Jean H. Gould

Transformation of plants via the shoot apex

SummaryWe 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.

Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissues for transformation.

A method of regenerating cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobacterium-mediated transformation. This method was developed to circumvent the problems of genotype restriction and chromosomal damage frequently encountered in cotton regeneration in tissue culture through somatic embryogenesis. In this procedure, the cells of the shoot meristem are targeted for transformation. Normal and fertile plants of Gossypium barbadense Pima S-6, and 19 cultivars of G. hirsutum were regenerated using this method. Shoot regeneration from these tissues was direct and relatively rapid. A MS based, hormone-free medium could be used with all the varieties tested.

A rapid and high yielding DNA miniprep for cotton (Gossypium spp.)

A rapid DNA minipreparation method was developed for cotton and yields 500–600 μg DNA from 1.0 g fresh leaf tissue. Cotton DNA extracted using this method is completely digested with restriction enzymes, supports PCR and Southern DNA analyses and was used successfully in these applications.

In vitro shoot-tip grafting improves recovery of cotton plants from culture

A rapid in vitro shoot-tip grafting (STG) technique was adapted to increase recovery of intact cotton plants from shoots developed in culture. Induction of root organogenesis in cotton shoots is genotype dependent and unreliable. The resulting loss of regeneration potential due to failure to form roots can vary from 30 to 80% according to genotype and represents a significant bottleneck in the overall recovery of plants from culture. If the non-rooting shoots are transgenic, the loss in regenerated plant material can be substantial. In vitro grafting of cotton shoots to seedling rootstock proved to be a simple and reliable method allowing 90–100% recovery of non-rooting shoots from culture. Success of any given graft was directly related to scion size (0.8–1.0 cm) and age (14–35 days) of the seedling rootstock. The method appeared to be genotype independent, and varietal differences between rootstock and scion did not effect the rate of plant recovery from culture.

Transformation and regeneration of loblolly pine: shoot apex inoculation with Agrobacterium

Loblolly pine (Pinus taeda L.) is the mostimportant tree species in US commerce and has much to gain through geneticengineering. This species can be transformed using particle bombardment andAgrobacterium; however, the regeneration of plants fromtransgenic tissues has been difficult and the recovery of transgenic plants hasbeen rare. A shoot-based and genotype-independent transformation methodemploying Agrobacterium tumefaciens was used to facilitaterecovery of plants and permit the transformation of elite germplasm. Shootsfrom4–6 week old seedlings and adventitious shoots from culture wereinoculated with A. tumefaciens EHA101 (pGUS3), or EHA105(pSSLa.3), subjected to selection and regenerated. Shoots that survivedexhibited expression of the uidA gene (GUS) in a patterncharacteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcSpromoter (pSSLa.3) transferred. Recovered plants were screened using PCRamplification. Southern DNA analyses and amplification of the T-DNA borderjunction confirmed genomic integration of both transferreduidA and nptII genes. In this proofofconcept study, the overall recovery of P. taeda shoots wasfair (10–20%), while recovery of intact rooted plants was poor (>1%)due to difficulty in rooting. Recovery of intact rooted plants from inoculatedshoots of P. eldarica and P. radiatawas more efficient (10–30%). The addition of a shoot multiplication stepand effective rooting protocols will improve the efficiency of this genotypeindependent transformation method in P. taeda, and inotherPinus spp.

A non-destructive assay for GUS in the media of plant tissue cultures

Abstractβ-glucuronidase (GUS) can be assayed in the spent media of plant tissues transformed with some GUS gene fusions (Jefferson, 1988). This approach is based on the presence of GUS in the media of transformed plant tissues expressing the gene and can be used to monitor the progress of transformation without destruction of the tissue under study.

A Simple Method for Identifying Plant/T-DNA Junction Sequences Resulting from Agrobacterium-mediated DNA Transformation

Genomic integration of transferred T-DNA is traditionally analyzed by Southern hybridization; however, these analyses often do not provide sufficient information pertaining to the transformation event. Analysis of the junction sequences spanning the region between the T-DNA borders and plant genomic DNA, give a clear demonstration of genomic integration. The procedures available for border junction analysis can be problematic, therefore a simplified method was developed for plants transformed by Agrobacterium tumefaciens harboring the binary vector with pBI121 backbone.

Analysis And Localization of the Water-Deficit Stress-Induced Gene (lp3)

LP3 is a water-deficit-induced protein, which is highly homologous to ASR (ABA, stress and ripening) proteins. Homology was found in the C-terminal region of the putative LP3 protein while lower homologies were found in the N-terminal region. The goal of this study was to investigate the function of the LP3 protein and the mechanism of the lp3 promoter in response to water-deficit stress (WDS) and other stresses. In regenerated transgenic tobacco (T0), expression of β-glucuronidase (GUS) from the lp3 promoter-GUS construct was observed in polyethylene glycol (PEG), abscisic acid (ABA), methyl-jasmonate (MeJa), and fluridone (Flu) treatments. GUS expression was not observed following gibberellin (GA3), 2-methyl-4-dichlorophenoxy acetic acid (2,4-D), silver nitrate, or ethephon (ethylene releasing agent) treatments. Germinated T1 seedlings containing the lp3 promoter-GUS construct exhibited GUS activity up to 40 days postgermination. Expression could be restored when 5-azacytidine was included in the culture media, indicative of a developmentally regulated silencing mechanism involving methylation. In transgenic tobacco, the LP3 protein localized in the cell nucleus was induced by WDS and appeared to be developmentally regulated.

Morphogenic substances released by plant tissue cultures: I. Identification of berberine in Nandina culture medium, morphogenesis, and factors influencing accumulation

The release of endogenous substances is a general phenomenon of plant tissue cultures, with some substances having significant developmental effects on the releasing tissues. Their systematic study was initiated with Nandina tissue cultures, and a yellow compound that accumulated in the culture medium was identified as the alkaloid, berberine. The rate of its release was related to the supplies of auxin, cytokinin, and nutrient salts. Addition of berberineHC1 to nutrient media did not inhibit Nandina tissues, but suppressed shoot formation in Nicotiana stem segments. Growth of Nandina and Nicotiana callus, as well as rooting of Nicotiana stem segments, was promoted by alkaloid addenda.

Shoot regeneration and ploidy variation in tissue culture of honeydew melon (Cucumis melo L. inodorus)

The Cucumis melo L. inodorus honeydew melon variant is one of the most consumed melons in the USA, and has important commercial and nutritional value. There is a need for improvement of several genetic traits in the US honeydew melon, such as nutrition, drought tolerance, and disease resistance. We investigated the existing regeneration media and optimized the medium composition for an elite honeydew diploid breeding line, “150”, using cotyledonary explants. Four combinations of three different plant growth regulators, 6-benzyladenine, abscisic acid, and indole-3-acetic acid (IAA), were tested in the shoot regeneration media. The presence of IAA in the medium caused the cotyledon explants to curl away from the medium, which made antibiotic selection problematic in our previous study. Omission of IAA from the culture media eliminated this problem and did not impact the shoot regeneration capacity of the cotyledonary explants. We also estimated the ploidy of regenerated plants using flow cytometry, and 50–60% were found to be polyploid (tetraploid or mixoploid). However, contrary to other studies, these polyploid plants did not show major morphological differences compared to the diploid plants.