Transformation experiments by pipetting Agrobacterium into the spikelets of wheat (Triticum aestivum L.)

Heß, Dieter; Dressler, K.; Nimmrichter, Rainer

doi:10.1016/0168-9452(90)90087-5

Cited by 78 publications

(29 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…5) Hybridization signals in all the three plants were of the same size indicating that these plants are derived from a single transformation event. However, six plants that were PCR positive were negative after southern analyses which may be due to the incomplete insertion, unstable integration of the transferred gene into plant genome and hence its loss (Hess et al 1990;Langridge et al 1992). Thus, southern analysis established the existence and integration of the transgene in the transformants.…”

Section: Southern Hybridizationmentioning

confidence: 97%

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

Sainger

Chaudhary

Dahiya

et al. 2015

Physiol Mol Biol Plants

View full text Add to dashboard Cite

An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B 5 vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2-3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron.The presence and integration of transgenes in putative T 0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T 1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their quantitative and qualitative improvements.

show abstract

Section: Southern Hybridizationmentioning

confidence: 97%

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

Sainger

Chaudhary

Dahiya

et al. 2015

Physiol Mol Biol Plants

View full text Add to dashboard Cite

show abstract

“…Hess et al (1990) pipetted A. tumefaciens into the spikelets of wheat, and several kanamycin-resistant grain progeny were obtained. However, the protocol was not reproducible and the Southern hybridization was not convincing in this study.…”

mentioning

confidence: 99%

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

et al. 1997

View full text Add to dashboard Cite

A rapid Agrobacferium fumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. l h e explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the p-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants i n soil was

show abstract

“…Putative wheat and barley transformants generated by injecting Agrobacterium into spikelets (Hess et a/., 1990) have been suggested to be artefacts, possibly due to gene transfer into an endophytic microorganism associated with these crop plants (Langridge et a/., 1992). The observations reported here, of ""A DNA in the transformed cell lines and of plasmid rescue from the same DNA preparations, support the possibility of transforming endophytes at the same time as transforming plant cells.…”

Section: Discussionmentioning

confidence: 99%

“…Wang et a/., 1992) or those methods mentioned above (Hess et a/., 1990;Kaepller et a/., 1992;Luo and Wu, 1988;Schnorf et a/., 1991). This type of pattern is usually interpreted as a concatemeric rearrangement of integrated foreign DNA (Czernilofsky eta/., 1986) or is attributed to favoured sites 429 for the integration of foreign DNA into the recipient genome (Hess et a/., 1990). However, no detailed investigation has been carried out to examine the somatic stability, origin and biological nature of the DNA that is represented by these similar hybridization patterns.…”

Section: Introductionmentioning

confidence: 99%

Stability of transgenes and presence of N⁶ methyladenine DNA in transformed wheat cells

et al. 1994

View full text Add to dashboard Cite

Summary A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post‐selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation‐sensitive enzymes revealed that the DNA represented by the common bands was N6‐adenine methylated (m6A DNA), and there was even m6A DNA in some 30 month samples. Although the source of m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma‐like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.

show abstract

Transformation experiments by pipetting Agrobacterium into the spikelets of wheat (Triticum aestivum L.)

Cited by 78 publications

References 35 publications

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

Stability of transgenes and presence of N⁶ methyladenine DNA in transformed wheat cells

Contact Info

Product

Resources

About

Transformation experiments by pipetting Agrobacterium into the spikelets of wheat (Triticum aestivum L.)

Cited by 78 publications

References 35 publications

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

Stability of transgenes and presence of N6 methyladenine DNA in transformed wheat cells

Contact Info

Product

Resources

About

Stability of transgenes and presence of N⁶ methyladenine DNA in transformed wheat cells