybrid zygote with one complete haploid chromosome set from each parent. Maize chromosomes are subsequently eliminated. This sytem has potential for wheat haploid production and may also allow segments of maize DNA, including transposable elements, to be transferred to wheat.Key words: wide crosses, wheat, maize, chromosome elimination, haploids.

... 小麦与玉米杂交, 因玉米染色体与小麦纺锤体的不亲和性, 杂合子在最初的几次细胞分裂中会使玉米染色体全部丢失, 产生仅有小麦21条染色体的单倍体, 经过幼胚培养获得单倍体苗, 再经染色体加倍, 就可获得纯合双单倍体DH (doubled haploid)^[1,2] ...

... 3 讨论小麦与玉米杂交诱导小麦单倍体后仅有极少部分胚有胚乳,而且胚乳总是异常并退化, 不能提供胚生长发育所需要的营养物质^[1,4], 如果不用生长素处理,让胚在植株上生长到胚培养时, 几乎所有的胚已退化消失^[9] ...

1991

1.596

0.0

. 1991, 106:182-189

High frequencies of fertilization and haploid seeding production in crosses between commercial hexaploid wheat varieties and maize

Laurie A D , Reymondie S

Nineteen commercial hexaploid wheat varieties were crossed with the maize F₁ hybrid ‘Seneca 60’. Fertilization frequencies ranged from 32.1 % to 47.5 % of pollinated florets (mean 39.5 %) in the 14 winter wheat varieties and from 40.7 % to 51.4 % (mean 47.8 %) in the five spring wheat varieties. In some cases only an endosperm was formed and the frequencies of embryo formation were therefore slightly lower, being 28.2 % to 45.9 % (mean 36.4 %) for winter wheats and 39.8 % to 48.6 % (mean 45.1 %) for spring wheats. Mean values were significantly higher in the spring wheats but no significant variation was found between varieties within the spring or winter categories. In the five spring wheats the mean yield of embryos, and hence the potential yield of haploid plants, was 3.4-fold higher than with the tetraploidHordeum bulbosum clone PB179. For the 14 winter wheats the figure was 10.9-fold higher. These differences were highly significant (p< 0.001) in all varieties. A single 2,4-D treatment given to spikes one day after pollination with maize enabled embryos to be recovered from all 19 varieties. A total of 311 embryos were recovered from 950 florets (an average of 7.3 embryos per spike) of which 191 germinated, giving an average yield of one haploid plant for every 5.0 florets pollinated (4.4 haploid plants per spike).

... 第二, 单倍体的产生对于小麦基因型无严格的选择性, 不同基因型的小麦均能获得单倍体^[2,5,6,7], 特别是花药培养不能产生愈伤组织或植株的小麦基因型, 在小麦×玉米中具有较高的胚和植株产生率^[8], 这一点优于小麦花药培养 ...

... 小麦与玉米杂交幼胚培养一般采用1/2 MS培养基, 也有的用Difco Orchid琼脂培养基和B₅培养基^[2,22] ...

1990

0.0

... 用这种方法产生单倍体有3个优点: 第一, 由于玉米对小麦可交配Kr基因的不敏感, 杂交得胚率远高于小麦×球茎大麦^[3,4] ...

1987

3.297

0.0

. 1987, 73:403-409

The effect of the crossability loci Kr1 and Kr2 on fertilization frequency in hexaploid wheat × maize crosses

Laurie D A , Bennett M D

Dominant alleles of theKr1 andKr2 genes reduce the crossability of hexaploid wheat with many alien species, including rye andHordeum bulbosum, withKr1 having the greater effect. However, a cytological study of wheat ovaries fixed 48 h after pollination showed that the wheat genotypes ‘Highbury’ (kr1, Kr2) and ‘Chinese Spring (Hope 5B)’ (kr1, kr2) were crossable with ‘Seneca 60’ maize, fertilization occurring in 14.4 and 30.7% of embryo sacs respectively. The latter figure was similar to the 29.7% fertilization found in ‘Chinese Spring’ (kr1, kr2). Most embryo sacs in which fertilization occurred contained an embryo but lacked an endosperm and where an endosperm was formed it was usually highly aberrant. All three wheat x maize combinations were karyotypically unstable and rapidly eliminated maize chromosomes to produce haploid wheat embryos.

