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1.
Rohit Jain Arunima Sinha Sumita Kachhwaha S. L. Kothari 《Journal of plant biochemistry and biotechnology.》2009,18(2):249-252
An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l?1), Kn (0.5 mg l?1) and PG (0.5 mg l?1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l?1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l?1) and PG (0.5 mg l?1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l?1) + PAA (0.5 mg l?1) + CC (2 mg l?1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each. 相似文献
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High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize 总被引:6,自引:0,他引:6
Zhao Zuo-yu Gu Weining Cai Tishu Tagliani Laura Hondred David Bond Diane Schroeder Sheryl Rudert Marjorie Pierce Dottie 《Molecular breeding : new strategies in plant improvement》2002,8(4):323-333
A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo3 in the infection medium and adding AgNo3 in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×109 c.f.u. ml–1 for bacterium inoculation; 100 mg l–1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T0 plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T1 plants were analyzed and transmission of transgenes to the T1 generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II. 相似文献
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Pythium guiyangense is a mosquito pathogen, and has been proved to be a promising agent for biological control of mosquitoes. In order to develop the strains adaptable to different ecological environment having stable virulence to mosquito larvae, and being able to prolong the shelf life, an effort was made on transforming the fungus by using homologous or heterologous virulence genes. In this paper, a genetic transformation experiment of P. guiyangense mediated by Agrobacterium tumefaciens is reported. As a result, an A. tumefaciens mediated genetic transformation system was established successfully. 相似文献
4.
根癌农杆菌介导的金边狗牙根遗传转化条件的优化 总被引:3,自引:0,他引:3
胡繁荣 《植物资源与环境学报》2005,14(2):15-18
为建立根癌农杆菌(Agrobacterium tumefaciens,菌(L.)Pers.]最佳遗传转化体系,以葡萄糖醛酸糖苷酶(GUS)基因的瞬间表达率为指标,从愈伤组织继代时间、根癌农杆菌侵染时间、负压条件及光照时间等方面进行了筛选.结果表明,根癌农杆菌介导的金边狗牙根最佳的转化体系是:以继代培养2周的愈伤组织为起始材料,根癌农杆菌介导感染10 min,经负压处理(抽拉20次)并在全光条件下共培养转化. 相似文献
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Efficient transformation of Agrobacterium tumefaciens by electroporation 总被引:16,自引:0,他引:16
High-voltage electroporation was used to transform Agrobacterium tumefaciens strains A136 and A348, reaching the efficiency of 1-3 x 10(8) transformants/micrograms DNA. Transformation frequency was dependent on the electrical field strength and the pulse length. No significant reduction in transformation efficiency was observed when the transforming DNA contained sites sensitive to endonuclease AtuCI of A. tumefaciens. 相似文献
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农杆菌介导的高效玉米遗传转化体系的建立 总被引:7,自引:0,他引:7
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%。 相似文献
8.
Prosanta Saha Karabi Datta Shuvobrata Majumder Chirabrata Sarkar Shyamsundar P. China Sailendra N. Sarkar Debabrata Sarkar Swapan K. Datta 《Plant Cell, Tissue and Organ Culture》2014,118(2):313-326
We have developed a reproducible method of Agrobacterium tumefaciens mediated stable genetic transformation of white jute (Corchorus capsularis cv. JRC 321) utilizing the shoot organogenesis potential of the shoot tip apical meristem. A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 was used in the transformation experiments. The explants were subjected to varying durations of preculture and cocultivation with A. tumefaciens in the presence of acetosyringone in order to optimize the conditions conducive for the highest expression of transgene. A schedule of 1 day preculture of shoot tips followed by 3 days cocultivation was optimized for Agrobacterium mediated stable genetic transformation of C. capsularis cv. JRC 321. The optimized lethal doses of the antibiotic hygromycin B for shoot tips (12 mg/L) and for 5 days old seedlings (14 mg/L) were employed in efficient selection of the transformed tissues. This method of transformation resulted in a mean transformation efficiency of 4.09 %. Stable expression of the intron harbored gusA transgene was observed in mature organs of the transformed plants and their progenies. Genomic integration and inheritance of the hpt transgene was further confirmed by Southern blot analysis. The transformed plants exhibited normal morphology and most of them produced viable progenies, many of which segregated in a 3:1 ratio following Mendelian inheritance for a single dominant locus. However, strong P value support for 3:1 segregation ratio was obtained in case of two lines of independent transformants. Nevertheless, the method of transformation mentioned in this protocol could be effectively implemented in genetic transformation of many other cultivars of jute due to the genotype independent regeneration potential of the shoot tip explants. 相似文献
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Ganeshan Sivanandhan Natesan Selvaraj Andy Ganapathi Markandan Manickavasagam 《Plant Cell, Tissue and Organ Culture》2014,119(1):221-225
The influence of Gracilaria edulis and Sargassum wightii extracts was investigated for the production of biomass and withanolides in the multiple shoot suspension culture of Withania somnifera. Supplementation of 40 % G. edulis extract in MS liquid medium for 24 h exposure time in the culture recorded the highest biomass accumulation [62.4 g fresh weight and 17.82 g dry weight (DW)] and withanolides production (withanolide A 0.76 mg/g DW; withanolide B 1.66 mg/g DW; withaferin A 2.80 mg/g DW and withanone 2.42 mg/g DW) after 5 weeks of culture, which were 1.45–1.58-fold higher than control culture. This naturally available G. edulis extract-treated multiple shoot suspension culture protocol offers a potential alternative for the optimum production of biomass and withanolides utilizing shake-flasks. 相似文献
12.
Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens 总被引:3,自引:0,他引:3
High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 g/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants. 相似文献
13.
Bilal Ahmad Mir Jabeena Khazir Khalid R. Hakeem Sushma Koul Don A. Cowan 《Journal of plant biochemistry and biotechnology.》2014,23(4):430-434
Withania somnifera (L) Dunal, commonly known as ashwagandha or Indian ginseng, is the source of large number of pharmacologically active withanolides. Withaferin-A (WS-3), a major withanolide of W. somnifera, has been proven to be an effective anti-cancer molecule. In this study, a liquid culture system for shoot proliferation, biomass accumulation and withaferin-A production of an elite accession (AGB002) of W. somnifera was investigated. The nodal explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with various concentrations of 6-benzyl adenine (BA) and Kinetin (Kn) elicited varied responses. The highest number of regenerated shoots per ex-plant (35?±?3.25) and the maximum average shoot length (5.0?±?0.25 cm) were recorded on MS medium supplemented with BA (5.0 μM). The shoots were further proliferated in half and full strength MS liquid medium supplemented with the same concentration BA. It was interesting to note that shoots cultured on MS half strength liquid medium fortified with 4 gL-1 FW (fresh weight) shoot inoculum mass derived from 5 week old nodal explants of W. somnifera showed highest accumulation of biomass and withaferin A content in 5 weeks. Withaferin A was produced in relatively high amounts (1.30 % and 1.10 % DW) in shoots cultured in half and full strength MS liquid media respectively as compared to natural field grown plants (0.85 % DW). A considerable amount of the withaferin A was also excreted in the culture medium. Successful proliferation of shoots in liquid medium and the synthesis of withaferin A in vitro opens new avenues for bioreactor scale-up and the large-scale production of the compound. 相似文献
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Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens 总被引:1,自引:0,他引:1
M. De Block 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1988,76(5):767-774
Summary Leaves of the in vitro grown potato cultivars Bintje, Berolina, Desiree, and Russet Burbank were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For Russet Burbank, it was necessary to include AgNO3 in the medium to obtain efficient shoot regeneration. The transformed plants have one to a few copies of the T-DNA, show NPT-II and PAT activities, and are resistant to high doses of the commercial preparation of phospinotricin (glufosinate). Almost no somaclonal variation was detected in trans-genic plants. 相似文献
15.
Echeverrigaray S. Fracaro F. Andrade L.B. Biasio S. Atti-Serafini L. 《Plant Cell, Tissue and Organ Culture》2000,60(1):1-4
Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation. 相似文献
16.
Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens 总被引:2,自引:0,他引:2
A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T0) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T0 and T1 generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved. 相似文献
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根癌农杆菌介导Bt基因转化水稻的研究 总被引:2,自引:0,他引:2
为了培育出无筛选标记基因的转基因水稻,试验将loxp-hpt-loxp基因与成基因连锁在-起转化水稻方法,得到loxp-hpt—loxp—Bt转基因水稻植株,再与同质的带有ere基因的水稻杂交,以定向删除潮霉素抗性筛选标记。试验表明以水稻品种“皖粳97”为供试材料,将成熟胚来源的愈伤组织用根癌农杆菌EHA105/pCAMBIA1305.1感染后,筛选出抗性愈伤组织并获得再生植株。经PCR验证,得到20棵转基因水稻植株。 相似文献
18.
Vladimir Orbović Esther Marie Göllner Patricia Soria 《Acta Physiologiae Plantarum》2013,35(5):1409-1419
A possible role of arabinogalactan proteins in control of shoot regeneration from stem explants of two citrus cultivars, Carrizo citrange and ‘Duncan’ grapefruit, was investigated. Treatment of explants with (β-d-Glc)3 Yariv phenylglycoside, able to bind specifically to AGPs, led to a decrease of cumulative regeneration potential of both Carrizo citrange and ‘Duncan’ grapefruit. For Carrizo, lower cumulative regeneration potential on (β-d-Glc)3 Yariv phenylglycoside-treated explants was the result of both lower number of shoots on the explants that had shoots (explant regeneration potential) and decreased percentage of explants with shoots. In the case of ‘Duncan’, treatment with (β-d-Glc)3 Yariv phenylglycoside reduced cumulative regeneration potential only by lowering the percentage of explants with shoots, but it did not affect the number of shoots on the explants with shoots. Citrus explants treated with (α-d-Man)3 Yariv phenylglycoside, which does not bind AGPs, responded similarly to untreated explants. Transformability of cells on the cut ends of explants was also lower for both cultivars following the treatment of explants with (β-d-Glc)3 Yariv phenylglycoside. Our data suggest that arabinogalactan proteins play important role in processes controlling differentiation and genetic transformation of citrus cells by Agrobacterium. 相似文献
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根癌农杆菌介导的日本曲霉转化体系的建立 总被引:1,自引:0,他引:1
【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。 相似文献
20.
Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R2 seedlings on media containing G418.Abbreviations IAA
indole acetic acid
- KIN
kinetin
- BA
benzyladenine
- NOS
nopaline synthase
- NPTII
neomycin phosphotransferase II
- RMNO
tobacco nutrient medium (Marton and Maliga, 1975)
- SH
Shenk & Hildebrandt nutrient medium (Shenk & Hildebrandt, 1972; Michelmore and Eash, 1985)
Present address: Agriculture Canada, P.O. Box 457, St. Jean-sur-Richelieu, Quebec, Canada, J3B 6Z8 相似文献