Sung Hun Park

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”.

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.

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.).

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the β-d-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6–8xa0days on co-cultivation medium supplemented with 0.1–0.001xa0mg/l l-α-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2–3xa0weeks of culturing on selection medium containing 2xa0mg/l dl-phosphinothricin. These shoots were rooted after 3xa0weeks of culturing on half-strength MS medium containing 0.1xa0mg/l indole acetic acid and 1xa0mg/l dl-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system

Abstractu2002In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3u2009μmol·l–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20u2009μmol·l–1) was evaluated. Multiple shoot induction medium with 1u2009μmol·TDZ l–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainungu20091, Tainungu20092, and Evergladesu200971). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1u2009μmol·TDZ l–1. Multiple shoot clumps formed after 2u2009weeks in culture without callus formation. Shoots elongated and rooted within 2u2009weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex.

Expression of an Arabidopsis CAX2 variant in potato tubers increases calcium levels with no accumulation of manganese

Previously, we made a chimeric Arabidopsis thaliana vacuolar transporter CAX2B [a variant of N-terminus truncated form of CAX2 (sCAX2) containing the “B” domain from CAX1] that has enhanced calcium (Ca2+) substrate specificity and lost the manganese (Mn2+) transport capability of sCAX2. Here, we demonstrate that potato (Solanum tuberosum L.) tubers expressing the CAX2B contain 50–65% more calcium (Ca2+) than wild-type tubers. Moreover, expression of CAX2B in potatoes did not show any significant increase of the four metals tested, particularly manganese (Mn2+). The CAX2B-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through the successive generations and showed no deleterious alternations on plant growth and development. These results demonstrate the enhanced substrate specificity of CAX2B in potato. Therefore, CAX2B can be a valuable tool for Ca2+ nutrient enrichment of potatoes with reduced accumulation of undesirable metals.

Transformation parameters enhancing T-DNA expression in kenaf (Hibiscus cannabinus)

Summary Kenaf( Hibiscus cannabinus )is a fast growing annual with tremendous potential as a source of fiber for ropes, textiles and paper. Kenaf is an environmentally friendly crop; however, commercial production of kenaf is hindered by weed competition at the seedling stage. Herbicide resistant kenaf cultivars would reduce seedling weed competion and make growing kenaf more profitable. Factors that are important in establishing a transformation system for kenaf were examined. The influence of Agrobacterium strain, temperature, host tissue wounding, acetosyringone, v irG/virE genes and host cell division on T-DNA expression in the kenaf shoot apex were investigated. Three Agrobacterium strains were tested, and A. tumefaciens LBA4404 significantly (α=0.05) yielded a high number of shoots surviving on selection medium; no shoots survived with EHA101S or Z707S. There was no significant difference (α=0.05) in transient T-DNA expression between 28 °C and 25 °C; however, shoots did not survive 16 °C or 19 °C co-cultivation temperatures. Shoot apex survival was increased significantly (α=0.05) when virulence genes and a cytokinin, TDZ, were combined. Sonicated shoots showed an increase in transient expression and shoot survival. Optimal conditions for shoot apex T-DNA transfer and expression were sonication for 5 s, co-cultivation with LBA4404 containing virG/virE at room temperature, and 200 μmol/L acetosyringone.

Shorter T-DNA or additional virulence genes improve Agrobactrium-mediated transformation.

Abstractu2002The effect of additional virulence (vir) genes and size of plasmid T-DNA in Agrobacterium tumefa- ciens was investigated for their impact on transformation efficiency. Transformation efficiency in tobacco, cotton, and rice was increased when the T-DNA was 4.3 kb compared to 8.4 kb in size. However, when additional virG, virGN54D,virE, or virE/virG plasmids were included with the 8.4-kb T-DNA, transformation frequencies in all cases were increased over that of the shorter T-DNA without additional vir plasmids. The use of virE, virG or virGN54D copies enhanced transformation efficiency; however, the most significant increase of transformation efficiency in all three plant species was observed when the virE/virG plasmid was used for infection. The virE/virG plasmid dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer; moreover, this plasmid appears to have broad efficiency since it was consistently effective on two different dicotyledon species as well as a monocotyledon species.

Herbicide and insect resistant elite transgenic rice

Summary Transgenic rice ( Oryza sativa L.) containing two agronomically useful genes was generated using Agrobacterium LBA4404 with an additional virulence plasmid, virG (pTiBo542)/ virE1virE2 (pTiA6). The plants were transformed with phosphinothricin acetyl transferase ( pat ) gene for herbicide resistance and Bacillus thuringiensis ( Bt ) crystal insecticidal protein gene for insect resistance. Three different sets of primary plants expressing (1) the pat and Bt genes, (2) the pat gene and a fragmented (nonfunctional) copy of the Bt gene, or (3) the pat gene only were produced. Stable integration, expression and transmission of the transferred genes in T 0 and T 1 generation plants were confirmed by both molecular analysis and phenotype expression. The herbicide application test of the progeny from the three sets of primary plants showed that the transferred pat gene was stably expressed in the T 1 generation. The insect feeding bioassay with T 1 generation plants conferring resistance to herbicide established that the transgenic plants having a complete Bt gene were toxic to tobacco budworm ( Heliothis virescens ) larvae. The insect feeding bioassay and herbicide application test results were clearly correlated with the molecular analysis.

Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expression.

Summary Additional copies of virulence ( vir ) genes do enhance transformation frequencies in multiple species; the mechanisms that play a role in this enhancement are not understood. In this report, transformants produced using additional copies of helper plasmids harboring pCH30 ( vir G), pCH32 ( vir G and vir E), and pCH42 ( vir E) were evaluated for gene expression, copy number, and complexity of integration patterns. The vir plasmids increased transformation efficiency 2-fold with pCH42, 3-fold with pCH30, and 4-fold with pCH32. One to six transgene copy numbers were detected, and irregular transgene integration patterns were observed in all plasmid combinations (pAGM280 only, pAGM280 + pCH30, pAGM280 + pCH32 and pAGM280 + pCH42). There were no differences in transgene copy number and/or integration patterns between transformants produced with or without additional vir genes. Additionally, no relationship between transgene copy number and expression or between transgene integration patterns and expression was observed. All transgenic plants resulting from the additional vir plasmids were able to express the foreign genes. In contrast, forty percent of transgenic plants regenerated without additional vir plasmids did not express the foreign gene even though the foreign genes were present. These results demonstrate a link between an increase in copy number of the vir genes and stable transgene expression and increased plant transformation efficiency.