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1.
A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were
transformed in the presence of 100 μM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l−1 was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with
13.3 μM 6-benzylaminopurine, 4.5 μM thidiazuron, 50 mg l−1 adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls
were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse.
This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash. 相似文献
2.
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
3.
Michal Moyal Ben Zvi Amir Zuker Marianna Ovadis Elena Shklarman Hagit Ben-Meir Shamir Zenvirt Alexander Vainstein 《Molecular breeding : new strategies in plant improvement》2008,22(4):543-553
As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility
of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment
of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA3-pretreated mother plants and a two-step selection scheme. The GA3 treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated
explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing
shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila
was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes
rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could
be beneficial to the ornamental industry. 相似文献
4.
A. Karthikeyan J. Shilpha S. Karutha Pandian M. Ramesh 《Plant Cell, Tissue and Organ Culture》2012,109(1):153-165
A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier
and Skoog (LS) medium supplemented with 2.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and
root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression
analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following
β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration
of the transgene was confirmed in four putatively transformed T0 plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among
the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and
containing 10 g l−1 (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l−1 timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%. 相似文献
5.
Wissam A. Abou-Alaiwi Shobha D. Potlakayala Stephen L. Goldman Puthiyaparambil C. Josekutty Deepkamal N. Karelia Sairam V. Rudrabhatla 《Plant Cell, Tissue and Organ Culture》2012,109(1):1-8
An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally
regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter.
A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including
PCR, Southern blotting and RT-PCR, were performed on T0 and T1 plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants.
Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay. 相似文献
6.
A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from
Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166
and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM
2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin
(Km) were then recovered on CIM + 25 mg l−1 Km + 250 mg l−1 timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per
explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM
α-naphthaleneacetic acid (NAA) + 25 mg l−1 Km + 250 mg l−1 timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and
18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration
of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot
analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production
of transgenic celery plants. 相似文献
7.
8.
Eva Boszoradova Jana Libantova Ildiko Matusikova Zuzana Poloniova Martin Jopcik Maria Berenyi Jana Moravcikova 《Plant Cell, Tissue and Organ Culture》2011,107(2):317-323
Oilseed rape is considered relatively recalcitrant for genetic modification. This work was performed to establish conditions
for effective transformation and regeneration of commercially used cultivars Campino, Haydn, Heros, Hunter and Topas (Brassica napus L.). Cotyledonary petioles and hypocotyls (obtained from the seedlings grown under dark conditions) as a source of explants
were used. Our experiments revealed that a lower selection pressure in combination with postponing of selection by 14 days
after co-cultivation resulted in recovery of transgenic plants from all cultivars. Transformants were obtained with efficiency
from 1 to 5.5%. Histochemically GUS-positive plants were analysed by PCR using primers corresponding to the internal fragments
of gus and nptII genes. The transgenic nature was confirmed by Southern blot analyses using a specific nptII probe. This work enriches the list of oilseed rape cultivars available for genetic modification. 相似文献
9.
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
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.
F. D. Espasandin M. M. Collavino C. V. Luna R. C. Paz J. R. Tarragó O. A. Ruiz L. A. Mroginski P. A. Sansberro 《Plant Cell, Tissue and Organ Culture》2010,102(2):181-189
A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which
carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented
with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml−1) and cefotaxime (400 μg ml−1) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during
the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots
per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS
strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and
Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines
were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90%
of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase
from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter
showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the
same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the
cloned materials was established. 相似文献
12.
<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
13.
14.
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
15.
