药用野生稻TAC克隆转化籼稻的体系初探

采用已构建的药用野生稻TAC文库,比较研究4个籼稻品种在不同培养基的诱导率及愈伤组织对潮霉素抗性。通过2种不同诱导方法产生的愈伤组织,将携带药用野生稻大片段DNA的TAC克隆转化到籼稻品种中的结果表明,除了‘粤香占’以外,其它3个品种在心诱导培养基中添加微量B,后,其愈伤诱导率最高。不同品种的潮霉素筛选适合浓度存在差异,其中‘华粳籼74’的适合筛选浓度为50mg．L-1,‘粤香占’为40mg．L-1。用预培养5d的愈伤组织进行遗传转化,在潮霉素筛选之前先以头孢拉定抑菌处理的,容易获得转基因植株。4个品种中以‘粤香占’的抗性愈伤组织筛选率和分化率最高,分别为22．58％和14．86％。分子检测11株转基因水稻的结果表明,其中8株含有抗性标记基因。据此认为,药用野生稻TAC文库在水稻创新育种中可能有一定的利用前景。     

不同抗生素对雪莲愈伤组织生长的影响

以雪莲叶片为外植体,探讨了卡那霉素(kanamycin,Kan)、潮霉素(hygromycin B,Hyg)、羧苄青霉素(car-benidillin,Car)3种抗生素对雪莲愈伤组织诱导、生长及分化的影响,以确定农杆菌介导的遗传转化研究中筛选剂和抑菌剂的最适浓度。结果表明:40mg/L的卡那霉素已抑制雪莲愈伤组织生长,当卡那霉素为50mg/L时,愈伤组织的生长基本停止;8.0mg/L潮霉素能够有效抑制雪莲愈伤组织的生长,当潮霉素为20mg/L时则生长的愈伤组织块较小、褐化、甚至死亡。同时,低浓度(0.5～2.0mg/L)的潮霉素可以提高雪莲愈伤组织的分化率;作为农杆菌抑菌剂,不同浓度羧苄青霉素对雪莲愈伤组织生长的影响差异极显著,当羧苄青霉素的浓度超过400mg/L时对雪莲愈伤组织的出愈及生长均有明显的抑制作用。     

羽衣甘蓝下胚轴农杆菌介导遗传转化体系的优化

以红鸥羽衣甘蓝下胚轴为试材,首先优化了不定芽分化体系,其次探讨了农杆菌介导遗传转化过程中农杆菌侵染浓度、侵染时间对不定芽分化率的影响及不定芽分化和生根过程中潮霉素B筛选压力,最后对抗性植株进行了潮霉素B筛选基因的PCR检测。结果表明,在MS+6-BA 5.0 mg/L+Ag NO3 9.0 mg/L培养基中不定芽分化率最高,为84.17%;农杆菌侵染浓度OD600值为0.5、侵染5 min时利于遗传转化,分化率为69.17%;不定芽分化和生根过程中潮霉素B筛选压力分别为4.0 mg/L和30.0 mg/L;潮霉素B筛选基因的PCR鉴定结果,在预期的602 bp处出现了条带,初步证明潮霉素B筛选基因已整合到羽衣甘蓝基因组中。     

浮萍遗传转化体系的建立和优化

以浮萍为实验材料,成功建立了其稳定的遗传转化体系。实验结果显示:EHA105农杆菌品系对浮萍愈伤组织的侵染效率较高;乙酰丁香酮的最佳作用浓度为100μM;抑菌抗生素的浓度为300 mg/L;潮霉素固体筛选浓度为60 mg/L,液体筛选浓度为1~10 mg/L;采用该体系对浮萍愈伤组织进行转化,得到3株抗性植株,分子鉴定结果表明该抗性植株为转基因植株,说明该遗传转化体系可行,为浮萍基因工程的研发提供参考。     

根癌农杆菌介导甘蔗遗传转化Bt(cry1Ab)基因北大核心CSCD

以自育甘蔗品种"川蔗23号"诱导的胚性愈伤为受体材料,首次用较低的根癌农杆菌侵染浓度(OD600=0.1左右)与较短的侵染时间(1.5 min)成功实现甘蔗遗传转化Bt(cry1Ab)基因。结果表明,胚性愈伤分化与小芽生根最适潮霉素筛选浓度分别为20 mg/L和30 mg/L;愈伤组织胚性的一致性是影响筛选阶段愈伤再生的重要因素,培养40 d的愈伤组织为转化最适受体;转化材料经连续的潮霉素抗性筛选后,获得65株抗性植株,通过特异性引物的PCR扩增检测,有2株获得阳性条带,初步表明目的基因已整合到甘蔗染色体基因组中。     

