A protocol with a high rate of transformation and regeneration of `Hibush' eggplant (Solanum melongena L.) has been developed. This protocol used leaves of in vitro-grown seedlings as a source of explants. The shoot regeneration culture medium contained 0.1 μm thidiazuron (TDZ) combined with 10 to 20 μm N6-[isopentyl] adenine (2iP). Adding TDZ significantly improved regeneration efficiency and produced a mean of 15 buds and 3 to 4 shoots per explant. When explants were cocultivated with Agrobacterium tumefaciens strains Q10, Q20, Q30, Q40, Q201, Q202, Q203, or Q204 containing the native cryIIIB Bacillus thuringiensis (Bt), neomycin phosphotransferase (NPTII), and β-glucuronidase (uidA) genes, a callus/bud regeneration frequency of 38.8% was observed on the selection medium. Kanamycin at 50 μg·mL-1 was most effective in selecting for transgenic buds and shoots. Augmentin at 300 μg·mL-1 was used to eliminate A. tumefaciens. Augmentin also enhanced shoot proliferation. A transformation/regeneration efficiency of 20.8% was observed for shoot production. More than 400 putative transgenic plants have been produced with this method. From 50 putative transgenic plants, gene integration has been confirmed with Southern blot analysis and progeny tests.
Sharon Billings, Gojko Jelenkovic, Chee-Kok Chin, and Jodi Eberhardt
Gojko Jelenkovic, Sharon Billings, Qi Chen, James Lashomb, George Hamilton, and Gerald Ghidiu
A population of 300 putative transgenic eggplants (Solanum melongena L.) carrying the syn cryIIIA gene was produced and tested for resistance to the Colorado potato beetle [CPB; Leptinotarsa decemlineata (Say)]. Toxicity tests in planta and in vitro demonstrated that 69% of the transformed plants were resistant to neonate larvae and adult CPB. Transgenicity of the plants was confirmed by studies of GUS expression and Southern and northern analysis. Primary transformants, having a single insert of the construct, upon selfing, produced progenies cosegregating for the uidA and syn cryIIIA genes at the expected 3:1 ratios with a few exceptions in which only one of the genes was expressed. The latter was attributed to the gene silencing phenomenon. The segregating resistant R1 seedlings showed the same level of resistance as the parental genotypes in growth chamber tests and under field conditions. One genotype carrying two copies of the construct, upon selfing, segregated at a 15:1 ratio for GUS expression and resistance to CPB, while Southern analysis revealed a 9:3:3:1 genotypic segregation ratio for individual copies of the construct.
Qi Chen, Gojko Jelenkovic, Chee-Kok Chin, Sharon Billings, Jodi Eherhardt, Joseph C. Goffreda, and Peter Day
Three constructs of a coleopteran toxic cryIIIB Bacillus thuringiensis gene were engineered and incorporated into eggplant (Solanum melongena L.). Southern blot analysis of the eight primary transformants and segregational analysis of their R, progenies indicated that the chimeric cryIIIB constructs in each of the transgenic plants were stably incorporated at a single locus or at multiple sites within the same linkage group and that they were regularly transmissible to the progeny. The results of Northern blot and RNase protection analyses demonstrated that transcription of the cryIIIB mRNA takes place in plant cells, but only a small amount of the expected entire length transcripts were produced. The amount of the 5' end mRNA fragment produced was at least 30 to 40 times more abundant than the amount of the 3' end mRNA fragment. This could be interpreted to mean that either the two ends of the mRNA are of different stability or that the transcription process is often interrupted and only a few mRNAs complete the entire process to the end. When the transgenic plant mRNA was reverse-transcribed, amplified by polymerase chain reaction, and hybridized to the cryIIIB probe, two smaller molecular weight mRNA species were identified. Thus, the preponderance of the cryIIIB mRNA in transgenic plants exists as a truncated species, a situation similar to that of cryI genes when expressed in transgenic plants. Seedlings from the eight independent transgenic plants were tested for Coleopteran insect resistance. However, they did not demonstrate any significant resistance to the first and second instar larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say).