黄瓜离体子叶切块培养直接分化花芽

４８小时龄黄瓜幼苗的子叶切块接种于附加２．０ｍｇ／ＬＢＡ及１．０ｍｇ／ＬＡｇＮＯ３的修改ＭＳ培养基上,培养约６０天后在子叶切块的近轴端可直接形成雄花芽,其频率达７．７％。     

Plant regeneration from different explants of neem

When different seedling explants, i.e. hypocotyl, epicotyl, cotyledonary node, root-shoot zone, cotyledon, leaves and roots from 7-day-old seedlings of neem were cultured on Murashige and Skoog's medium supplemented with 2 mg l⁻¹ benzyladenine and 0.1 mg l⁻¹indole-3-acetic acid, shoot buds were initiated from all the explants tested, with leaf explants producing the highest average number of shoots/explant. The regenerated shoots were further subcultured and later could be rooted on a medium supplemented with indole butyric acid (1 mg l⁻¹) and complete plants could be obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean)

Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.     

Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L.

Summary An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.Abbreviations BA benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog basal medium - LSD least significant difference - NOS nopaline synthase promoter - NPTII neomycin phosphotransferase (EC 2.7.1.95) - CaMV35S cauliflower mosaic virus promoter - GUS beta-glucuronidase (EC 3.2.1.31) - LB Luria Broth base - CTAB hexadecil trimethyl ammonium bromide - PCR polymerase chain reaction - X-gluc 5-bromo-4-chloro-3-indolyl-glucuronide     

Regeneration of transgenic plants expressing the GFP gene from rape cotyledonary and leaf explants: Effects of the genotype and ABA

Cotyledons of five-day-old seedlings and leaves of 6-week-old plants of two rape cultivars (Brassica napus L., cvs. Westar and Podmoskovnyi) were co-cultured with the culture of Agrobacterium tumefaciens cells comprising the genetic construct with the marker gfp gene, on Murashige and Skoog nutrient medium supplemented with benzyladenine, NAA, and ABA in various combinations. A capacity for regeneration on both types of explants was rather high, but leaf explants produced weakly differentiated shoots and most of them were vitrificated. On cotyledonary explants of transformed rape plants of both cultivars expressing the gfp gene, regeneration frequency was 70%. On leaf explants, it was much lower (47% in cv. Westar and 28% in cv. Podmoskovnyi). The gfp gene was expressed on all stages of shoot development. On primary, starting differentiation calli, we observed the strongest fluorescence of GFP in meristematic and vascular tissues. On leaf blades, GFP fluorescence was much brighter in old than young leaves; often it was observed only in the cell groups; it. PCR analysis of seed generation of transformants showed that some plants did not follow the Mendelian inheritance of a monogenic trait (transgene) in self-pollinated plants. This phenomenon could be explained as a result of meiotic recombination or production of genotypic chimeric organisms at regeneration.     

PSAG12-ipt嵌合基因转化马铃薯体系的研究

以根癌农杆菌介导法将PSAG12-ipt嵌合基因导入马铃薯栽培品种,对影响马铃薯遗传转化的多种因素进行系统研究.结果表明:马铃薯茎段分化效率高于叶片,马铃薯愈伤诱导和芽分化最适培养基为MS+6-BA 0.25mg/L+NAA 0.25mg/L+2,4-D 0.25mg/L,添加1%Na2SO3能有效防止褐化;茎段愈伤诱导和分化苗生根最适的Kan浓度分别为50mg/L和75mg/L;外植体预培养2d,OD600为0.2～0.5的农杆菌浓度侵染8min、共培养3d后进行选择培养能有效地提高植株再生能力.用PSAG12和ipt双重PCR检测再生植株,阳性转化率为65.8%.Southern blotting结果表明,转基因植株多以单拷贝形式整合进马铃薯基因组中.     

Shoot Regeneration from Cultured Leaf Explants of Lycium barbarumand Agrobacterium-Mediated Transformation1

A method for fast plant regeneration via organogenesis directly from Lycium barbarumleaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2 mg/l BA and 0.5 mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1 mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n = 24). Using the optimized regeneration system, the genetic transformation of L. barbarumwas carried out mediated by Agrobacterium tumefaciensEHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens.The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptIIgene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2 mg/l 2,4-D and 0–100 mg/l kanamycin.     