影响根癌农杆菌介导水稻转化的因素分析

根癌农杆菌与来自水稻成熟种子盾片的愈伤组织共培养,将GUS基因导入水稻愈伤组织,并获得了转基因植株。通过比较影响根癌农杆菌转化频率的各种因素,表明激素配比为2,4-D1mg/L、TDZ0.5mg/L、NAA1mg/L时,可以大大促进籼稻愈伤组织的分化能力;酚类化合物的加入使农杆菌的转化频率提高8.9%-23.5%;共培养时农杆菌的稀释方式及适当调整潮霉素（hygB）的使用浓度影响到农杆菌的转化频率。     

‘光叶蔷薇’器官间接再生体系的建立及GUS基因转化的初步研究

以‘光叶蔷薇’(Rosa wichuriana‘Basye's thornless’)无菌苗的顶生幼嫩小叶为外植体,探讨了其愈伤组织诱导及植株再生的方法。结果表明,高浓度的生长素NAA能诱导外植体产生愈伤组织;由NAA诱导的愈伤组织在附加TDZ的MS培养基上,先暗培养再进行光照培养可直接分化出不定芽。诱导愈伤组织的最佳NAA浓度是7.0 mg/L、暗培养时间为10 d,而最佳分化培养基是MS+5.0 mg/L TDZ+30 g/L葡萄糖+2.5 g/L GEL,分化率达18.34%。以诱导产生的愈伤组织为侵染受体,初步建立了‘光叶蔷薇’GUS基因转化体系。农杆菌菌液浓度OD600值为0.5、侵染30 min、共培养2 d、乙酰丁香酮的浓度为50μmol/L是‘光叶蔷薇’愈伤组织转基因的最优条件。     

不同抗生素对金发草愈伤组织生长分化的影响

该研究以金发草愈伤组织为材料,通过分析比较不同抗生素种类(卡那霉素、潮霉素、头孢噻呋钠和氨苄青霉素)和浓度对金发草愈伤组织生长分化的影响,来确定适用于金发草遗传转化体系中的抗性筛选剂和抑菌剂。结果表明：(1)金发草愈伤组织对卡那霉素很敏感,且其分化率随着卡那霉素浓度的增加显著减少( P＝0.01)。当卡那霉素浓度为10 mg·L-1时,金发草愈伤组织的生长分化受到明显抑制,且有大量的白化苗形成,但分化率仍有36.56％;当卡那霉素浓度为15 mg·L-1时,金发草愈伤组织的分化率为11.94％,只有很少部分的愈伤分化出绿色的丛生苗;当卡那霉素浓度为20 mg·L-1时,金发草愈伤组织基本褐化死亡,分化率仅为2.26％。因此,浓度为15 mg·L-1的卡那霉素适合作为金发草遗传转化体系中的抗性筛选剂。(2)金发草愈伤组织对潮霉素的敏感性要比卡那霉素弱,且潮霉素对金发草愈伤组织分化率的影响小,但毒害作用大。因此,潮霉素不适合作为金发草遗传转化体系中的抗性筛选剂。(3)300 mg·L-1的头孢霉素和氨苄青霉素对金发草愈伤组织生长分化影响很小且能有效抑制杂菌的生长,较高浓度的氨苄青霉素对金发草愈伤组织的抑制作用不太明显。因此,300 mg·L-1的头孢霉素和较高浓度的氨苄青霉素均可作为金发草遗传转化体系中的抑菌剂。该研究确定了适用于农杆菌介导的金发草遗传转化体系中的抗性筛选剂和抑菌剂,为金发草的遗传改良及功能性基因的研究奠定了基础。     

金山绣线菊遗传转化体系的建立

以金山绣线菊愈伤组织为受体材料,采用组织培养的方法,在附加不同浓度TDZ的1／2MS培养基上诱导培养,获得再生植株。在附加0．03mg·L^-1 TDZ的培养基上获得了92．5％不定芽的再生率,且再生芽发育良好。选择抗生素筛选试验的结果表明：金山绣线菊愈伤组织对潮霉素较为敏感,在培养基中添加浓度为5～35mg·L^-1的潮霉素均对愈伤组织分化影响较大,潮霉素浓度为5mg·L^-1时,4N时间可使外植体全部褐化死亡;在培养基中添加0～100mg·L^-1的卡那霉素,不同浓度卡那霉素均对愈伤组织分化产生一定程度的影响,当卡那霉素浓度为80mg·L^-1时,愈伤组织基本不发生分化。由此确定卡那霉素为绣线菊遗传转化中适用的选择抗生素,最适选择压为80mg·L^-1。抑菌抗生素的筛选试验结果表明：200mg·L^-1的头孢霉素和200mg·L^-1。的羧苄霉素都能有效抑制农杆菌菌株LBA4404的生长,却对金山绣线菊愈伤组织的芽分化影响不大,可确定为适宜的抑菌抗生素。利用农杆菌介导法对金山绣线菊愈伤组织进行遗传转化,得到卡那霉素抗性植株164株,并初步确定预培养1d、菌液稀释10倍、侵染4min、共培养2d为金山绣线菊最优遗传转化体系,为金山绣线菊的基因工程育种奠定基础。     

