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William C. Mitchell and Gojko Jelenkovic

Assays of enzyme activity, in vivo inhibition studies and the developmental analysis of strawberry (Fragaria × ananassa Duch.) fruit alcohol dehydrogenases (ADH) suggest that both the NAD-(E.C. 1.1.1.1) and the NADP-dependent (E.C. 1.1.1.2) forms of ADH enzymes play integral roles in the development and ripening of fruits. One role of ADH enzymes appears to be the evocation of changes in sugar, soluble solids, acidity and volatile compounds necessary for the normal organoleptic character of strawberry fruits. The data presented includes: 1.) The wide substrate specificity of both ADH enzymes for the “fragrance and flavor alcohols and aldehydes” synthesized by ripe strawberry fruits, 2.) the effect of inhibitors of ADH activity upon strawberry fruit ripening, and 3.) the comparative regulation of NAD- and NADP-ADH enzymes including 4.) the developmental control of ADH enzymes in strawberry fruits.

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William C. Mitchell and Gojko Jelenkovic

The NAD-dependent and NADP-dependent alcohol dehydrogenase activities of strawberries (Fragaria xananassa Duch.) were found to have broad substrate specificities including those alcohols and aldehydes responsible for strawberry aroma and flavor either directly or through their ester products. NAD-dependent activities were greatest against short-chained alcohols, whereas the NADP-dependent activities were most active against aromatic and terpene alcohols. Differences were seen in substrate specificity between receptacle and achene alcohol dehydrogenase activities. Alcohol dehydrogenase activities were found to be developmentally regulated in receptacle tissue and increased during the period of fruit maturation and ripening. Isoelectric focusing of NAD-dependent ADH activities showed that several isozymes of this enzyme exist, that they differ between receptacle and achene tissues, and that they vary among specific genotypes. Our results suggest that NAD- and NADP-dependent ADH activities are integral components of flavor and fragrance volatile production in ripening strawberries.

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Joseph A. Fiola, Gojko Jelenkovic, and Gene Galletta

The major objective of the NJUS Strawberry Breeding Program is the development of early ripening cultivars with excellent fruit flavor and size for production under conventional matted-row, and high density annual production systems. In the 1993 replicated Step 3 trials (1991; 1992 planted), sixteen selections had higher yield than `Earliglow' (8127, 11312 kg/Ha), ranging from 8433 kg/Ha to 13334 kg/Ha. Thirty-one had higher weighted average fruit weight (WAFW) over the season than `Earliglow' (8.8 g; 8.4 g), ranging from 9.0 g to 12.3 g.

Selection for phenotype best suited for annual stem includes: low runnering, strong vigor, earliness, and large fruit size. In 1993 harvested Step III, four selections had comparable or higher yield (range: 12,866 to 27,128 kg/Ha) than `Chandler' (12,950 kg/Ha), as well as larger primary and WAFW (range: 13.5 to 16.4 g). All selections were significantly earlier than `Chandler'. In summary, the NJUS Strawberry Breeding Program has selections for the matted-row and annual production systems which are early, with excellent fruit flavor, size, and firmness for fresh market production.

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Arlen D. Draper, Gene J. Galletta, Nicholi Vorsa, and Gojko Jelenkovic

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Sharon Billings, Gojko Jelenkovic, Chee-Kok Chin, and Jodi Eberhardt

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.

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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.

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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).