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1.
High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics 总被引:2,自引:0,他引:2
Oosumi T Gruszewski HA Blischak LA Baxter AJ Wadl PA Shuman JL Veilleux RE Shulaev V 《Planta》2006,223(6):1219-1230
Fragaria vesca L., a diploid (2n=2x=14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6–7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l−1 hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14–15 weeks to complete its life cycle in different seasons in the greenhouse. 相似文献
2.
Summary Translational genomics is defined as the application of molecular-genetic principles derived from model systems to species
of experimental or economic interest. The past 20 years of research in plant model systems such as Arabidopsis thaliana have relinquished vast amounts of information regarding gene function, the integration of genetic components into pathways,
and the interrelationships between pathways to control form and function in plants and plant-products alike. At present, the
challenge is to relate these paradigms to other species of economic or scientific interest. Apart from being an important
and valuable crop, strawberry (Fragaria spp.) is a member of the Rosaceae, a plant family containing fruit, nut, ornamental and wood-bearing species. Strawberry is unique within the Rosaceae in that it is a rapidly growing herbaceous perennial with a small genome and the ability to thrive in a laboratory setting.
Strawberry species may also be transformed and regenerated in a time scale of weeks or months instead of years. For these
reasons, strawberry has been recognized as the translational genomics model for the Rosaceae family. This review summarizes and synthesizes the technical reports of strawberry regeneration and transformation, consolidating
the large body of information regarding genetic modification of this important genus. 相似文献
3.
Transformation of plants via the shoot apex 总被引:4,自引:0,他引:4
E. C. Ulian R. H. Smith J. H. Gould T. D. McKnight 《In vitro cellular & developmental biology. Plant》1988,24(9):951-954
Summary We have transformed petunia byAgrobacterium tumefaciens containing genes for kanamycin resistance and beta-glucuronidase using isolated shoot apices from seedling tissue. Regeneration
of transformed plants in this model system was rapid. The technique of shoot apex transformation is an alternative for use
inAgrobacterium-mediated transformation of dicotyledonous crop species for which a method of regeneration via protoplasts, leaf disks, or
epidermal strips does not exist. This approach offers direct and rapid regeneration of plants and low risk of tissue-culture-induced
genetic variation.
Texas Agricultural Experiment Station Technical Article No. 23317. 相似文献
4.
Dipankar Chakraborti Anindya Sarkar Sampa Das 《Plant Cell, Tissue and Organ Culture》2006,86(1):117-123
Lacking of an efficient regeneration protocol for the recalcitrant crop chickpea is a limiting factor for adapting genetic engineering approaches for its improvement. The present study describes a rapid and efficient method for multiple shoot regeneration for three Indian cultivars, B115, C235, ICCV89314, using single cotyledons with half embryos as explant. Modified MS medium with 1.5 mg l−1 6-benzyladenine (BA) and 0.04 mg l−1 α-naphthaleneacetic acid (NAA) induced a maximum of 26 shoots from a single explant after 20 days of culture. When cultured in modified MS medium containing 0.2 mg l−1 indole-3-acetic acid (IAA), 80% of the shoots from each regenerating explant elongated in another 20–25 days. Following a root-grafting protocol, 90–95% of the elongated shoots survived in soil which subsequently produced seeds. The regeneration process from explant preparation to complete plants took 55–60 days. The presently optimized rapid regeneration method holds promise for facilitating the deployment of agronomically important components through genetic transformation for betterment of this important food crop. 相似文献
5.
Neftali Ochoa-Alejo Rafael Ramirez-Malagon 《In vitro cellular & developmental biology. Plant》2001,37(6):701-729
Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is
a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have
lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation
aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of
chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied
to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques
have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures
have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but
no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper
fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications
of chili pepper biotechnology are discussed. 相似文献
6.
