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1.
Eline Kirk Mørk Karin Henriksen Henrik Brinch-Pedersen Kell Kristiansen Karen Koefoed Petersen 《Plant Cell, Tissue and Organ Culture》2012,108(3):501-512
A protocol for adventitious shoot formation in Symphyotrichum novi-belgii was developed after investigating the effects of cultivar and hormone combinations. A Murashige and Skoog medium with 1.0 mg l−1 6-benzyladenine induced adventitious shoot formation in 15 out of 19 cultivars. Addition of 0.1 mg l−1 indole-3-acetic acid or naphthaleneacetic acid increased the total number of shoots per explant, but not the number of shoots
longer than 1 cm. Addition of dichlorophenoxyacetic acid (2,4-D) promoted callus formation, but inhibited shoot elongation.
A transformation system for the two cultivars Victoria Fanny and Victoria Jane was developed by co-cultivation of leaf explants
with Agrobacterium tumefaciens. Three bacterial strains (LBA 4404, A281 and C58) all carrying the binary vector, p35S-GUS-INT, and harbouring the uidA gene coding for β-glucuronidase (GUS) were used. Regeneration of transgenic plants after co-cultivation with A281 was independent
of cultivar, and all explants produced callus followed by indirect shoot formation. In ‘Victoria Fanny’ shoots were formed
faster and without a callus phase after co-cultivation with LBA 4404 or C58. The highest number of potentially transformed
shoots was regenerated after co-cultivation of ‘Victoria Fanny’ leaf explants with LBA 4404. Integration of the transgenes
in the plant genome was confirmed using PCR and Southern blot hybridisation. To verify that the transgenes could be transferred
to offspring, crosses were conducted between three transgenic lines of ‘Victoria Fanny’ and two wild type pollen donors. It
was demonstrated that viable seeds were produced and that the uidA gene was inherited. 相似文献
2.
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
3.
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
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6.
Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants
placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum
shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented
with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and
without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants
in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important
medicinal plant. 相似文献
7.
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
8.
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
9.
Guirong Qiao Jing Zhou Jing Jiang Yuehua Sun Luanyin Pan Honggai Song Jingmin Jiang Renying Zhuo Xiaojuan Wang Zongxiu Sun 《Plant Cell, Tissue and Organ Culture》2010,102(2):163-170
Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal
properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious
shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn),
and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l−1 induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious
shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l−1 α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l−1 TDZ and 0.5 mg l−1 NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on
the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with
3 mg l−1 TDZ and 0.5 mg l−1 NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly
increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant
(14.6) was observed on MS medium with 3 mg l−1 TDZ, 0.5 mg l−1 NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium
with 3 mg l−1 BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with
4 mg l−1 indole-3-butyric acid (IBA) and 1.5 mg l−1 activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro. 相似文献
10.
A protocol for Agrobacterium-mediated transformation was developed for embryogenic callus of an excellent climber species, Parthenocissus tricuspidata. A. tumefaciens strain EHA105 or C58 harboring the pCAMBIA2301 binary vector with the neomycin phosphotransferase (nptII) and β-glucuronidase (uidA) gene was used. Factors affecting the transformation efficiency, including the Agrobacterium strains, co-cultivation time, Agrobacterium concentration, and infection time, were evaluated. Strain EHA105 proved to be significantly better than C58, and 4 days of
co-culture was critical for transformation. An Agrobacterium suspension at a concentration of 0.5–0.7 × 108 cells ml−1 (OD600 = 0.5–0.7) and an infection time of 40 min was optimal for transformation. By applying these optimized parameters, we recovered
six independent transformed shoots that were kanamycin-resistant and contained the nptII gene, as verified by polymerase chain reaction (PCR) analysis. Southern blot analysis confirmed that T-DNA was stably integrated
into the genome of three out of six PCR-positive lines. Furthermore, histochemical GUS assay revealed the expression of the
uidA gene in kanamycin-resistant calli, somatic embryos, and leaves of transgenic plants. 相似文献
11.
