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1.
<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis> 总被引:3,自引:0,他引:3
A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies. 相似文献
2.
An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes. 相似文献
3.
An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine
(BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration
medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l
BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots
were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong
β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear
genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective
transformation and direct regeneration of E. tereticornis Sm. 相似文献
4.
5.
Summary
In vitro adventitious shoot bud regeneration systems are considered most suitable for Agrobacterium-and biolisticsmediated genetic transformation to obtain transgenic plants. In the present investigation, multiple adventitious
shoot buds could be induced directly from Feronia limonia hypocotyl explants inoculated on Murashige and Skoog (MS) medium containing different growth regulators. During the initial
phase, the hypocotyl segments nearer to the cotyledons responded quickly compared to those closer to the root. The response,
however, was comparable in both the segments in subsequent subculture. Of the various cytokinins, 2.22 μM 6-benzylaminopurine (BA) proved to be more effective compared to kinetin (Kn). The two-way interaction of BA and Kn significantly
influenced shoot regeneration and contributed the most among the interactions studied. The best response, however, was obtained
when 2.22 μM BA and 2.32 μM Kn were combined. Although the effect of auxins like α-naphthaleneactic acid (NAA) combined with cytokinins evoked a significant
responsein terns of number of shoot buds, this response did not supersede the effect of combined cytokinins. Vone of the polyamines
tested induced shoot buds on hypocotyl segments. Adventitious shoots were multiplied on MS medium containing 2.22 μM BA, 6.96 μM Kn, and 0.05 μM NAA. More than 60% of the shoots produced roots when cultured on medium containing one-quarter strength MS salts, 10% suerose,
0.6% agar, and 7.36μM indole-3-butyric acid. The adventious origin of shoot buds showing continuous vascular connections was confirmed through
histological investigations. 相似文献
6.
Qi Zhu Fengtao Wu Feng Ding Dong Ye Yongqin Chen Yi Li Yang Zhifan 《Plant Cell, Tissue and Organ Culture》2009,96(3):317-324
Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important
steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and
female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive
to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation
medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three
days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for
formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh
selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of
the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to
produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated
from initial infected callus explants. 相似文献
7.
Herbicide-resistant transgenic Panax ginseng plants were produced by introducing the phosphinothricin acetyl transferase (PAT) gene that confers resistance to the herbicide Basta (bialaphos) through Agrobacterium tumefaciens co-cultivation. Embryogenic callus gathered from cotyledon explants of P. ginseng were pre-treated with 0.5 M sucrose or 0.05 M MgSO(4 )before Agrobacterium infection. This pre-treatment process markedly enhanced the transient expression of the beta-glucuronidase (GUS) gene. Embryogenic callus was initially cultured on MS medium supplemented with 400 mg/l cefotaxime for 3 weeks and subsequently subcultured five times to a medium containing 25 mg/l kanamycin and 300 mg/l cefotaxime. Somatic embryos formed on the surfaces of kanamycin-resistant callus. Upon development into the cotyledonary stage, these somatic embryos were transferred to a medium containing 50 mg/l kanamycin and 5 mg/l gibberellic acid to induce germination and strong selection. Integration of the transgene into the plants was confirmed by polymerase chain reaction and Southern analyses. Transfer of the transgenic ginseng plantlets to soil was successfully accomplished via acclimatization in autoclaved perlite. Not all of the plantlets survived in soil that had not been autoclaved because of fungal infection, particularly in the region between the roots and leaves. Transgenic plants growing in soil were observed to be strongly resistant to Basta application. 相似文献
8.
Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO3 under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO3 were diploid. 相似文献
9.
10.
<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>) 总被引:1,自引:1,他引:0
Byoung-Kyu?Lee Seung-Hee?Yu Yul-Ho?Kim Byung-Ohg?Ahn Han-Sun?Hur Sang-Chul?Lee Zhanyuan?Zhang Jang-Yong?Lee
Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l−1 phosphinothricin (PPT) and 150 mg l−1 kanamycin, respectively, for shoot induction and 1 mg l−1 PPT and 125 mg l−1 kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T0 and T1 plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T0 and T1 transgenic plants. 相似文献
11.
T. Sretenović-Rajičić S. Ninković B. Uzelać B. Vinterhalter D. Vinterhalter 《Russian Journal of Plant Physiology》2007,54(5):653-658
Two inbred lines of Brassica oleracea L. var. capitata were transformed with two Agrobacterium tumefaciens strains harboring resistance to herbicide Basta: AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Inoculated cotyledons and hypocotyls
provided equally good explants and manifested a high percentage of shoot regeneration on MS medium supplemented with 1 mg/l
benzyladenine and 0.5 mg/l indole-3-butyric acid. The P34I5 genotype was superior to P22I5 in shoot regeneration (48.1 vs. 26.9%), multiplication, and acclimation in the greenhouse (76 vs. 40%). A. tumefaciens AGL1/pDM805 provided more regenerated shoots per explant, especially in the case of cotyledon explants, and the higher transformation
rate (up to 35% vs. up to 12%) as compared to LB5-1. Putative transformants survived spraying with 10–30 mg/l phosphinothricin.
Transformation was confirmed by GUS assay and PCR analysis in T0 and T1 generations.