... 用这种方法产生单倍体有3个优点: 第一, 由于玉米对小麦可交配Kr基因的不敏感, 杂交得胚率远高于小麦×球茎大麦^[3,4] ...

1996

0.0

1.8267

Acta Agron Sin. 1996, 22(4):437 - 441

Difference of Haploid Production in Crosses between different Wheat F1 and Maize

不同小麦组合与玉米杂交产生单倍体的差异

Chen Xinmin;Lai Guixian;Chen Xiao;Zhou Juenfang;Sun Fanghua;Liu Junxiu

陈新民;赖桂贤;陈孝;周俊芳;刘俊秀;孙芳华

Institute of Crop Breeding and Cultivation,CAAS,Beijing,100081

1995

0.0

1989

2.274

0.0

1993

1.596

0.0

. 1993, 110:96-102

Production of doubled haploids by anther culture and wheat × maize method in a wheat breeding programme

Kisan N S , Nkongolo K K , Quick J S , Johnson D L

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.

1988

3.297

0.0

. 1988, 76:393-397

The production of haploid wheat plants from wheat × maize crosses

Laurie A D , Bennett M D

Hybrid embryos from hexaploid wheat x maize crosses rapidly lose the maize chromosomes to produce haploid wheat embryos. Such embryos almost always aborted when left to develop on the plant, and only 1 was recovered from 2440 florets (0.17% of the expected number). Embryos had greater viability in spikelet culture, 47 (26.5% of the expected number) being recovered from 706 ovaries. Thirty-two of these embryos germinated to give green plants, 31 of which were haploid (21 wheat chromosomes) and 1 of which was euploid (42 wheat chromosomes). Spikelet culture enabled 17.1% of the expected number of embryos to be recovered as haploid plants, a 100-fold improvement on allowing embryos to develop in vivo. Ten haploid plants of ‘Chinese Spring’ (kr1, kr2), 13 plants of ‘Chinese Spring (Hope 5A)’ (kr1, Kr2), and 8 of ‘Hope’ (Kr1, Kr2) were recovered. The potential of wheat x maize crosses for wheat haploid production and for gene transfer from maize to wheat is discussed.

... 自Laurie等^[9]首次获得小麦×玉米单倍体植株后, 国内外许多学者均进行了有关研究并应用于育种实践^[10,11] ...

1998

0.0

... 自Laurie等^[9]首次获得小麦×玉米单倍体植株后, 国内外许多学者均进行了有关研究并应用于育种实践^[10,11] ...

2006

0.0

. 2006, 26(4):154-157

Advance on producing double haploid of wheat by wide hybridization between wheat and maize

Cai H , 蔡华), Ma C-X , 马传喜), Si H-Q , 司红起), Qiao Y-Q , 乔玉强), Lu W-Z

利用小麦（Triticum aestivum L．）与玉米（Zea mays L．）进行远缘杂交，通过玉米染色体的消失而获得小麦单倍体胚，再经幼胚离体培养及染色体加倍诱导双单倍体（Double haploid，DH），是小麦单倍体育种的一种重要途径，用这种方法创建的DH群体是进行小麦分子标记研究和QTL分析的重要遗传材料。本文对DH群体创建过程中影响单倍体胚产生、单倍体及双单倍体植株形成等因素以及双单倍体的应用进行了综述，重点探讨了亲本选配原则、花期调节技术、2，4-D处理、玉米花粉和小麦柱头发育状况以及环境因素等对诱导单倍体胚效果的影响等方面的研究状况，同时对利用小麦与玉米杂交诱导小麦双单倍体技术进行了展望。

... 自Laurie等^[9]首次获得小麦×玉米单倍体植株后, 国内外许多学者均进行了有关研究并应用于育种实践^[10,11] ...