Osvaldo A. Castellanos-Hernández Araceli Rodríguez-Sahagún Gustavo J. Acevedo-Hernández Benjamín Rodríguez-Garay José Luis Cabrera-Ponce Luis Rafael Herrera-Estrella 《Plant Cell, Tissue and Organ Culture》2009,99(2):175-181
A biolistic protocol for the stable genetic transformation of the hardwood tree Paulownia elongata was developed. Leaf explants were bombarded using the PDS-1000/He system with plasmid pBI121. The introduced DNA contained
the β-glucuronidase (GUS) reporter gene and neomycin phosphotransferase (nptII) as a selection marker. Transformed calli were induced and selected on medium supplemented with 50 mg L−1 kanamycin, and transgenic plants were regenerated through indirect organogenesis. Complete plants were successfully transferred
to soil and established under greenhouse conditions. Different helium pressures and explant positions were used and the transformation
frequency was calculated. Optimal conditions for genetic transformation were bombardment of the abaxial leaf surface at a
pressure of 450 psi. The integration of the transgenes in the plant genome and their stable expression was demonstrated by
fluorometric GUS assay, determination of NPTII activity and PCR analysis. This method allows the production of transgenic
trees of P. elongata in a relatively short time. 相似文献
12.
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
13.
Sesbania drummondii (Rydb.) Cory is a source for phytopharmaceuticals. It also hyperaccumulates several toxic heavy metals. Development of an
efficient gene transfer method is an absolute requirement for the genetic improvement of this plant with more desirable traits
due to limitations in conventional breeding methods. A simple protocol was developed for Agrobacterium-mediated stable genetic transformation of Sesbania. Agrobacterium
tumefaciens strain EHA 101 containing the vector pCAMBIA 1305.1 having hptII and GUS plus genes was used for the gene transfer experiments. Evaluation of various parameters was carried out to assess
the transformation frequency by GUS expression analysis. High transformation frequency was achieved by using 7-day-old precultured
cotyledonary node (CN) explants. Further, the presence of acetosyringone (150 μM), infection of explants for 30–45 min and
3 days of cocultivation proved to be critical factors for greatly improving the transformation efficiency. Stable transformation
of S. drummondii was achieved, and putative transgenic shoots were obtained on medium supplemented with hygromycin (25 mg l−1). GUS histochemical analysis of the putative transgenic tissues further confirmed the transformation event. Genomic Southern
blot analysis was performed to verify the presence of transgenes and their stable integration. A transformation frequency
of 4% was achieved for CN explants using this protocol. 相似文献
14.
Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally
important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength
Murashige and Skoog (MS) medium containing 1.0 mg l−1 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to
expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted
on 1/4×MS mediumcontaining 1.0 mg l−1 indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots
were successfully acclimatized in soil and were normal phenotypically. 相似文献
15.
Waleerat Banyai Chalermpol Kirdmanee Masahiro Mii Kanyaratt Supaibulwatana 《Plant Cell, Tissue and Organ Culture》2010,103(2):255-265
Transgenic plants of Artemisia annua L., a medicinal plant that produces the compound artemisinin which has an anti-malarial activity, were developed following
Agrobacterium tumefaciens-mediated transformation of leaf explants. A. tumefaciens strain EHA105 carrying either pCAMBIA1301 or pCAMBIAFPS was used. Both plasmids harbored the hygromycin phosphotransferase
II (hptII) gene as a selectable gene, but the latter plasmid also harbored the gene encoding for farnesyl pyrophosphate synthase
(FPS), a key enzyme for artemisinin biosynthesis. Shoot regeneration was observed either directly from leaf sections or via intervening
callus when explants were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 0.1 mg l−1 α-naphthaleneacetic acid (NAA), 1 mg l−1 N6-benzyladenine (BA), 30 mg l−1 meropenem and 10 mg l−1 hygromycin. Applying vacuum infiltration dramatically increased transformation efficiency up to 7.3 and 19.7% when plasmids
with and without FPS gene were used, respectively. All putative transgenic regenerants showed positive bands of hptII gene following Southern blot analysis. Expression of FPS was observed in all transgenic lines, and FPS over-expressed lines exhibited higher artemisinin content and yield, of 2.5- and 3.6-fold, respectively, than that detected
in wild-type plants. A relatively high correlation (R
2 = 0.78) was observed between level of expression of FPS and artemisinin content. However, gene silencing was detected in some transgenic lines, especially for those lines containing
two copies of the FPS transgene, and with some lines exhibiting reduced growth. 相似文献
16.