Published in Russian in Fiziologiya Rastenii, 2007, vol. 54, No. 5, pp. 738–743.
The text was submitted by the authors in English. 相似文献
12.
Mingjia Yang Xiangming Xie Caixia Zheng Fangqiu Zhang Xiaoqing He Zhiru Li 《Plant Cell, Tissue and Organ Culture》2008,95(2):141-147
A protocol was developed for Agroacterium-mediated genetic transformation of Acacia crassicarpa via organogenesis by using in vitro phyllode (leaf) as the explant. Phyllode (leaf) explants were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI101 (harboring antisense Pt4CL1 with respect to the Pt4CL1P promoter). The selection for transgenic shoots was performed through two consecutive steps on
Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators and antibiotics in the
following order: 0.5 mg/l thidiazuron (TDZ), 0.5 mg/l α-naphthaleneacetic acid (NAA), 300 mg/l carbenicillin (Car) and 20 mg/l
kanamycin (Km) for 10 days; 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days; 0.5 mg/l indole-3-butyric acid (IBA), 100 mg/l
Car and 20 mg/l Km 50 days. 21.7% of nodules produced multiple adventitious shoot buds, of which 27.7% survived in initial
selection. The shoot buds were subjected to repeated selection on MS medium supplemented with 0.1 mg/l TDZ, 200 mg/l Car and
20 mg/l Km for 60 days. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.5 mg/l
IBA, 100 mg/l Car 20 mg/l Km 50 days. Genomic PCR analysis confirmed the incorporation of the antisense Pt4CL1 with respect to the Pt4CL1P promoter fragment into the host genome. 相似文献
13.
14.
Semiha Erisen Mustafa Yorgancilar Emine Atalay Mehmet Babaoglu 《Plant Cell, Tissue and Organ Culture》2010,100(2):229-233
Prolific shoot regeneration via organogenesis was induced from leaf and leaf petiole explants of the endemic Astragalus cariensis species on Murashige and Skoog (MS) medium with α-naphthaleneacetic acid (NAA) and benzyladenine (BA) within 8 week. The
highest number of shoots (23/explants) was obtained from leaf explants cultured on MS with 0.5 mg/l NAA and 4 mg/l BA. Elongated
shoots were successfully rooted in MS medium with 0.5 mg/l indole-3-butyric acid. Rooted plantlets were acclimatized in pots
containing 1:1 mixture of peat and perlite. 相似文献
15.
Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent
cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with
emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region
of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis
on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm
in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation
period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants
or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An
optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl−1 hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl−1 hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature
cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols
for transformation of soybean should be established under both Agrobacterium and selection conditions. 相似文献
16.
Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L−1 BA, 150 mg L−1 Ads, 0.1 mg L−1 NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of
shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of
the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment
explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation.
Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L−1 IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the
soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement
prograrmme. 相似文献
17.
We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS
gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria
and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l
sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin.
The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes
were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and
segregation of the transgenes were demonstrated by molecular and genetic analyses in the T0 and T1 generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants
from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the
frequency of transformation was lower and sterility was more frequent. 相似文献
18.
In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM
6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l−1 casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots)
to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop
into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented
with 4.4 μM BA, 0.29 μM gibberellic acid (GA3), and 500 mg l−1 CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration
(16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with
0.44 μM BA and 0.29 μM GA3. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response
and shoot numbers per explant were recorded on WPM supplemented with BA and GA3, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA3 were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20
shoots within 12 wk. Rooted plantlets were successfully acclimatized. 相似文献
19.
In vitro regeneration from leaf, cotyledon and hypocotyl explants of six cultivars belonging to three species of Capsicum was achieved by direct organogenesis. The cultivar Umorok showed the best response while Meiteimorok, Haomorok, Mashingkha
and Uchithi showed intermediate response and the cultivar Chiengpi was the least responsive. Leaf and cotyledon explants regenerated
more shoots than hypocotyl explants and the maximum number of shoots were produced on Murashige and Skoog (1962) medium containing
8.8 μM 6-benzylaminopurine (BAP) with 11.4 μM indole-3-acetic acid (IAA). Elongation of shoot buds derived from different
explants was achieved on medium containing 2.8 μM IAA and the elongated shoots were rooted on medium containing 2.8 or 5.7
μM IAA and 2.4 or 4.9 μM indole-3-butyric acid (IBA). Four-week old rooted plantlets were hardened and transplanted to the
soil. The plantlets showed 90 % survival during transplantation. 相似文献
20.
Meiru Li Hongqing Li Huawu Jiang Guojiang Wu 《Plant Cell, Tissue and Organ Culture》2008,93(3):249-255
Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful
genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with
a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively.
For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473,
1962) (MS) medium supplemented with 1.5 mg l−1 benzyladenine (BA) and 0.05 mg l−1 indole-3-butyric acid (IBA) (CI medium) containing 5 mg l−1 hygromycin and 500 mg l−1 cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots
were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l−1 BA, 0.05 mg l−1 IBA, and 0.5 mg l−1 gibberellic acid (GA3) (SI medium), 5 mg l−1 hygromycin and 250 mg l−1 cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l−1 hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay.
Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as
44%. 相似文献