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1.
We have generated putative promoter tagged transgenic lines inArachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated byAgrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4 mg/l in combination with 0.1 mg/l α-napthaleneacetic
acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters
enhancing genetic transformation viz. seedling age,Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation
with CN explants from 6-day-old seedlings co-cultivated withAgrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation
frequency was achieved with p35S GUSINT in Β-glucuronidase (GUS) assays. Among thein vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS
percentage. We have generated over 141 putative T0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the
green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase
chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression
or blue spots in at least one plant part. The progeny of 15 T0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression
patterns were more or less similar in both T0 and corresponding T1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic
lines in the present communication. 相似文献
2.
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
3.
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
4.
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
5.
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
6.
Pooja Jha Shashi Anjana Rustagi Pankaj Kumar Agnihotri Vishvas M. Kulkarni Vishnu Bhat 《Plant Cell, Tissue and Organ Culture》2011,107(3):501-512
A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions
for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system,
without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin
phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A
number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration,
co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The
highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30 min with Agrobacterium O.D.600 = 1.2 under a negative pressure of 0.5 × 105 Pa and co-cultivated for 3 days in medium containing 400 μM acetosyringone. Histochemical GUS assay and polymerase chain
reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of
the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid
and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed
protocol will facilitate the insertion of desirable genes of useful traits into pearl millet. 相似文献
7.
Diane E. Darlington Chiu-Yueh Hung Jiahua Xie 《Plant Cell, Tissue and Organ Culture》2009,99(2):157-165
Agrobacterium
tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The
optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l−1 and thus all putative transgenic plants were obtained on induction medium containing 50 mg l−1 kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the
addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes
responsible for Se-accumulation in A. racemosus. 相似文献
8.
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
9.
10.
The effect of chemical additives (acetosyringone, AS; L-cysteine, CYS; dithiothreitol, DTT; glutathione, GSH; cellulase, CEL;
pectinase, PEC) and light regimes (16/8 light/dark photoperiod, 16L/8D; continuous light, 24L; continuous dark, 24D) applied
during cocultivation procedure of pea explants with Agrobacterium tumefaciens on transformation efficiency was studied. A hypervirulent strain of A. tumefaciens EHA 105 with two plasmids, namely pGT89 and pBIN19, both carrying reporter gus-int gene, and bar or nptII selectable marker gene, respectively, was used for genetic transformation of cotyledonary node explants of three dry seed
pea cultivars Adept, Komet and Menhir. The focus was laid on cocultivation step (48 h) of transformation protocol. After chemical
or physical treatments, transient GUS expression was recorded 20 days after cocultivation as a measure of successful transformation,
using a four category scale (0 – without GUS expression, 1 – weak, 2 – medium and 3 – strong GUS expression) for calculation
of IGE (Intensity of GUS Expression). Of the tested chemical cocultivation additives, 100 μM AS and 50 mg CYS significantly
improved GUS expression (IGE value), while DTT, GSH and both macerating enzymes (CEL, PEC used either separately or in combination)
either had no positive effect or were even negative. There were no statistically significant differences between the light
regimes tested. Nevertheless, cocultivation in 24L, without chemical additives, reproducibly resulted in the highest frequency
of explants scored in category 3 of GUS expression (followed by 24D and 16L/8D treatment). However, application of 100 μM
AS reverted this trend. Cv. Adept yielded higher transformation frequencies than cvs. Menhir and Komet. Plasmid pGT89 produced
a higher IGE value than pBIN19. Based on our results, the improved cocultivation step for pea consists of 48 h cocultivation
at 20 ± 2°C, with 50 mg l−1 CYS and 100 μM AS, 16L/8D photoperiod (or without AS in continuous light). 相似文献
11.
X. Niu X. Li P. Veronese R. A. Bressan S. C. Weller P. M. Hasegawa 《Plant cell reports》2000,19(3):304-310
Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent
of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications
were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic
evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted
in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher
concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration
medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the
number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average
transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein,
osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is
a viable alternative to plant breeding and clonal selection for improvement of this crop.
Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999 相似文献
12.
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
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.
Xiuping Zou Demou Li Xiaoying Luo Keming Luo Yan Pei 《In vitro cellular & developmental biology. Plant》2008,44(3):169-177
Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected
with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable
marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation
efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic
acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy
was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied
on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency
and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically
reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical
assay, PCR, and Southern blot analysis. 相似文献
15.
