CN113215161A - 利用单碱基编辑技术创造除草剂抗性植物的方法 - Google Patents
利用单碱基编辑技术创造除草剂抗性植物的方法 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及除草剂技术领域,且公开了利用单碱基编辑技术创造除草剂抗性植物的方法,包括以下步骤:1)GhEPSP基因的sgRNA设计:选择陆地棉5‑烯醇式丙酮酰莽草酸‑3‑磷酸合酶(5‑Enolpyruvylshikimate‑3‑phosphatesynthase,EPSP合酶)为验证基因,利用在线软件CRISPR‑P(http://cbi.hzau.edu.cn/cgi‑bin/CRISPR)(Lei et al 2014)在基因外显子区域设计sgRNA靶标序列,并且在编辑窗口内C‑T突变改变氨基酸功能的靶点,最终选择1个sgRNA用来构建单碱基系统植物表达载体。该利用单碱基编辑技术创造除草剂抗性植物的方法,在棉花中首次成功应用棉花基因组特性的单碱基编辑系统对棉花GhEPSP实现单碱基突变,突变位点未见其他作物报道,通过叶片草甘膦涂抹实验,表现出一定的草甘膦耐受性,提高了棉田除草效率,降低了人工除草成本,防范棉田超级杂草。
Description
技术领域
本发明涉及除草剂技术领域,具体为一种利用单碱基编辑技术创造除草剂抗性植物的方法。
背景技术
除草剂抗性是作物育种的重要方向,抗除草剂作物的种植面积在全球总种植面积比例达到47%,抗除草剂育种一方面通过物理或化学等手段处理(包括体细胞诱变、辐照和化学诱变剂等)作物后获得抗性基因突变体植株,另一方面通过基因工程导入外源基因来获得抗除草剂作物,也是目前最重要手段之一,最近,随着基因编辑技术的发展,在其他作物中利用CRISPR-Cas9系统定向改造抗除草剂靶标基因,获得抗除草剂材料,为农作物分子育种提供了一条全新途径。
草甘膦自1974年在美国注册登记以来,已成为当今世界上销售量最大且使用最广的农药品种,草甘膦与5-烯醇式丙酮酰莽草酸-3-磷酸合酶(5-Enolpyruvylshikimate-3-phosphate synthase,EPSP合酶)的作用机制是:草甘膦与磷酸烯醇丙酮酸(Phosphoenolpyruvate,PEP)在结构上非常相似,在莽草酸途径中草甘膦占据了EPSP合酶和PEP的连接位点,形成EPSP合酶-S3P-glyphosate的复合物,使EPSP合酶催化烯醇式丙酮酸基团从PEP转化到S3P上形成EPSP的过程停止,竞争性抑制EPSP合酶的活性从而阻断植物内辅酶Q、叶酸和芳香族氨基酸等化合物的生物合成,使植物细胞分裂、叶绿素合成、蒸腾、呼吸及蛋白质等代谢受到影响,最终导致一些关键性代谢物和激素如酚类化合物和木质素代谢失调,使生物体不能进行正常的氮代谢而死亡,而且EPSP合酶进入叶绿体的过程也受草甘膦的抑制。
抗草甘膦EPSP合酶基因来源于1983年Comai等从鼠伤寒沙门菌中分离克隆出的抗草甘膦突变基因——aroA基因,此后许多研究者对抗草甘膦基因进行了深入广泛的研究,随着分子生物学技术的快速发展和对EPSP合酶的深入研究,1996年,美国Monsanto公司利用CP4农杆菌的抗草甘膦EPSP合酶的编码基因研发出第抗草甘膦作物-抗草甘膦大豆(Roundup ready),目前获得抗草甘膦EPSP合酶编码基因的途径主要有两种:第1种是从天然抗草甘膦的物种或突变体中克隆分离出抗草甘膦基因或突变基因,在天然抗草甘膦基因中,土壤农杆菌CP4-EPSP合酶基因是应用最广泛的抗草甘膦EPSP合酶基因,并形成商业化生产,除此之外,研究者从一些物种的突变体中克隆分离出抗草甘膦突变基因,如研究者克隆大豆、油菜、马铃薯及拟南芥抗草甘膦的EPSP合酶突变基因发现,大豆EPSP合酶基因编码的104Gly突变为Ala,油菜EPSP合酶基因编码的Gly96突变为Ala,马铃薯和拟南芥EPSP合酶第101位上Gly变为Ala,Shah等从筛选出的抗草甘膦矮牵牛突变体MP4-G细胞系中克隆出EPSP合酶的cDNA,将其cDNA导入野生型矮牵牛叶片后,对草甘膦的抗性明显提高,Scott等发现牛筋草的EPSP合酶基因发生突变,第101位脯氨酸突变为色氨酸后,对草甘膦具有很低的敏感度,表现出抗草甘膦,第2种是利用遗传学方法改造EPSP合酶编码基因,提高对草甘膦的抗性,定点突变可以减小酶与草甘膦的亲和力或提高酶与底物的亲和力,从而提高对草甘膦的抗性,Tian等利用定点突变使苹果的EPSP合酶基因编码的Thr101变为Ala,Ala187变为Thr,通过动力学分析证实两个氨基酸突变后提高了对草甘膦的抗性,Ming等使用易错倾向PCR随机突变技术使来源于大肠杆菌和沙门氏菌的aroA基因发生突变和重组,得到aroM1、aroM2、aroM3和aroM4四种突变体,通过EPSP合酶活性的动力学分析表明,4种突变体的4个突变基因编码的EPSP合酶的活性比突变前提高了2-10倍,与烯醇式丙酮酸的亲和性提高了2.5-19倍,与草甘膦的抑制常数Ki值提高了0.4-8倍。
2016年美国哈佛大学Komor等利用APOBEC1和dCas9创建出第一代的碱基编辑器BE1(POBEAC1-XTEN-dCas9),发现BE1碱基编辑器有5个碱基的活性编辑窗口(靶标序列上PAM远端的第4-8位核苷酸)。
单碱基编辑技术对作物基因组也可以进行定点编辑,进而提高作物基因编辑的精准性,目前CRISPR-Cas9系统已经在拟南芥、水稻、小麦、玉米和棉花等植物中成功实现了基因编辑,为植物遗传研究和作物遗传改良提供了平台,Zong等在CRISPR-Cas9系统的基础上,创造两种碱基编辑系统:nCas9-PBE和dCas9-PBE,它们由大鼠胞苷脱氨酶APOBEC1,Cas9变体(nCas9或dCas9)和尿嘧啶糖基化酶抑制剂(UGI)组成,这个融合载体根据谷物植物密码子偏好进行密码子优化,并克隆在玉米泛素(Ubi)基因启动子下产生pnCas9-PBE和pdCas9-PBE,在相同的C位点突变,pnCas9-PBE产生的编辑效率明显高于pdCas9-PBE,他们使用野生型Cas9(pCas9)产生缺失或插入突变(indel)作为对照,发现pnCas9-PBE和pdCas9-PBE产生的缺失或插入相同且比较低,但是都比pCas9少很多,他们通过深测序来评估各植物的原生质体中的基因编辑,发现pnCas9-PBE在第7个目标位点及临近位置引起C-T替换效率最高,胞嘧啶核苷脱氨酶的活性窗口为7个核苷酸,比动物中的编辑窗口(5个)更广,与此同时,Li和Lu等也分别独立研究了单碱基编辑系统在水稻中的应用,并表明碱基编辑系统可以有效地进行单个碱基的编辑,任斌等成功开发出水稻单碱基编辑系统rBE3和rBE4,在此基础上引入人源AID胞嘧啶脱氨酶,开发了单碱基编辑系统的升级版系统rBE5和rBE9,成功实现了对水稻中多个靶基因的高效定点编辑,该系统避免了APOBEC1对CT偏好性的问题,对AC,GC,CC同样具有较高的编辑效率,极大的推动水稻功能基因组学和和植物现代分子育种研究进程,这些研究表明,该技术可以在农作物中高效编辑单个碱基,而且编辑范围更大,进而改良作物品质及获得优良性状的农作物,上海植物逆境生物学研究中心朱健康团队通过利用SpCas9和SaCas9的变体开发了新的腺嘌呤和胞嘧啶碱基编辑器,很大程度上扩展了基因组中的可靶向位点,此外,该研究还将胞嘧啶和腺嘌呤碱基编辑器在水稻中同时进行基因编辑,并同时取得C-T和G-A的编辑类型,为实现棉花自身单碱基突变(非转外源基因)创制草甘膦抗性的棉花材料,提高棉田除草效率,降低人工除草成本,防范棉田超级杂草,故提出一种利用单碱基编辑技术创造除草剂抗性植物的方法。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了利用单碱基编辑技术创造除草剂抗性植物的方法,具备突变棉花材料具有较好的草甘膦耐受的优点,解决了棉花自身单碱基突变(非转外源基因)创制的棉花材料不具备草甘膦抗性的问题。
(二)技术方案
为实现上述突变棉花材料具有较好的草甘膦耐受的目的,本发明提供如下技术方案:利用单碱基编辑技术创造除草剂抗性植物的方法,包括以下步骤:
1)GhEPSP基因的sgRNA设计:选择陆地棉5-烯醇式丙酮酰莽草酸-3-磷酸合酶(5-Enolpyruvylshikimate-3-phosphate synthase,EPSP合酶)为验证基因,利用在线软件CRISPR-P(http://cbi.hzau.edu.cn/cgi-bin/CRISPR)(Lei et al 2014)在基因外显子区域设计sgRNA靶标序列,并且在编辑窗口内C-T突变改变氨基酸功能的靶点,最终选择1个sgRNA用来构建单碱基系统植物表达载体;
2)sgRNA与GhBE3载体的连接:靶标插入GhBE3载体序列为tRNA-sgRNA-gRNA的重复序列,需要中间载体转换,第一次PCR的引物如下:
pRGEB32-7/S:AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG,将sgRNA加到反向引物的接头上,GhEPSP/AS:TTCCACCGGGAAGACCACCCTGCACCAGCCGGGAAT,下划线的碱基是sgRNA序列,以PGTR载体(Xie et al.2015)为模板进行PCR扩增tRNA序列,获得tRNA+sgRNA利用一步克隆试剂盒(Vazyme,C112-01/02)将tRNA+sgRNA片段分别连接到pGREB32-GhU6-7载体的BsaI酶切位点处,利用HpaI和SbfI双酶切pGREB32-GhU6-7,将目的片段连接到GhBE3载体的HpaI和SbfI双酶切位点处;
3)农杆菌介导的遗传转化:
A、将剥好的棉花种子(品种为Jin668,专利申请号201510833618.0)用0.1%升汞杀菌,无菌水清洗数次后放入无菌苗培养基中,28℃暗培养1天,挑去种皮,将苗扶正,在28℃,暗培养4d;
B、将下胚轴切成小茎段,用活化后的农杆菌侵染,弃菌液,并吹干;
C、将下胚轴平铺在放有滤纸的共培养培养基中,于20℃,暗培养1d;
D、将下胚轴转入到附加2,4-D的愈伤组织诱导培养基中,放入光照培养室,25d左右用新鲜愈伤组织诱导培养基继代培养一次;
E、当愈伤组织长成米粒状颗粒,转入分化培养基中,进一步分化成胚状体;
F、将分化出的小苗继代到生根培养基中,直至长成生根良好健康的小苗;
G、将小苗转到清水中,进行炼苗,一周后,转移到到温室;
4)测序检测编辑:提取的棉花嫩叶阳性基因组DNA,以阳性DNA为模板,扩增GhEPSP基因,然后将PCR片段连入pGEM-T easy载体,将连接产物热激转化大肠杆菌感受态TOP10,挑取的单克隆进行阳性检测并进行Sanger测序,将测序结果与靶标序列比对;
5)叶片涂抹草甘膦:将农达(孟山都)稀释200倍,涂抹棉花叶片,一周后观察叶片药害情况。
优选的,所述转化所用的培养基组分及配比如下:
无菌苗培养基:1/2MS大量元素,15g/L葡萄糖,2.5g/L的Phytagel;pH:6.1-6.2。
愈伤组织诱导培养基:MSB+24-D 0.1mg/L+KT 0.1mg/L+3%Glucose+0.3%Phytagel;pH:5.85-5.95。
农杆菌活化培养基:胰化蛋白胨5g/L+NaCl 5g/L+MgSO4.7H2O 0.1g/L+KH2PO4+0.25g/L+甘露醇5g/L+甘氨酸1.0g/L;pH:5.85-5.95。
共培养培养基:MSB+2,4-D 0.1mg/l+KT 0.1mg/l+50mg/l AS+3%Glucose+0.25%Phytagel,pH5.8。
选择培养基:MSB+2,4-D 0.1mg/L+KT 0.1mg/L+3%Glucose+0.3%Phytagel,卡那霉素50mg/L和头孢霉素400mg/L;pH:5.85-5.95。
分化培养基:MSB培养基中去掉NH4NO3,将KNO3用量加倍+Gln 1.0g/L+Asn 0.5g/L+IBA 0.5mg/L+KT 0.15mg/L+3%Glucose+0.25%Phytagel,pH:6.1-6.2。