2种菊苣再生体系及遗传转化效率的比较 总被引:1,自引:0,他引:1
以普那菊苣和将军菊苣子叶为材料,通过植物组织培养的方法,探讨了不同激素浓度配比对二者愈伤组织诱导、芽分化以及根再生的影响,并通过农杆菌介导法将编码獐茅液泡膜Na+/H+逆向转运蛋白基因(AlNHX)导入菊苣中,比较普那菊苣和将军菊苣的遗传转化效率。结果表明:不同基因型的菊苣愈伤组织诱导和芽分化条件不同,普那菊苣最佳培养基为MS+1.5mg/L 6-BA+0.2mg/L IBA;将军菊苣最佳培养基为MS+1.0mg/L 6-BA+0.5mg/L NAA;二者最佳生根培养基均为1/2MS+0.1mg/L NAA。获得的抗性芽经PCR检测,初步证实AlNHX已插入到菊苣基因组中,且普那菊苣转化效率为10.0%,将军菊苣转化效率为13.3%。 相似文献
7.
Medicago
truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes. 相似文献
8.
An in vitro propagation system for Artemisia vulgaris L., a traditional medicinal plant, has been developed. The best organogenic response, including adventitious shoot number
and elongation, was obtained when hypocotyl segments were cultured onto MS medium supplemented with 4.54 μM TDZ (N-phenyl-N′-(1,2,3-thidiazol-yl) urea). Up to 28 shoots formed per explant for an optimal duration of exposure of 48 days. Regenerated
shoots formed roots when subcultured onto a medium containing 8.56 μM IAA (indole-3-acetic acid). Healthy plantlets were transferred
to a garden soil:farmyard soil:sand (2:1:1) mixture for acclimatization, which was successful, and subsequent maturity was
achieved under greenhouse conditions over a six-month period. The survival rate of the plantlets varied under acclimatization.
The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation
of medicinally active constituents of A. vulgaris. This optimized protocol has been successfully employed for genetic transformation studies in A. vulgaris, which are currently underway in our laboratory. 相似文献
9.
Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve
this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration
from rice shoot apical meristem. By use of MS medium containing 4 mg L−1 thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage.
All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil
within 5–8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either
250 mg L−1 carbenicillin or 250 mg L−1 cefotaxime to kill the rice shoot apical meristem was 50 mg L−1 and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency
of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient
GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T0 transformant plantlets. 相似文献
10.
With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based
biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation
procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are
suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media.
Transformation was verified through β-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification
of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes.
The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic
selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials
and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3–4 months, rather than the 6–12 months
required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes
them better suited for large-scale functional genomics studies in poplars. 相似文献
11.
Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth
regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS)
medium was found to be best for promoting shoot regeneration, followed by Gamborg's B5 and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented
with 2.0 mgl−1 (8.9 μM) 6-benzyladenine and 1.0 mgl−1 (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per
explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial
for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl−1 (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration
procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage. 相似文献
12.
Summary A temporary immersion bioreactor system (TIB system) provides a convenient and efficient way to propagate plant material in vitro while requiring significantly lower labor input than conventional methods. The applicability of a TIB system for adventitious
shoot regeneration from strawberry leaf explants was studied. Five commercial cultivars, i.e. Bounty, Jonsok, Korona, Polka,
and Zephyr, were propagated in regeneration medium in commercially available TIB bioreactors (RITA?) and, for comparison, on the same medium solidified with agar. The TIB system proved to be well suited for shoot propagation
and for subsequent subculture of the developing plantlets. Regeneration frequencies were 70±8 to 94±2% and 83±5 to 92±3% in
the TIB system and on semi-solid medium, respectively. The labor time taken by the TIB system was less than half of the time
required for handling plant material for cultivation on semi-solid medium. This system thus provides a convenient method that
could be adopted for commercial in vitro propagation or for regeneration of transgenic strawberry cultivars. 相似文献
13.
J. R. Myers J. W. Grosser N. L. Taylor G. B. Collins 《Plant Cell, Tissue and Organ Culture》1989,19(2):113-127
Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.Research supported by USDA/CRGO Grant No. 81 CRCR-1-0613 相似文献
14.
Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future. 相似文献
15.
16.
Pramila Shah N. K. Singh Neeraj Khare Meenal Rathore S. Anandhan M. Arif Rupesh Kumar Singh S. C. Das Z. Ahmed Narendra Kumar 《Plant Cell, Tissue and Organ Culture》2008,95(3):363-371
Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore,
experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation.