Gaurab Gangopadhyay Subhash Kanti Roy Sangita Basu Gangopadhyay Kalyan Kumar Mukherjee 《Plant Cell, Tissue and Organ Culture》2009,97(3):295-302
A protocol for Agrobacterium-mediated transformation of a local ‘elite’ Indian variety (Queen) of pineapple [Ananus comosus (L.) Merr, family Bromeliaceae] has been established using a standard transformation vector (pCAMBIA 1304). High transformation
efficiency, expressed as the mean percentage of transgenic micro-shoots regenerated from initial callus explants (20.6%) was
achieved using a novel encapsulation-based, antibiotic selection procedure. The Agrobacterium-infected micro-shoots derived from callus explants survived selection in high concentration of hygromycin (60 mg l−1 and beyond) in encapsulated alginate beads. The integration of transgene in hygromycin-resistant shoots and plants was confirmed
by histochemical GUS assay, PCR amplification and Southern hybridization. It is possible to eliminate false antibiotic positives
in pineapple transformation program to a large extent following this procedure. 相似文献
16.
Winter jujube, a species that originated in China, is the most prominent elite variety of jujube (Zizyphus jujuba Mill.). Due to its economic value and its recalcitrance to improvements through traditional plant breeding approaches, genetic
transformation techniques may have a great potential in providing the means to transfer one or more selected desirable traits
into the plant genome. We reported here an improved protocol for the Agrobacterium-mediated transformation of shoot tips of winter jujube. We have identified a set of optimum transformation conditions that
take into account Agrobacterium inoculum density, Agrobacterium incubation period, co-cultivation conditions, and vacuum (use of a vacuum pump to create a negative-pressure environment).
The highest transformation frequency (5.2%) was obtained when the shoot-tip explants were infected for 10 min and co-cultured
for 4 days with Agrobacterium at OD600 0.8 under a negative pressure of 0.5 × 105 Pa. PCR and southern blot analyses confirmed the presence of transgenic plants and the stable integration of the target gene
into the genome of regenerated plants. A histochemical staining analysis for GUS activity in the transgenic shoot tips also
validated the efficiency of the transformation system. 相似文献
17.
Rocío Torreblanca Sergio Cerezo Elena Palomo-Ríos José A. Mercado Fernando Pliego-Alfaro 《Plant Cell, Tissue and Organ Culture》2010,103(1):61-69
Olive tree, Olea europaea L., is one of the most commercially important oil crops. A reliable protocol for the genetic transformation of this species
has been developed. Embryogenic calli were infected with different Agrobacterium tumefaciens strains harboring pBINUbiGUSint or pGUSINT binary plasmids. These vectors contain the nos-nptII and the uidA gene driven by the maize polyubiquitin Ubi1 and CaMV35S promoter, respectively. Inoculated explants were cocultured for 2 days, and later selected in the presence of 200 mg l−1 paromomycin. The inclusion of a 3 weeks selection period in liquid medium supplemented with 50 mg l−1 paromomycin was critical for elimination of chimaeric calli. Agrobacterium strain AGL1 containing pBINUbiGUSint plasmid yielded higher transformation frequencies than EHA105 or LBA4404. Globular somatic
embryos (SE), 1–2 mm diameter, cultured in the selection medium in groups of three, were the best explant for transformation.
Using this protocol, transformation frequencies in the range of 20–45%, based on the number of infected explants proliferating
in the selection medium, have been obtained. More than 100 independent transgenic lines were generated, and 16 of them converted
to plants. Transgenic plants were acclimated and grown in the greenhouse, being phenotypically similar to wild type plants.
The uidA gene was strongly expressed in transgenic material during the in vitro regeneration phase; however, β-glucuronidase (GUS)
activity in pBINUbiGUSint transgenic plants was neither detected in shoots growing in vitro nor in acclimated plants. Transgenic
leaves, however, contained high levels of NPTII protein. By contrast, plants transformed with the pGUSINT plasmid showed a
strong GUS activity in leaves. The protocol here described will allow the genetic improvement of this traditional crop. 相似文献
18.
19.
Petra Krejčí Petra Matušková Pavel Hanáček Vilém Reinöhl Stanislav Procházka 《Acta Physiologiae Plantarum》2007,29(2):157-163
Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation
procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots
were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred
to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis
of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene
in the analysed plants. 相似文献