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1.
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
2.
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
3.
An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA3 Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz 相似文献
4.
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
5.
Wenhao Dai Zong-Ming Cheng Wayne Sargent 《In vitro cellular & developmental biology. Plant》2003,39(1):6-11
Summary An efficient regeneration and transformation system was developed for two elite aspen hybrid clones (Populus canescens × P. grandidentada and P. tremuloides × P. davidiana). Callus was induced from in vitro leaf explants on modified Murashige and Skoog medium (MSA) and woody plant medium (WPM) containing four different combinations
of cytokinins and auxins. Callus tissues regenerated into shoots on WPM medium supplemented with 2.0 mgl−1 (9.12 μM) zeatin or 0.01 mgl−1 (0.045 μM) thidiazuron. P. canescens × P. grandidentata exhibited the higher callus and shoot production. In vitro leaf explants from the two hybrid clones were cocultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uidA gene encoding for β-glucuronidase (GUS) and the npt II gene encoding for neomycin phosphotransferase II. Transformation was confirmed by GUS assays, polymerase chain reaction,
and Southern blot analyses. Agrobacterium concentration, acetosyringone, and pH of the cocultivation medium were evaluated for enhancing transformation efficiency
with the clone P. canescens × P. grandidentata. 相似文献
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
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
14.
London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering
could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots
from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene β-glucuronidase (GUS), we have followed the
transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of
leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD600 of 0.8–1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer.
Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established.
By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained
the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines,
the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome. 相似文献
15.
Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants,
leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types
began first with culture on 50 mg l−1 of Km for 6 wk and then transfer to 30 mg l−1 of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl−1 cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m−2s−1. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%,
and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment
for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four. 相似文献
16.
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
17.
Y. Bao P. Dharmawardhana R. Arias M. B. Allen C. Ma Steven H. Strauss 《Plant cell reports》2009,28(6):947-962
We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs
to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common
in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems
as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were
placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary
growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response
regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events
recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended
to stimulate meristem development to promote clonal propagation and genetic transformation.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient -glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were obtained at a frequency of up to 30% from all the explants tested. However, transgenic shoots were obtained only from the hypocotyl of plantlets derived from pollen embryoids. Transformation was confirmed by the ability of leaf segments to produce kanamycin resistant calluses, -glucuronidase histochemical and flurometric assays, polymerase chain reaction and Southern blot analysis. The results show that pollen embryoid-derived explants may be an alternative source for both efficient transformation and regeneration of transgenic plants in recalcitrant species. 相似文献
19.
Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were
obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l−1 phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT
(2–6 mg l−1). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l−1) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern
analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants
followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency
as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew
on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization. 相似文献
20.
<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