6-benzylaminopurine at 0.05 mg l−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets
in indole-3-aceticacid 0.5 mg l−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after
co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by
polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent. 相似文献
17.
广藿香是重要的芳香药用植物,利用基因工程技术对广藿香进行品种改良,需要建立一个高效的广藿香植株再生体系。该研究以广藿香无菌苗叶片为材料,将叶盘外植体分别置于不同条件下培养,观察、统计其再生植株的数量及生长状况。通过研究15~50 d的苗龄、第2~4节上的叶及培养基中2,4-D、NAA、BA和KT的浓度和配比等因素对广藿香叶盘再生植株的影响,在此基础上优化培养条件,建立广藿香高效再生体系。结果表明:广藿香无菌苗的苗龄、叶片在茎上的着生位置以及培养基中的植物生长调节物质浓度和配比都对广藿香的植株再生有显著影响;优化培养条件为以培养30 d的广藿香无菌苗顶芽下第2对展开叶片切割的叶盘为外植体,在含0.1 mg·L-1NAA和0.5 mg·L-1BA的MS培养基中培养28 d,叶盘的不定芽发生频率达到100%,单个叶盘的平均再生芽数为96.5个,经生根培养及温室炼苗,再生植株的移栽成活率达到96%。广藿香叶盘植株再生体系的建立为其基因转化研究及优良品种的快速繁育奠定了基础。 相似文献
18.
A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6–7 weeks through
a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg l−1) and kinetin (0.1 mg l −1) was used for induction of friable embryogenic calli and somatic embryos. Germination of somatic embryos was achieved about
5 weeks after transfer onto Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (2 mg l−1) and indole-3-acetic acid (0.5 mg l −1) under light. Seeds from in vitro-regenerated plants produced a normal crop in a field trial, and were comparable to the crop grown with the seeds of the mother
plant used to initiate tissue culture. The simplicity of the protocol and possible advantages of the system for transformation
over other protocols using different explants are discussed.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
19.
Amanda Ferreira da Silva Mendes Luciana Cardoso Cidade Maria Luiza Peixoto de Oliveira Wagner Campos Otoni Walter Dos Santos Soares-Filho Marcio Gilberto Cardoso Costa 《Plant Cell, Tissue and Organ Culture》2009,97(3):331-336
Identification of beta-lactam antibiotics that have negligible effects on plant regeneration is a critical step towards the
establishment of a reliable Agrobacterium-mediated transformation protocol for perennial trees. In the present report, we have evaluated the effects of the novel beta-lactam
antibiotics meropenem and timentin on plant regeneration of a perennial woody fruit plant, Citrus sinensis, in comparison with the commonly used beta-lactam cefotaxime. It was observed that, in contrast to cefotaxime, meropenem
and timentin had a positive or no detrimental effect on the shoot regeneration from epicotyl explants. Residual effects of
the beta-lactams from shoot regeneration medium also affected the subsequent ability of the roots to elongate. The addition
of meropenem and/or timentin in the rooting medium mostly improved or did not affect the rooting ability of the adventitious
shoots. These data indicated that meropenem and timentin can positively replace cefotaxime in Agrobacterium-mediated transformation of C. sinensis. 相似文献
20.
We have achieved high-frequency shoot regeneration in radish(Raphanus sativus). Cotyledon explants from four-day-old seedlings were suitable for the effective induction of shoots on Murashige and Skoog’s
(MS) medium containing 3.0 mg/L kinetin. We also determined that it was essential to include 1- to 2-ram petiole segments
with the cotyledons for efficient induction. When the regenerated shoots were transferred to an MS liquid medium containing
0.1 mg/L NAA, roots formed within four weeks, and normal plant development ensued. We established a transformation protocol
using anAgrobacterium binary vector that carries the GUS reporter gene. Preculturing the explants for I d in an MS medium containing 3.0 mg/L kinetin
also increased efficiency. Five days of cocultivation proved best for delivering T-DNA into radish. Transformation frequencies
of up to 52% were obtained in shoot induction media that contained 3.0 mg/L kinetin. 相似文献