共查询到20条相似文献,搜索用时 31 毫秒
1.
Taskin Kemal Melik Turgut Kenan Gulhan Ercan A. Scott Rod J. 《Plant Cell, Tissue and Organ Culture》2003,72(2):173-180
An efficient method for adventitious shoot regeneration for Arabis drummondii and a transformation protocol for A. gunnisoniana from hypocotyl explants are described. Hypocotyl explants from 7-day-old aseptically grown seedlings were cultured on MS medium containing plant growth regulators (6-benzylaminopurine, 1-phelyl-3- (1,2,3-thiadiazol-5-yl) urea, -naphthaleneacetic acid and 2,4-dichlorophenoxy-acetic acid). After 4 weeks in culture, high frequency of adventitious shoot regeneration was observed. Regenerated shoots were rooted on half-strength MS basal medium supplemented 1% (w/v) sucrose, with or without NAA. This protocol was then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS 21 medium supplemented with 50 mg l kanamycin. PCR analysis verified the presence of the nptII gene in the plant DNA isolated from kanamycin resistant shoots. 相似文献
2.
Genotypic control of peanut somatic embryogenesis 总被引:2,自引:0,他引:2
The protocol for obtaining a high frequency of plant development via somatic embryogenesis from mature zygotic embryo-derived
leaflets of peanut (Arachis hypogaea L.) involves multiple stages; these include the induction of embryogenic masses, development of embryos, radicle emergence/conversion
of embryos and the development of plants from rooted abnormal embryos. Sixteen genotypes were subjected to this protocol by
exposing mature zygotic embryo-derived leaflets to the common media sequence and comparing responses. Although the protocol
was effective for all the genotypes, variation in frequency of response at each stage of development indicated that, with
the exception of root meristem differentiation and subsequent radicle emergence, the whole process of somatic embryogenesis
depended on the genotypic constitution of the original plant. The failure of somatic embryos to undergo conversion to plantlets
could be a genotype-dependent characteristic.
Received: 5 June 1997 / Revision received: 2 December 1997 / Accepted: 12 December 1997 相似文献
3.
Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium 总被引:1,自引:0,他引:1
The present study describes a simple and efficient protocol for plant regeneration from scutellar-derived embryogenic calli of an elite basmati indica rice (Oryza sativa L., cv Pusa Basmati 1) transformed with Agrobacterium. A supervirulent plasmid pTOK233 as well as a non-supervirulent plasmid pJB90GI containing -glucuronidase (gus) and hygromycin phosphotransferase (hpt) chimeric genes were used to assess transformation and regeneration efficiency. The effects of some factors like the bacterial density and inclusion of sorbitol in the medium on the co-culture and transformation have been evaluated; the procedure for selection and regeneration from transformed calli was found to be critical. Furthermore, co-culture and selection on regeneration medium was found to be better than callus medium and led to minimal media manipulations. Regeneration medium supplemented with 3% maltose was found to be better for regeneration as compared to 3% sucrose. The transformed calli were subjected to three cycles of regeneration, thus converting a higher number of transformation events into regenerants. The selected calli as well as leaf sections and roots of the transformants were GUS positive. The stable integration of the transgene was confirmed by polymerase chain reaction and Southern blot analysis of the transformants. Interestingly, the presence of three additional vir genes in supervirulent plasmid pTOK233 was not required for transformation as transformation was successful with non-supervirulent plasmid pJB90GI, although the transformation and regeneration frequency was higher with the former. This effective protocol for regeneration from transformed calli resulted in a relatively high transformation frequency. 相似文献
4.
Jayanand B. Sudarsanam G. Sharma Kiran K. 《In vitro cellular & developmental biology. Plant》2003,39(2):171-179
Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived
from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting
of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige
and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion
of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency
of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA3) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the
regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed
by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency
of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement. 相似文献
5.
Sairam Reddy P. Rodrigues Rosaline Rajasekharan Ram 《Plant Cell, Tissue and Organ Culture》2001,66(3):183-188
A high frequency shoot organogenesis and plant establishment protocol has been developed for Coleus forskohlii from leaf derived callus. Optimal callus was developed from mature leaves on Murashige and Skoog (MS) medium supplemented
with 2.4 μM kinetin alone. Shoots were regenerated from the callus on MS medium supplemented with 4.6 μM kinetin and 0.54
μM 1-naphthalene acetic acid. The highest rate of shoot multiplication was achieved at the sixth subculture and more than
150 shoots were produced per callus clump. Regenerated shootlets were rooted spontaneously on half-strength MS medium devoid
of growth regulators. The in vitro raised plants were established successfully in soil. The amount of forskolin in in vitroraised plants and wild plants was estimated and found that they produce comparable quantity of forskolin. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this plant.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
Manoj K. Sharma Amolkumar U. Solanke Dewal Jani Yogendra Singh Arun K. Sharma 《Journal of biosciences》2009,34(3):423-433
We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and
indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation
frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and β-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa
Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone. 相似文献
7.