2011

0.532

0.0

... 如在加拿大, 从1997年开始利用DH育种技术, 至2011年已审定小麦品种25个, 其中23个是应用小麦×玉米DH技术培育的, 2009年DH品种种植面积占全国小麦面积的1/3^[12] ...

2011

0.0

. 2011, 31(3):427-429

New wheat variety Zhongmai 533 developed by wheat × maize cross

Chen X-M , 陈新民), He Z-H , 何中虎), Liu C-L , 刘春来), Wang D-S , 王德森), Zhang Y

利用小麦与玉米杂交方法进行单培体育种，可缩短育种年限，提高育种效率。本文报道了用小麦×玉米方法培育小麦新品种中麦533的过程。该品种的突出特点是高产、节水，条锈病中抗至高抗，白粉病免疫。北京市节水区域试验平均产量5 598 kg·hm_-2，比对照京冬8号增产5.2%；生产试验平均产量5 502 kg·hm_-2，比对照增产9.7%。同时介绍了利用小麦×玉米单倍体育种的体会。

... 我们从1992年开始小麦×玉米产生小麦单倍体和双单倍体的技术研究, 虽然在国内率先建立了利用小麦与玉米杂交产生小麦双单倍体技术体系, 并利用该技术首先培育了小麦新品种^[13], 但是由于实验条件和一些技术方面的问题, 单倍体胚和植株产生频率以及单倍体加倍效率还不稳定, 在育种实际应用中存在一些问题 ...

1994

1.596

0.0

. 1994, 113(2):125-129

Improved Techniques for Haploid Production in Wheat using Chromosome Elimination

F. Matzk,A. Mahn

Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466 Gatersleben, Germany.

Wheat × maize and wheat × pearl millet crosses have proved efficient for haploid production using various genotypes of wheat; 22 and 27 % of florets produced embryos. In favourable conditions 6—9 haploid plants per spike were produced. The following simplifications or improvements in technique are recommended: 1. Only a single treatment with an aqueous solution of dicamba or 2,4-D (50–100 ppm) for embryo stimulation in vivo; 2. Application by spraying or dipping the spikes; 3. Application time two to four days after pollination; 4. Embryo rescue 15 to 18 days after pollination; 5. Crosses without emasculation are possible if pollination occurs 1–2 days before anthesis. More than 450 haploids and some doubled haploid (DH) lines (after colchicine treatmentin vitro) were produced using these methods. No hybrid plants, chromosome additions or substitutions were found.

... 一般常用生长素2,4-D, 但也有人用Dicamba^[14,15] ...

1998

1.014

0.0

... 一般常用生长素2,4-D, 但也有人用Dicamba^[14,15] ...

2000

1.596

0.0

. 2000, 119:289-298

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Knox R E , Clarke J M , DePauw R M

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat.Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4-D the number of doubled haploids per spike increased from 0.2 for the best 2,4-D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4-D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4-D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.

... Knox等^[16]在硬粒小麦与玉米杂交中发现Dicamba处理较2,4-D处理得胚率显著提高, 但二者使用了不同浓度 ...

1998

0.0

... 研究表明^[17], 小麦在未通暖气的冷温室生长条件下与玉米杂交的平均得胚率(29 ...

2008

0.0

... 割穗培养比大田生长条件下能显著提高得胚率^{[18,19,20,21]} ...