生根培养基:1/2MS无机盐+B5有机物,15g/L葡萄糖,2.5g/L的Phytagel;pH:5.90-5.95;MSB的成分如下:MS培养基+B5维生素。
(三)有益效果
与现有技术相比,本发明提供了利用单碱基编辑技术创造除草剂抗性植物的方法,具备以下有益效果:
该利用单碱基编辑技术创造除草剂抗性植物的方法,在棉花中首次成功应用棉花基因组特性的单碱基编辑系统对棉花GhEPSP实现单碱基突变,突变位点未见其他作物报道,通过叶片草甘膦涂抹实验,表现出一定的草甘膦耐受性,提高了棉田除草效率,降低了人工除草成本,防范棉田超级杂草。
附图说明
图1为本发明提出的利用单碱基编辑技术创造除草剂抗性植物的方法表达载体GhBE3的构建图;
图2为本发明提出的利用单碱基编辑技术创造除草剂抗性植物的方法GhBE3的电泳图;
图3为本发明提出的利用单碱基编辑技术创造除草剂抗性植物的方法GhEPSP突变的遗传转化图;
图4为本发明提出的利用单碱基编辑技术创造除草剂抗性植物的方法突变位点示意图;
图5为本发明提出的利用单碱基编辑技术创造除草剂抗性植物的方法草甘膦耐受性检测示意图。
具体实施方式
下面将结合本发明的实施例,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。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参照图1-5,图2中泳道1、2是GhBE3检测,泳道CK是阴性对照,泳道M是5K的marker,图3中的罗马字编号分别是:Ⅰ.共培养阶段、Ⅱ.选择培养阶段、Ⅲ.愈伤阶段、Ⅳ.分化培养阶段、Ⅴ.生根培养阶段、Ⅵ.营养液培养和Ⅶ-Ⅸ.转基因植株在温室生长。
序列表SEQ ID NO.1是陆地棉基因组高效转化载体GhBE3的核苷酸序列,序列长度为17150bp。
序列表SEQ ID NO.2是靶标sgRNA的核苷酸序列,序列长度为20bp。
利用单碱基编辑技术创造除草剂抗性植物的方法,包括以下步骤:
1)GhEPSP基因的sgRNA设计:选择陆地棉5-烯醇式丙酮酰莽草酸-3-磷酸合酶(5-Enolpyruvylshikimate-3-phosphate synthase,EPSP合酶)为验证基因,利用在线软件CRISPR-P(http://cbi.hzau.edu.cn/cgi-bin/CRISPR)(Lei et al 2014)在基因外显子区域设计sgRNA靶标序列,并且在编辑窗口内C-T突变改变氨基酸功能的靶点,最终选择1个sgRNA用来构建单碱基系统植物表达载体;
sgRNA的序列如下:
sgRNA | 序列 |
sgRNA1 | GGGTGGTCTTCCCGGTGGAA |
2)sgRNA与GhBE3载体的连接:靶标插入GhBE3载体序列为tRNA-sgRNA-gRNA的重复序列,需要中间载体转换,第一次PCR的引物如下:
pRGEB32-7/S:AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG,将sgRNA加到反向引物的接头上,GhEPSP/AS:TTCCACCGGGAAGACCACCCTGCACCAGCCGGGAAT,下划线的碱基是sgRNA序列,以PGTR载体(Xie et al.2015)为模板进行PCR扩增tRNA序列,获得tRNA+sgRNA利用一步克隆试剂盒(Vazyme,C112-01/02)将tRNA+sgRNA片段分别连接到pGREB32-GhU6-7载体的BsaI酶切位点处,利用HpaI和SbfI双酶切pGREB32-GhU6-7,将目的片段连接到GhBE3载体的HpaI和SbfI双酶切位点处;
PCR体系如下:
组分 | 体积 |
10×taq Buffer | 2 |
dNTP | 0.3 |
Inf pRGEB32-7S | 0.2 |
Inf CLA 2As | 0.2 |
Taq | 0.2 |
PCR1 | 0.5 |
PCR2 | 0.5 |
ddH2O | 16.1 |
PCR条件如下:
