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1.
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
2.
<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
3.
AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at
27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L
and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic
embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR
amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus
far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants. 相似文献
4.
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
5.
We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin
for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic
embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7
d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in
regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L
giberellic acid (GA3). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern
hybridization to confirm gene integration. 相似文献
6.
Silvia Flores-Benítez Juan F. Jiménez-Bremont Sergio Rosales-Mendoza Gerardo R. Argüello-Astorga Rosalba Castillo-Collazo Ángel Gabriel Alpuche-Solís 《Plant Cell, Tissue and Organ Culture》2007,91(3):215-224
Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and
embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants
was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving
a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive
with the GUS assay after 14 months on selective medium while still undergoing regeneration. 相似文献
7.
E. A. Popowich A. P. Firsov T. Y. Mitiouchkina V. L. Filipenya S. V. Dolgov V. N. Reshetnikov 《Plant Cell, Tissue and Organ Culture》2007,90(3):237-244
Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP
and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning
preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l−1 kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l−1 kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants
expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines
were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same
time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation. 相似文献
8.
<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
9.
Huang Heping Gao Shanlin Chen Lanlan Jiao Xiaoke 《In vitro cellular & developmental biology. Plant》2008,44(5):448-455
Dioscorea zingiberensis is an important medicinal plant and a source of diosgenin in China. We report research on the induction, characteristics,
and chemical assays of polyploid plants of D. zingiberensis. Immersing calli in 0.3% colchicine solution for 16 h prior to culture induced a high number of autotetraploid plants. The
induction rate reached as high as 36.7% of treated calli. More than 50 lines of autotetraploid plants were obtained. All tetraploid
plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination
of morphological characteristics and for chemical assays. Six elite lines have been selected for further selection and breeding
into new varieties for commercial production. 相似文献
10.
Hai Ping Hong Hongyi Zhang Paula Olhoft Steve Hill Hunt Wiley Effie Toren Helke Hillebrand Todd Jones Ming Cheng 《In vitro cellular & developmental biology. Plant》2007,43(6):558-568
A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes
as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced
on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with
various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect
on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a
low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside,
and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction
medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected
calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been
regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including
leaf, stem, and roots. Southern and T1 plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy
number ranging from 1–5 copies. 相似文献
11.
Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 °C on kanamycin free shoot
regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm−3 kanamycin and 300 mg dm−3 cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm−3 kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl
explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival
rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on
transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved. 相似文献
12.
Anindita Banerjee Sharmila Chattopadhyay 《In vitro cellular & developmental biology. Plant》2009,45(1):57-64
Phyllanthus amarus Schum & Thonn. is a source of various pharmacologically active compounds such as phyllanthin, hypophyllanthin, gallic acid,
catechin, and nirurin, a flavone glycoside. A genetic transformation method using Agrobacterium tumefaciens was developed for this plant species for the first time. Shoot tips of full grown plants were used as explants for Agrobacterium-mediated transformation. Transgenic plants were obtained by co-cultivation of shoot tips explants and A. tumefaciens strain LBA4404 containing the pCAMBIA 2301 plasmid harboring neomycin phosphotransferase II (NPT II) and β-glucuronidase
encoding (GUS) genes in the T-DNA region in the presence of 200 μM acetosyringone. Integration of the NPT II gene into the
genome of transgenic plants was verified by PCR and Southern blot analyses. Expression of the NPT II gene was confirmed by
RT-PCR analysis. An average of 25 explants was used, out of which an average of 19 explants produced kanamycin-resistant shoots,
which rooted to produce 13 complete transgenic plants. 相似文献
13.
Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic
plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic
plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved.
The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines,
the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated
one or two copies of the uidA gene.
C. Gao and J. Liu contributed equally to the work. 相似文献
14.
Agrobacterium rhizogenes was used for efficient transformation of chrysanthemum. Two types of Agrobacterium, A. rhizogenes (A-13) and A. tumefaciens (LBA4404), which harbor pIG121-Hm, were employed for infection. In the A. rhizogenes-infected explants, hairy roots were not observed on any tested medium or culture condition. When explants were cultured on shoot induction medium, calli were formed at the cutting edge within 4–6 weeks of culture, and shoot primordia were observed on the callus surface after 2 weeks of callus formation. Consequently, with gus introduction, a significantly higher transformation rate was observed for A. rhizogenes (6.0%) compared with A. tumefaciens (3.3%). However, only 0.6% of the frequency of rol insertion was exhibited in A. rhizogenes mediation. These results indicate that A. rhizogenes effectively introduces T-DNA of the binary plasmid into the chrysanthemum genome by introducing Ri T-DNA at a low frequency. It also indicates that the system is a useful alternative for the transformation of chrysanthemum. 相似文献
15.
Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following
co-cultivation, leaf explants were cultured on selective medium containing 10 mg l−1 hygromycin and 500 mg l−1 cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved
after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength
MS basal medium supplemented with 10 mg l−1 hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical
assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%. 相似文献
16.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
17.
Elena Palomo-Ríos Araceli Barceló-Muñoz José A. Mercado Fernando Pliego-Alfaro 《Plant Cell, Tissue and Organ Culture》2012,109(2):201-211
Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of
avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for
selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l−1 kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l−1 partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth.
For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l−1 kanamycin and 250 mg l−1 timentin for 2 months. Then, kanamycin level was increased to 100 mg l−1 for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant
calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was
confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic
embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA3 for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l−1 agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar
to control plants. 相似文献
18.
To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting
transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated
on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected
the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed
hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene
integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding
of this very common indoor plant. 相似文献
19.
A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l−1 BAP and 0.5 μmol l−1 NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection
medium (co-culture medium supplemented with 60 mg l−1 kanamycin and 200 mg l−1 cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l−1 kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil.
The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical
assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this
procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to
soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced
in our lab in the past two years. 相似文献
20.
Indrajit Dutta Prasenjit Saha Sampa Das 《In vitro cellular & developmental biology. Plant》2008,44(5):401-411
Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige
and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver
nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency
of stable transformation was found to be approximately 19% in the T
0 generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern
blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of
T
1 seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency
over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea. 相似文献