2005

0.0

0.8529

J Trit Crops. 2005, (6):20 - 24

Study on Increasing Frequency of Haploid Embryo Production through Wheat × Maize

提高小麦×玉米产生的单倍体成胚率的研究

CAIHua^[1];MAChuan-xi^[2];SIHong-qi^[2];QIAOYu-qiang^[2];LUWei-zhong^[3]

蔡华^[1];马传喜^[2];司红起^[2];乔玉强^[2];陆维忠^[3]

为了提高小麦×玉米产生单倍体的频率,采用人工控制环境条件的室内离体培养法和田间常规杂交法诱导小麦×玉米单倍体,研究了不同生长环境下的成胚率、环境温度对离体培养法成胚率以及T>10℃有效积温对常规杂交法成胚率的影响效果.结果表明,就平均成胚率而言,室内离体培养法(23.6%)高于常规杂交法(18.1%),且以20℃、光照3 000 Lx、12 h/d、相对湿度70%条件下离体培养杂交穗14 d获得的成胚率最高(28.6%);授粉后足够的T>10℃有效积温是高成胚率的必要条件,获得最高成胚率的T>10℃有效积温在187.9～254.9℃之间;授粉后环境温度是影响成胚速率的重要因素,以20℃最佳.

... 割穗培养比大田生长条件下能显著提高得胚率^{[18,19,20,21]} ...

1997

0.392

0.0

... 割穗培养比大田生长条件下能显著提高得胚率^{[18,19,20,21]} ...

2000

1.596

0.0

. 2000, 119:31-36

Durum wheat × maize crosses for haploid wheat production: influence of parental genotypes and various experimental factors

Cherkaoui S , Lamsaouri O , Chlyah A , Chlya H H

To improve haploid plant production in durum wheat, the haplomethod involving intergeneric crossing with maize followed by embryo rescue was used. The influence of parental genotypes and various experimental factors were studied. Ten cultivars ofTriticum turgidum ssp.durum (female parent) were crossed with eight genotypes ofZea mays. After pollination, plant stems were either maintainedin situ or cut near the base and kept in a 2,4-dichlorophenoxyacetic acid (2,4-D)-sucrose solution. Ten to 18 days after pollination, embryos were excised from developed ovaries and cultured on one of MS, MS/2, or B5 media. Haploid embryos and plants were obtained (78 green haploid plants regenerated in 0 year). The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation. Detailed results of all crosses showed the best crossing partner for each wheat genotype. Cutting the plant stems after pollination gave better results than maintaining themin situ. The optimal stage for embryo rescue was 14 days and B5 and MS/2 media were more efficient than MS for embryo culture.

... 割穗培养比大田生长条件下能显著提高得胚率^{[18,19,20,21]} ...

... 在硬粒小麦与玉米杂交胚培养时也得到了相似结果^[21] ...

1996

3.297

0.0

. 1996, 93:1267-1273

Doubled haploids of wheat from wheat × maize crosses: genotype influence, fertility and inheritance of the 1BL/1RS chromosome

Lefebre D , Devaux P

The wheat x maize cross as a technique for haploid induction in wheat was evaluated in a replicated block design comprising 18 wheat F₁ hybrids and fiveZea mays L. parents. Haploid plants were regenerated at an average of 9.1 (4.4–14.7) plants per 100 florets processed. Genotypic differences for haploid production efficiency were recorded for both wheat andZea mays L. Interaction between parents was significant for number of plants/100 florets. All 610 of the 1,703 regenerated plantlets that were analyzed by flow cytometry were haploid. At maturity, 70% (60–81 %) of the colchicinetreated haploid plants were fertile, but the frequency of fertile and sterile plants was not consistent over the wheat hybrids from which they were derived. Flow cytometry performed using the first tiller which arose following colchicine treatment enabled prediction of fertility. The 1BL-1RS chromosome was found at the expected ratios in the F₂ and in the haploid progenies produced through the wheat x maize cross but deviated from the 1∶1 ratio in the haploid progenies produced by anther culture.

... 小麦与玉米杂交幼胚培养一般采用1/2 MS培养基, 也有的用Difco Orchid琼脂培养基和B₅培养基^[2,22] ...

2002

0.449

0.4431

... 壮苗是提高小麦与玉米杂交单倍体植株加倍效率的关键^[23] ...