温度 | 时间 | |
预变性 | 95℃ | 4min |
变性 | 95℃ | 30s |
退火 | 55℃ | 30s |
延伸 | 72℃ | 20s |
循环 | 3℃ | |
变性 | 95℃ | 30s |
退火 | 60℃ | 30s |
延伸 | 72℃ | 20s |
循环 | 27℃ | |
最后延伸 | 72℃ | 5min |
保存 | 4℃ | ∞ |
3)农杆菌介导的遗传转化:
A、将剥好的棉花种子(品种为Jin668,专利申请号201510833618.0)用0.1%升汞杀菌,无菌水清洗数次后放入无菌苗培养基中,28℃暗培养1天,挑去种皮,将苗扶正,在28℃,暗培养4d;
B、将下胚轴切成小茎段,用活化后的农杆菌侵染,弃菌液,并吹干;
C、将下胚轴平铺在放有滤纸的共培养培养基中,于20℃,暗培养1d;
D、将下胚轴转入到附加2,4-D的愈伤组织诱导培养基中,放入光照培养室,25d左右用新鲜愈伤组织诱导培养基继代培养一次;
E、当愈伤组织长成米粒状颗粒,转入分化培养基中,进一步分化成胚状体;
F、将分化出的小苗继代到生根培养基中,直至长成生根良好健康的小苗;
G、将小苗转到清水中,进行炼苗,一周后,转移到到温室;
转化所用的培养基组分及配比如下:
无菌苗培养基:1/2MS大量元素,15g/L葡萄糖,2.5g/L的Phytagel;pH:6.1-6.2。
愈伤组织诱导培养基:MSB+24-D 0.1mg/L+KT 0.1mg/L+3%Glucose+0.3%Phytagel;pH:5.85-5.95。
农杆菌活化培养基:胰化蛋白胨5g/L+NaCl 5g/L+MgSO4.7H2O 0.1g/L+KH2PO4+0.25g/L+甘露醇5g/L+甘氨酸1.0g/L;pH:5.85-5.95。
共培养培养基:MSB+2,4-D 0.1mg/l+KT 0.1mg/l+50mg/l AS+3%Glucose+0.25%Phytagel,pH5.8。
选择培养基:MSB+2,4-D 0.1mg/L+KT 0.1mg/L+3%Glucose+0.3%Phytagel,卡那霉素50mg/L和头孢霉素400mg/L;pH:5.85-5.95。
分化培养基:MSB培养基中去掉NH4NO3,将KNO3用量加倍+Gln 1.0g/L+Asn 0.5g/L+IBA 0.5mg/L+KT 0.15mg/L+3%Glucose+0.25%Phytagel,pH:6.1-6.2。
生根培养基:1/2MS无机盐+B5有机物,15g/L葡萄糖,2.5g/L的Phytagel;pH:5.90-5.95;MSB的成分如下:MS培养基+B5维生素。
4)测序检测编辑:提取的棉花嫩叶阳性基因组DNA,以阳性DNA为模板,扩增GhEPSP基因,然后将PCR片段连入pGEM-T easy载体,将连接产物热激转化大肠杆菌感受态TOP10,挑取的单克隆进行阳性检测并进行Sanger测序,将测序结果与靶标序列比对;
5)叶片涂抹草甘膦:将农达(孟山都)稀释200倍,涂抹棉花叶片,一周后观察叶片药害情况。
本发明的有益效果是:该利用单碱基编辑技术创造除草剂抗性植物的方法,在棉花中首次成功应用棉花基因组特性的单碱基编辑系统对棉花GhEPSP实现单碱基突变,突变位点未见其他作物报道,通过叶片草甘膦涂抹实验,表现出一定的草甘膦耐受性,提高了棉田除草效率,降低了人工除草成本,防范棉田超级杂草,解决了棉花自身单碱基突变(非转外源基因)创制的棉花材料不具备草甘膦抗性的问题。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (2)
1.利用单碱基编辑技术创造除草剂抗性植物的方法,其特征在于,包括以下步骤:
1)GhEPSP基因的sgRNA设计:选择陆地棉5-烯醇式丙酮酰莽草酸-3-磷酸合酶(5-Enolpyruvylshikimate-3-phosphate synthase,EPSP合酶)为验证基因,利用在线软件CRISPR-P(http://cbi.hzau.edu.cn/cgi-bin/CRISPR)(Lei et al 2014)在基因外显子区域设计sgRNA靶标序列,并且在编辑窗口内C-T突变改变氨基酸功能的靶点,最终选择1个sgRNA用来构建单碱基系统植物表达载体;
