Roberta H. Smith

Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis H+/Ca2+ transporters.

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H+/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H+/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth.

Efficient and genotype-independent Agrobacterium - Mediated tomato transformation

An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation. Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A. tumefaciens significantly reduced the callus induction and regeneration. From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested. In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal. Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis. This procedure represents a simple, efficient and general means of transforming tomato.

T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices

An incorrect description of the results of Gould et al. published in Plant Physiol. 95: 426–434, 1991, “Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot apex” appeared in S.H. Park et al., Plant Molecular Biology 32: 1134–1148, 1996, “TDNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices”. Gould et al. [1991] reported the presence of Gus and NPT II genes in progeny of inoculated and regenerated plants. Unfortunately, Park et al. described the work of Gould et al. as “reporting NPT and Gus expression in primary and R1 progeny of Zea mays”.

Somatic embryogenesis in suspension cultures of Gossypium klotzschianum anderss.

Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3–4 weeks of culture in a liquid medium containing glutamine (optimally, 10–15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.

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.

TRANSFORMATION OF A TEXAS COTTON CULTIVAR BY USING AGROBACTERIUM AND THE SHOOT APEX

Abstract Transgenic cotton (Gossypium hirsutum L.) plants of a Texas cultivar CUBQHRPIS were obtained using Agrobacterium-mediated transformation coupled with the use of shoot-apex explants. After inoculation with A. tumefaciens strain LBA 4404 containing the pBI121 plasmid, regeneration of primary plants was carried out in a medium containing kanamycin (100  mg l-1). Progeny obtained by selfing were germinated in the greenhouse and selected for expression of the T-DNA marker gene encoding neomycin phospho-transferase II (NPTII) by painting kanamycin (2%) on the leaves. Plants that survived the leaf painting were analyzed by DNA blots. Evidence for integration of the transgene (GUS) was observed in two successive generations from the regenerants (T0). The transformed plants appeared to have more than one copy of the T-DNA.

Increased calcium in carrots by expression of an Arabidopsis H+/Ca2+ transporter

We demonstrate that carrots expressing the Arabidopsis H+/Ca2+ transporter CAX1 (Cation Exchanger 1) contained up to 50% more calcium (Ca) than plants transformed with control vectors. The CAX1-expressing carrots were fertile, and robust plant growth was seen in the majority of the transgenic plants. CAX1-expressing carrots were crossed to a commercial carrot variety to confirm that the increased Ca accumulation was mediated by CAX1-expression, and the increased Ca content was clearly correlated with the transgene. This study suggests that modulation of ion transporters could be an important means of increasing the Ca content of agriculturally important crops. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of an agriculturally important crop.

Temperature influence on stable T-DNA integration in plant cells

Abstract. Four co-cultivation temperatures (15°C, 19°C, 25°C, and 32°C) were evaluated to determine their effects on T-DNA transfer and stable integration. Tobacco leaf explants were co-cultivated with Agrobacterium tumefaciens LBA4404 containing plasmids encoding resistance to the herbicide phosphinothricin, and Bt for insect resistance. Transgenic plants were evaluated for insect and herbicide resistance as well as at the molecular level for foreign gene integration. Even though 19°C has been reported as the optimal temperature for Agrobacterium-mediated gene transfer, co-culture at 25°C led to the highest number of stable transformed plants. Although 19°C may be the best temperature for the Agrobacterium transfer machinery, co-culture at 25°C appears beneficial for plant cell susceptibility to infection and for stable T-DNA insertion into the plant chromosomes.

Somatic embryogenesis from shoot tip and immature inflorescence of Phoenix dactylifera cv. Barhee

SummaryA method of clonal propagation via somatic embryogenesis of date palm, cultivar Barhee, which has potential for large scale commercial application as well as for developmental studies on embryos is described. Cultures were initiated from shoot tip and immature inflorescence explants, both of which were capable of development into embryogenic callus. When the embryogenic callus was cultured in liquid suspension on a rotary shaker, hundreds of embryos developed from milligram quantities of callus in a fairly synchronous manner. Scanning electron microscopy showed globular, heart-shaped and torpedo-shaped embryos. Green leaves emerged from a white cotyledonary sheath.