An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.)

An efficient transformation and regeneration system was established for the production of transgenic spinach (Spinacia oleracea L.) plants. Cotyledon explants were infected with Agrobacterium tumefaciens strain LBA4404 carrying the selectable marker gene, neomycin phosphotransferase II (nptII), and the reporter gene smgfp, encoding soluble-modified green-fluorescent protein, driven by the cauliflower mosaic virus 35S promoter. The infected explants were cultured on Murashige and Skoog medium, containing 1 mg/l benzyladenine and 0.4 mg/l naphthaleneacetic acid. Shoots were regenerated on selection medium containing 50 mg/l kanamycin. Regenerated kanamycin-resistant shoots were rooted on medium containing 1 mg/l indolebutyric acid and subsequently grown in soil in the greenhouse. Southern blot analysis indicated that the smgfp gene had been integrated into the spinach genome. Northern and Western blots showed that the smgfp gene was expressed in progeny plants. Received: 31 March 1998 / Revision received: 27 September 1998 / Accepted: 10 Ocotber 1998     

Tissue Cultures of Phaseolus vulgaris L.

Abstract

Callus production and plant regeneration from different explants of Phaseolus vulgaris L. cv. Giza are reported. Calli cultures were induced from leaf, hypocotyl, embryo and root explants. Rapid growth of callus was achieved by leaf explants cultured on MS salts, B5 vitamins and supplemented with 2,4— dichlorophenoxyacetic acid (2, 4—D)+0.5 mg/l kinetin (kin). Addition of casein hydrolysate at 2 g/l to maintenance medium enhanced callus growth and hindered the early appearance of necrotic parts. This report also provides a detailed method for production of multiple shoots directly from the wounded edges of immature cotyledon explant via organogenesis on 1 mg/l benzyladenine (BA) or indirectly on 0.5 mg/l naphthaleneacetic acid (NAA)+2 mg/l BA. The regeneration of bean plants through the two ways described here (direct or indirect) may be of use in genetic improvement of bean.     

Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants

Transgenic muskmelon (Cucumis melo L.) plants were produced efficiently by inoculating cotyledon explants with Agrobacterium tumefaciens strain LBA4404 bearing a Ti plasmid with the NPT II gene for kanaymcin resistance. After co-cultivation for three days, expiants were transferred to melon regeneration medium with kanamycin to select for transformed tissue. Shoot regeneration occurred within 3–5 weeks; excised shoots were rooted on medium containing kanamycin before transferring to soil. Morphologically normal plants were produced in three months. Southern blot analysis confirmed that ca. 85% of the regenerated plants contained the NPT gene. Dot blot analysis and leaf callus assay of progeny of transgenic plants verified transmission of the introduced gene(s) to the next generation. Factors affecting transformation efficiency are discussed.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - IAA indole 3 acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NPT II neomycin phosphotransferase II     

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T₀ generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T₁ seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T₁ generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Adventitious shoot regeneration from cotyledonary explants of rapid-cycling fast plants of <Emphasis Type="Italic">Brassica rapa</Emphasis> L

Rapid-cycling fast plants (Brassica rapa; RCBr) is also known as Wisconsin Fast Plant and is widely used in K-12 and undergraduate studies. RCBr has a short generation time (seed-to-seed in 30–60 days), which allows for the completion of experiments in a semester. Previous studies have shown that cotyledonary explants with attached petioles are capable of generating shoots. However, there is no published adventitious shoot regeneration protocol to date. Sterile cotyledonary explants were excised; all edges and petioles were removed. Five-day-old cotyledonary explants produced shoots on a Murashige and Skoog medium containing 1.5 mg/L thiadiazuron (TDZ) and 0.5 mg/L 1-naphthaleneacetic acid (NAA) (FPM I) at a mean rate of 8.8%. This rate increased to 14.8% in explants placed on FPM I medium supplemented with 5.0 mg/L silver nitrate (AgNO₃) (SRM 2). The rate increased to 32.5% when 5-day-old explants, excised from the part of the cotyledon nearest to the petiole, were placed adaxial side up on SRM 2 medium. The shoot regeneration rate increased to 44.5% using 4-day-old cotyledonary explants. A shoot regeneration rate of 23% was observed among 9-day-old leaf explants. Shoots from cotyledonary explants were elongated on basal medium with 0.5 mg/L NAA, rooted on basal medium, and later acclimatized. This is the first report of shoot regeneration from cotyledonary explants of rapid-cycling Brassica rapa without pre-existing meristematic tissues.     