Trifolium alexandrinum L. (Egyptian clover) is one of the most important forage crops in the world. Its regeneration in tissue culture has been
described in a few reports but the efficiency, accurate time scales and applicability to various genotypes of the described
procedures are uncertain. Therefore their suitability for genetic transformation is unclear. In this study, were report new
fast procedures for regeneration of Egyptian clover that are applicable to the regeneration of various genotypes (Mescawi-ahaly,
Sakha3 and Sakha4). Shoots were regenerated from intact and wounded cotyledons as well as hypocotyls of Mescawi-ahaly on naphthaleneacetic
acid/benzyladenine (NAA/BA) and naphthaleneacetic acid/thidiazuron (NAA/TDZ) media. The highest shoot regeneration frequencies
were obtained from intact cotyledons on NAA/BA (0.05 mg l−1 NAA combined with 2.0 mg l−1 BA) and NAA/TDZ (0.05 mg l−1 NAA combined with 1.0 mg l−1 TDZ) media (66.2 and 43.1% respectively) compared to 18.4 and 10.1% for wounded cotyledons on NAA/BA and NAA/TDZ respectively.
21.0% shoot regeneration frequency was observed for hypocotyls on NAA/BA (2.0 mg l−1 NAA combined with 0.5 mg l−1 BA) medium but no regeneration was obtained on NAA/TDZ medium. Rooting of the regenerated shoots was induced on indole butyric
acid (IBA: 0.24 mg l−1) or NAA (2.0 mg l−1) media where IBA medium supported significantly higher frequencies of rooting as well as survival of the whole plantlets
after transfer to soil. However, the rooting and survival frequencies also depended on the type of explant and the medium
used for shoot regeneration. The two cultivars Sakha3 and Sakha4 were regenerated using the culture conditions optimized for
Mescawi-ahaly with comparable efficiencies, indicating that the described procedure is not genotype dependent. The time scale
of whole plantlet regeneration ranged from 7.5 weeks for intact and wounded cotyledons to 10 weeks for hypocotyl explants. 相似文献
16.
An improved protocol for genetic transformation of juvenile explants of Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.), Duncan (Citrus paradisi Macf.), Hamlin (Citrus sinensis (L.) Osbeck) and Mexican Lime (Citrus aurantifolia Swingle) cultivars using a vector containing a bifunctional egfp-nptII fusion gene is described. Several parameters were investigated to optimize genetic transformation of these four cultivars.
It was determined that a short preincubation in hormone rich liquid medium and subculture of Agrobacterium for 3 h in YEP medium containing 100 μM acetosyringone were required for improvement of transformation efficiency. Co-cultivation
duration as well as addition of acetosyringone to co-cultivation medium also played an important role in transformation efficiency
as did OD600 value of the Agrobacterium suspension used for transformation. We regenerated numerous EGFP expressing transgenic lines from all four cultivars. Based
on these results, we conclude that genetic transformation of citrus is cultivar specific and optimization of conditions for
maximum transgenic production are required for each individual cultivar. 相似文献
17.
We have established a shoot regeneration system and genetic transformation of cockscomb (Celosia cristata and Celosia plumosus). The best results in terms of frequency of shoot regeneration and number of shoot buds per explant are observed on media
supplemented with 0.5 mg l−1 6-BA (for explants of apical meristems of C. cristata) or 2.0 mg l−1 6-BA, 0.5 mg l−1 NAA and 0.5 mg l−1 IAA (for hypocotyls explants of C. plumosus). We use apical meristems of C. cristata and hypocotyls of C. plumosus as the starting material for transformation. A novel KNOTTED1-like homeobox1 (KNOX), PttKN1 (Populus tremula × P. tremuoides
knotted1) isolated from the vascular cambial region of hybrid aspen, is introduced into cockscomb by Agrobacterium. A series of novel phenotypes are obtained from the transgenic cockscomb plants, including lobed or rumpled leaves, partite
leaves and two or three leaves developed on the same petiole, on the basis of their leaf phenotypes. Transformants are selected
by different concentrations of kanamycin. Transformants are confirmed by PCR of the NptII gene and PCR or RT-PCR of PttKN1 gene. Furthermore, RT-PCR shows that 35S:: PttKN1 RNA levels do not correlate with phenotypic severity. It is discussed that our results bring elements on possible function
of PttKN1 gene. To our knowledge, genetic transformation of cockscomb is first reported. 相似文献
18.
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
19.
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
20.
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