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
8.
Jesús Arellano Sara Isabel Fuentes Patricia Castillo-España Georgina Hernández 《Plant Cell, Tissue and Organ Culture》2009,96(1):11-18
A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic
axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production
was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred
to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots
per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of
the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development
and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure. 相似文献
9.
Nan Gao Weishou Shen Yu Cao Yanhua Su Weiming Shi 《Plant Cell, Tissue and Organ Culture》2009,98(3):321-330
An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar (Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density
of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and
resuspended in a liquid cocultivation medium-I, adjusted to OD600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This
improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency
is stable and highly efficient. 相似文献
10.
K. Balaraju P. Agastian S. Ignacimuthu Kyungseok Park 《Acta Physiologiae Plantarum》2011,33(6):2501-2510
An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used
for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in
vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA3). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on
Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron
(TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot
tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were
transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of
the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based
DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that
there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future. 相似文献
11.
Zheng Si-Jun Khrustaleva Ludmila Henken Betty Sofiari Eri Jacobsen Evert Kik Chris Krens Frans A. 《Molecular breeding : new strategies in plant improvement》2001,7(2):101-115
This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology. 相似文献
12.
Winthrop B. Phippen James E. Simon 《In vitro cellular & developmental biology. Plant》2000,36(4):250-254
Summary An efficient plant regeneration protocol was successfully developed for basil (Ocimum basilicum L.). Explants from 1 mo. old seedlings yielded the highest frequency of 85% regeneration with an average of 5.1 shoots per
explant. The regeneration protocol was performed on three basil varieties (Sweet Dani; methylcinnamate; Green Purple Ruffles).
Callus and shoot induction was initiated on Murashige and Skoog basal medium supplemented with thidiazuron (16.8 μM) for approximately 30 d. Shoot induction and development were achieved by refreshing the induction medium after 14 d. The
most morphogenetically responsive explants were from the first fully expanded true leaves of greenhouse-grown basil seedlings.
All developing bud tissue demonstrated temporary anthocyanin expression; however, anthocyanin expression in Green Purple Ruffles
remained stable until maturity. Developing shoots were rooted in the dark on media with thidiazuron removed. Within 20 d,
rooted plantlets were transferred and acclimatized under greenhouse conditions where they developed normal morphological characteristics.
This is the first report of a successful in vitro regeneration system for basil through primary callus. 相似文献
13.
G. -X. Tang K. Knecht X. -F. Yang Y. -B. Qin W. -J. Zhou D. Cai 《Biologia Plantarum》2011,55(1):21-26
A two-step protocol for improving the frequency of shoot regeneration from oilseed rape (Brassica napus L.) hypocotyl explants was established. The protocol consists of a pre-culture on callus induction medium (CIM) and a subsequent
shoot regeneration on shoot induction medium (SIM). The SIM was Murashige and Skoog medium supplemented with different concentrations
of 6-benzylaminopurine (BA; 2–5 mg dm−3) and naphthaleneacetic acid (NAA; 0.05–0.15 mg dm−3). Maximum frequency of shoot regeneration (13 %) was on the SIM medium containing 4 mg dm−3 BA and 0.1 mg dm−3 NAA, but it increased to 24.45 % when 20 μM silver thiosulphate (STS) was added. Strikingly, an extremely high frequency
of shoot regeneration up to 96.67 % was reached by a two-step protocol when hypocotyl explants had been pre-cultured for 7
d on a CIM medium containing 1.5 mg dm−3 2,4-dichlorophenoxyacetic acid. In addition, the shoot emergence was also 7 d earlier than that observed by use of the one-step
protocol. The two-step protocol was also applied for regeneration of transgenic plants with cZR-3, a nematode resistance candidate gene. As a result, 43 plants were generated from 270 shoots and from these 6 plants proved
to be transgenic. 相似文献
14.