Summary Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from
seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from
leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest
number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels
of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other
explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of
shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from
all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed
into plants that were morphologically identical to the source material. 相似文献
17.
Katarzyna Sykłowska-Baranek Agnieszka Pietrosiuk Anna Gawron Anna Kawiak Ewa Łojkowska Małgorzata Jeziorek Ioanna Chinou 《Plant Cell, Tissue and Organ Culture》2012,108(2):213-219
The effects of medium exchange and methyl jasmonate addition on growth and production of shikonin and its derivatives acetylshikonin
and isobutyrylshikonin in hairy root cultures of Lithospermum canescens were investigated. Responses varied depending on the transgenic line and stage of growth at which these lines were subjected
to treatment. Shikonin itself was not detected, irrespective of the transgenic line and culture treatment used. A eightfold
increase in acetylshikonin and isobutyrylshikonin accumulation was achieved when 32-day-old transgenic roots of Lc1D line
were transferred from LS to M9 medium for the subsequent 3 weeks of culture. Methyl jasmonate exerted a detrimental effect
on red naphthoquinones production. The extracts obtained from roots cultivated in M9 medium for 3 weeks were subjected to
a cytotoxicity assay and displayed cytotoxic activity against human promyelocytic leukemia cells (HL-60) at the dose of 4 μg ml−1. 相似文献
18.
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
19.
A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium
supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l−1 timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog
medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l−1 timentin, and 30 mg l−1 kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase
chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested.
The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence
of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization
of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black
cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry
genotypes. 相似文献
20.
A. V. Loskutov G.-Q. Song K. C. Sink 《In vitro cellular & developmental biology. Plant》2008,44(4):239-245
Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery.
Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for
2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and Skoog (MS) medium + 2,4-Dichlorophenoxyacetic
acid (2,4-D) (2.3 μM) + kinetin (2.8 μM) + timentin (300 mg·l−1). Calluses 4- to 6-wk-old were selected for glufosinate (GS) resistance by a two step method. First, calluses were transferred
to C medium + GS 0.35, 0.5, 1, 2, 5, or 10 mg·l−1; calluses formed only with 0, 0.35 and 0.5 mg·l−1 GS. All growing calluses from 0 and 0.35 mg·l−1 and a few from 0.5 mg·l−1, were divided and placed back on C + GS 0.35–0.5 mg·l−1 for another 5–6 wk. Second, tolerant clones were again divided and placed on C + GS 1–50 mg·l−1. When cultivar XP85 was inoculated with both strains, using pGPTVBAR, 19 glufosinate resistant (GR) callus clones were selected,
but shoots regenerated only for strain EHA105 inoculations. When both of the strains (each with pDHB321.1) were inoculated
on cv. XP166, 3 and 12 GR calluses occurred for EHA105 and GV3101, respectively. Using CS, a total of 34 GR callus clones
were selected, and shoots were regenerated from over 50% of them on Gamborg B5 medium + 6-(γ, γ-dimethylallylamino) purine
2ip (4.9 μM) + naphthaleneacetic acid (NAA; 1.6 μM) and rooted on MS in 5–6 mo total time. Conversely, using FB with inoculation
by GV3101/pDHB321.1 on cv. XP166 yielded putative transgenic celery plants confirmed by polymerase chain reaction (PCR) in
just 6 wk. Transformation of the bar gene into celery was confirmed by PCR for 5 and 6 CS and FB lines, respectively. Southern blot analyses indicated 1–2 copies
in CS lines and 1 copy in FB lines. Herbicide assays on whole plants with 100 and 300 mg·l−1 glufosinate indicated a range of low to high tolerance for lines derived by both methods. The bar gene was found to be Mendelian inherited in one self-fertile CS derived line. 相似文献