中国传统菊花品种‘小林静’再生及转化体系的建立

以中国传统菊花品种‘小林静’叶片为外植体,建立了‘小林静’较好的再生体系及遗传转化体系.结果表明,‘小林静’叶盘最适不定芽分化培养基为MS+2.0 mg/L 6-BA+1.5 mg/L NAA,不定芽分化率为92.76％,平均再生不定芽数为2.3767个;试管苗最佳生根培养基为1/2MS,生根率达100％.移栽采用灭菌的蛭石,再生苗移栽成活率达90％以上.采用根癌农杆菌C58C1介导的叶盘转化法进行‘小林静’的遗传转化试验,农杆菌OD600=0.5-0.6,侵染10 min后,将叶片外植体接种到MS+2.0 mg/L 6-BA+1.5mg/L NAA的培养基中黑暗共培养2d,之后转接到附加10 mg/L硫酸卡那霉素和400 mg/L羧苄青霉素的分化筛选培养基中进行转化细胞的筛选,待长出抗性芽后转接至生根培养基中进行培养,最终建立了菊花品种‘小林静’的遗传转化体系.     

Effects of hygromycin on cotton cultures and its application in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated cotton transformation

We have evaluated the effects of the antibiotic hygromycin B on cotton (Gossypium hirsutum L.) callus induction, callus proliferation, and seed germination. Nontransgenic cotyledon and hypocotyl showed obvious variance in tolerance to hygromycin. Cotyledons were more sensitive to hygromycin than hypocotyls. Hygromycin at 7.5 and 20 mg l⁻¹ completely inhibited callus initiation from cotyledon and hypocotyl explants, respectively. Nontransformed calli did not grow on media supplemented with 10 mg l⁻¹ hygromycin and were killed at 15 mg l⁻¹. In seed germination assay, the presence of 20 mg l⁻¹ hygromycin significantly suppressed shoot and root elongation of seedlings. This hygromycin concentration was applied to select regenerated transgenic plantlets and their progenies. Based on these results, we developed an efficient hygromycin selection protocol for Agrobacterium-mediated cotton transformation and regeneration.     

Development of transgenic rice pure lines by particle bombardment‐mediated transformation and genetic analysis‐based selection

Mature seed‐derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β‐glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)‐containing medium, resistant callus was transferred to hygromycin (30 mg/l)‐containing regeneration medium for plant regeneration. Twenty‐three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin‐containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment‐mediated transformation and through genetic analysis‐based selection.     

大麦转化体系的改进及TrxS基因的转化

以啤酒大麦品种“晋引6号”的幼胚为转化起始材料,用基因枪法将分别携带有目的基因(TrxS)和除草剂基因(筛选基因,Bar)的两个质粒进行了共转化,同时对基因转化的相关技术和植株再生的培养方案进行了优化。结果表明,受体材料宜选用预培养15d的幼胚;在培养前2周添加1mg／L ABA可抑制胚芽萌发而且有助于胚性愈伤组织的形成;1．0mg／L ZT与0．1mg／L IAA激素配比可有效促进愈伤组织的分化。利用优化的培养条件,经在含3～5mg／L筛选剂PPT的培养基上筛选、再生及生根培养。共在178块抗性愈伤组织上获得11株再生植株,再生率达到6．2％,经对T0、T1、T2代PCR、nested PCR和Southern杂交检测表明,TrxS基因已经稳定整合到大麦基因组中且遗传稳定、结构完整。     

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed‐derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β‐glucuronidase gene (gusA) and the snow‐drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin‐containing medium, resistant callus was transferred to hygromycin‐containing regeneration medium for plant regeneration. Twenty‐six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy‐three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis‐based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.     