Monique F. van Wordragen Jan de Jong Hans B. M. Huitema Hans J. M. Dons 《Plant cell reports》1991,9(9):505-508
Summary To develop an Agrobacterium mediated transformation protocol for chrysanthemum we studied the transformation efficiency of commonly used A.tumefaciens strains on 14 genotypes by comparing tumour size and frequency. One genotype was analyzed in detail using 14 strains of both A.tumefaciens and A.rhizogenes. Only a few genotype/strain combinations resulted in significant tumour formation. Especially 0-type strains were highly efficient. An 0-type strain was used to transfer genes for neomycine phosphotransferase (NPT II) and ß-glucuronidase (GUS) to a susceptible cultivar. Transfer of the GUS gene was confirmed by using the Polymerase Chain Reaction (PCR). 相似文献
15.
Harsh Chauhan Srinivas A. Desai Paramjit Khurana 《Plant Cell, Tissue and Organ Culture》2007,91(3):191-199
An efficient genotype independent, in vitro regeneration system was developed for nine popular Indian wheat cultivars, three
each of Triticum aestivum L. viz., CPAN1676, HD2329 and PBW343, Triticum durum Desf. viz., PDW215, PDW233 and WH896, and Triticum dicoccum Schrank. Schubl. viz., DDK1001, DDK1025 and DDK1029, by manipulating the concentration and time of exposure to the growth
regulator, thidiazuron (TDZ). A total of 18 (for immature inflorescence and embryo explant) and six (for mature embryo explant)
different combinations of growth regulators were tried for callusing and regeneration, respectively. Media combination with
low concentration of TDZ (2.2 μM) in combination to auxin and/or cytokinin (depending upon culture stage), was found to be
effective for immature and mature explants. Compact, nodular and highly embryogenic calli were obtained by using immature
embryo, immature inflorescence and mature embryo explants, and regeneration frequency up to 25 shoots/explant with an overall
80% regeneration was achieved. Comparable regeneration frequency was achieved for mature embryo explants. No separate hormone
combination for rooting was required and plantlets ready to transfer to soil could be obtained in a short period of 8–10 weeks.
This protocol can be used for raising transgenic plants for functional genomics analysis of agronomically important traits
in the three species of wheat. 相似文献
16.
Khanna Harjeet Becker Doug Kleidon Jennifer Dale James 《Molecular breeding : new strategies in plant improvement》2004,14(3):239-252
Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group (AAA) cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars (Cavendish and Lady Finger) and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) and gusA (-glucuronidase) or nptII (neomycin phosphotransferase) and a modified gfp (green fluorescent protein) gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome. T-DNA:banana DNA boundary sequences were amplified and sequenced in order to study the integration profile. 相似文献
17.
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
18.
C. P. Kaviraj G. Kiran R. B. Venugopal P. B. Kavi Kishor Srinath Rao 《In vitro cellular & developmental biology. Plant》2006,42(2):134-138
Summary This study reports a protocol for plant regeneration from cultured explants of green gram [Vigna radiata (L.) Wilezek] via somatic embryogenesis. Somatic embryos were induced from nature cotyledons of var., TAP-7 and Pusa Baisaki
when cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 2,4-dichlorophenoxyacetic acid,
α-naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid singly or in combination with 2.22–8.88 μM N
6-benzylaminopurine (BAP) or 2.32–9.38 μM kinetin. The type and concentration of auxin and plant genotype influenced the frequency of somatic embryogenesis. NAA was
the most effective auxin for somatic embryo induction. The well-developed, cotyledonary shaped embryos of var. TAP-7 germinated
into plantlets at a frequency of 56.6% on MS medium supplemented with 1.88 μM abscisic acid and 6.66 μM BAP. Regenerated plants were transferred to soil and grown to maturity with 90% survival. The protocol described here offers
a good potential for genetic improvement using gene transfer techniques and the production of synthetic seeds of V. radiata. 相似文献
19.
Pei‐ling Song Małgorzata Jȩdryczka Witold Irzykowski Meng‐jiao Yan Hai‐yan Huangfu Li‐fen Hao Yu‐ying Bao Zi‐qin Li 《Journal of Phytopathology》2016,164(11-12):1097-1104
An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape . 相似文献
20.
Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system 总被引:1,自引:0,他引:1
Narender S. Nehra Ravindra N. Chibbar Kutty K. Kartha Raju S. S. Datla William L. Crosby Cecil Stushnoff 《Plant cell reports》1990,9(6):293-298
An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94). 相似文献