2)sgRNA与GhBE3载体的连接:靶标插入GhBE3载体序列为tRNA-sgRNA-gRNA的重复序列,需要中间载体转换,第一次PCR的引物如下:
pRGEB32-7/S:AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG,将sgRNA加到反向引物的接头上,GhEPSP/AS:TTCCACCGGGAAGACCACCCTGCACCAGCCGGGAAT,下划线的碱基是sgRNA序列,以PGTR载体(Xie et al.2015)为模板进行PCR扩增tRNA序列,获得tRNA+sgRNA利用一步克隆试剂盒(Vazyme,C112-01/02)将tRNA+sgRNA片段分别连接到pGREB32-GhU6-7载体的BsaI酶切位点处,利用HpaI和SbfI双酶切pGREB32-GhU6-7,将目的片段连接到GhBE3载体的HpaI和SbfI双酶切位点处;
3)农杆菌介导的遗传转化:
A、将剥好的棉花种子(品种为Jin668,专利申请号201510833618.0)用0.1%升汞杀菌,无菌水清洗数次后放入无菌苗培养基中,28℃暗培养1天,挑去种皮,将苗扶正,在28℃,暗培养4d;
B、将下胚轴切成小茎段,用活化后的农杆菌侵染,弃菌液,并吹干;
C、将下胚轴平铺在放有滤纸的共培养培养基中,于20℃,暗培养1d;
D、将下胚轴转入到附加2,4-D的愈伤组织诱导培养基中,放入光照培养室,25d左右用新鲜愈伤组织诱导培养基继代培养一次;
E、当愈伤组织长成米粒状颗粒,转入分化培养基中,进一步分化成胚状体;
F、将分化出的小苗继代到生根培养基中,直至长成生根良好健康的小苗;
G、将小苗转到清水中,进行炼苗,一周后,转移到到温室;
4)测序检测编辑:提取的棉花嫩叶阳性基因组DNA,以阳性DNA为模板,扩增GhEPSP基因,然后将PCR片段连入pGEM-T easy载体,将连接产物热激转化大肠杆菌感受态TOP10,挑取的单克隆进行阳性检测并进行Sanger测序,将测序结果与靶标序列比对;
5)叶片涂抹草甘膦:将农达(孟山都)稀释200倍,涂抹棉花叶片,一周后观察叶片药害情况。
2.根据权利要求1所述的利用单碱基编辑技术创造除草剂抗性植物的方法,其特征在于,所述转化所用的培养基组分及配比如下:
无菌苗培养基:1/2MS大量元素,15g/L葡萄糖,2.5g/L的Phytagel;pH:6.1-6.2。
愈伤组织诱导培养基:MSB+24-D 0.1mg/L+KT 0.1mg/L+3%Glucose+0.3%Phytagel;pH:5.85-5.95。
农杆菌活化培养基:胰化蛋白胨5g/L+NaCl 5g/L+MgSO4.7H2O 0.1g/L+KH2PO4+0.25g/L+甘露醇5g/L+甘氨酸1.0g/L;pH:5.85-5.95。
共培养培养基:MSB+2,4-D 0.1mg/l+KT 0.1mg/l+50mg/l AS+3%Glucose+0.25%Phytagel,pH5.8。
选择培养基:MSB+2,4-D 0.1mg/L+KT 0.1mg/L+3%Glucose+0.3%Phytagel,卡那霉素50mg/L和头孢霉素400mg/L;pH:5.85-5.95。
分化培养基:MSB培养基中去掉NH4NO3,将KNO3用量加倍+Gln 1.0g/L+Asn 0.5g/L+IBA0.5mg/L+KT 0.15mg/L+3%Glucose+0.25%Phytagel,pH:6.1-6.2。
生根培养基:1/2MS无机盐+B5有机物,15g/L葡萄糖,2.5g/L的Phytagel;pH:5.90-5.95;MSB的成分如下:MS培养基+B5维生素。
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