农杆菌介导的大豆高频遗传转化

大豆(Glycine max(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题.为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2 mg/L 6-BA和5 mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01 mg/L TDZ和2mg/L glufosinate的液体培养基中筛选,并每周更换一次培养液.得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1～2个基因拷贝数.该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论.     

强德勒红心柚离体培养与植株再生体系的建立

以强德勒红心柚（Citrus grandis Osbeckcv. Chandler）种子萌发的无菌苗为材料,选取子叶、子叶节段、上胚轴、带芽的茎段进行离体培养研究。结果表明：子叶节段是诱导丛生芽的最佳外植体,诱导率100%;诱导丛生芽的最佳培养基为MS＋6-BA2.0mg/L＋NAA0.05mg/L＋蔗糖30g/L＋活性炭0.4g/L,丛生芽增殖可达11.2倍;最适生根培养基为1/2MS＋NAA0.5mg/L,生根率达100%,移栽15d后成活率100%。     

农杆菌介导的大豆高频遗传转化

大豆(Glycinemax(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题。为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2mg/L6-BA和5mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01mg/LTDZ和2mg/Lglufosinate的液体培养基中筛选,并每周更换一次培养液。得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1~2个基因拷贝数。该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论。     

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.)

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.     

一种快速有效的741杨离体叶片再生芽方法

在对杂交品种741杨(Populus alba(P.davidiana×P.simonii)×P.tomentosa)进行农杆菌介导法转基因的试验中发现了一种快速有效的叶片再生芽的方法.首先叶片外植体在培养基Ⅰ(MS培养基添加0.5 mg/L BA和1.0 mg/L 2,4-D)上培养2～3 d,再转移到培养基SH(MSmedium containing 2.0 mg/L of BA and 0.1 mg/L of NAA)上培养10 d,然后再转移到培养基Ⅱ(MSmedium with 0.5 mg/L of BA)上,培养大约5 d之后86.7%的叶片外植体产生的芽,每片叶片外植体(1 cm×1 cm)可产生40～50个芽.但是,如果叶片外植体在培养基Ⅰ上培养的时间长于5 d,再依次转移到培养基SH和Ⅱ上,则叶片会产生大量根.     

Enhanced anthocyanin synthesis in foliage plant Caladium bicolor

A protocol was developed for Agrobacterium-mediated genetic transformation of monocotyledon foliage plant Caladium bicolor cv. Jackie Suthers using leaf disc and petiole as the explants. The explants were inoculated with Agrobacterium strain LBA4404 harboring a binary vector with the maize anthocyanin regulatory gene Lc under the control of the cauliflower mosaic virus promoter. Callus formation was induced in MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (6-BA), 0.1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose and kanamycin 50 mg/l for selection. Resistant calli were induced for shoot generation in MS medium with 2 mg/l 6-BA and 0.2 mg/l alpha-naphthaleneacetic acid. As much as 10% of the explants gave rise to kanamycin-resistant shoots with our procedure. Transformed plants had enhanced anthocyanin accumulation in the roots, leaves and stems (epidermis and vascular bundles). Integration of the transgene into the host genome was confirmed by genomic Southern blot hybridization, and RNA blot hybridization analysis indicated that the expression of the transgene correlated with anthocyanin accumulation. This investigation illustrates the utility of anthocyanin regulatory genes in the genetic manipulation of the color of foliage plants. It also supports the premise that the Lc gene can be used as a powerful non-destructive cell autonomous visual marker in a wide variety of plants, as exemplified by the perfect symmetrical half-green/half-red plant presumably derived from the symmetrical division of one transgenic and one non-transgenic precursor meristematic cell.