农杆菌介导的高效玉米遗传转化体系的建立

为了建立玉米高频再生及高效遗传转化体系, 对影响玉米胚性愈伤组织诱导的11个因素及影响胚性愈伤分化的9个因素用正交实验方法进行研究。结果显示, 基因型对胚性愈伤诱导有极显著影响。6-BA、培养基、AgNO3、2,4-D、ABA对胚性愈伤诱导的影响达到显著水平。多重比较分析显示ABA 2 mg/L每间隔1代添加对胚性愈伤诱导率有显著影响。在影响分化的因素中, 基因型和6-BA浓度表现出极强的主效应, NAA、培养基、KT、2,4-D对分化产生显著影响。Southern blotting 分析表明, 25 mg/L潮霉素选择压下抗性愈伤率作为转化体系优化指标是可靠的。在影响转化效率的因素中, acetosyringone (AS)使用浓度因基因型不同而表现出敏感度差异, 共培养温度24~25℃、农杆菌浓度和浸泡时间0.7 OD×15 min, 以及pH值5.5~6.2是最高转化率的优选组合。在整合后的玉米遗传转化体系中, 黄早4和综31自交系以抗性愈伤率为指标的GUS基因稳定转化率分别达到48.6%和46.2%。     

水稻悬浮细胞再生及根癌农杆菌介导转化的影响因素

已经成功报道的农杆菌介导的水稻遗传转化多以活力较高的胚性愈伤为材料,很少以水稻悬浮细胞作为受体．另外,利用农杆菌转化多数都是通过浸泡的方式进行侵染．本实验利用滴加浸染法进行农杆菌介导转化水稻悬浮细胞,探讨影响 DNA 转化效率的因素．研究显示,在转化前,将水稻悬浮细胞在愈伤诱导培养基上培养1～2周,诱导产生直径为2～3 mm的微小愈伤组织对转化非常重要.微小愈伤组织大小不应小于 2 mm;对悬浮细胞短时间培养不但会缩短植株再生时间,而且会提高转化效率．此外,侵染农杆菌的浓度、侵染时间和不同侵染方法也影响 T-DNA 插入基因组的效率．用 1 ml Ａ600值为 0.5 浓度的农杆菌悬液滴加在水稻悬浮细胞诱导的愈伤,培养3 d或直到可见农杆菌菌落,此方法可以得到较高转化效率．将再生的潮霉素抗性的转化植株在含有 50 mg/L 潮霉素的分化和生根培养基中筛选得到,并对转化植株 gus 基因的表达进行 PCR 检测.结果显示,用 Ａ600值为 0.5 浓度的农杆菌浸泡侵染 20 min和滴加浸染法,分别得到PCR阳性植株率为 70% 和92%.     

Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato

Genetic transformation of tomato was first accomplished around 30 years ago. However, variability in transformation efficiency of distinct cultivars exists and to some extent remains a bottleneck for transgenic research. This study reports strategies to improve transformation efficiency in tomato and investigates regeneration capacity of transgenic plants under different selection regimes and hormonal applications. Tomato cv. Rio Grande was used as plant material and hygromycin and phosphinothricin (PPT) were used as selection agents. We found that cv. Rio Grande inherently produced a significant number of abnormal (“blind”) shoots lacking an apical meristem. Replacing cytokinin zeatin riboside with 6-benzylaminopurine (BAP) reduced the number of blind shoots although it slightly prolonged regeneration time. Survival rate of calli and shoots was very low using PPT as selection, whereas regeneration was achieved using hygromycin. Delayed application of hygromycin selection following co-cultivation with Agrobacterium tumefaciens improved the overall callus and shoot production. In vivo GFP fluorescence was detected to investigate the development of transgenic tissues using different hygromycin selection regimes. Higher transformation frequency was achieved when explants were continuously exposed to selection agents immediately following the pre-selection stage. Reducing the selection period followed by a non-selection stage increased the number of shoots, but these shoots were mostly non-transgenic. Thus, although less stringent selection, as expected, encouraged regeneration of shoots from calli, it did not improve transformation efficiency. Omitting selection altogether greatly reduced the efficiency of transformation. It was concluded that BAP is more suitable for normal shoot development, and that delayed selection followed by continuous selection results in higher transformation frequency.

     

以根为外植体建立大蒜的组织培养体系

以国内4个大蒜栽培品种为材料,建立了以根为外植体的再生体系。将蒜瓣去皮后灭菌消毒,萌发后选取苗龄为5~7 d的无菌苗的根接种到含不同激素配比的培养基中进行愈伤组织诱导,发现MS+2,4-D 1 mg/L+2ip 0.1 mg/L组合愈伤诱导效率最高,平均为56.06%;愈伤组织经过2~3次继代培养,选取胚性愈伤组织置于不同分化培养基上进行培养,2~3个月后可见小芽产生,分化培养基为MS+KT 1 mg/L时,植株再生效率最高,平均达到35.01 %。本研究建立了一种以根为外植体的高效的大蒜愈伤诱导和再生体系,为大蒜遗传转化体系的建立打下良好基础。