Phytochelatins (PCs) are heavy metal binding peptides that play important roles in sequestration and detoxification of heavy metals in plants. To develop transgenic plants with increased tolerance and/or accumulation of heavy metals from soil, an Arabidopsis thaliana FLAG–tagged AtPCS1 cDNA encoding phytochelatin synthase (PCS) under the control of a 35S promoter was expressed in Indian mustard (Brassica juncea). Four transgenic Indian mustard lines, designated pc lines, with different levels of AtPCS1 mRNA accumulation and correspondent AtPCS1 protein levels were selected and analyzed for tolerance to cadmium (Cd) and zinc (Zn). Heavy metal tolerance was assessed by measuring root length of 10-day-old seedlings grown on agar medium supplemented with different concentrations of Cd (0, 100, 150, and 200 μm CdCl2) and Zn (200, 400, 600, and 800 μm ZnCl2). All transgenic lines showed significantly longer roots when grown on a medium supplemented with 100 μm CdCl2. No significant differences were observed between transgenic lines and wild type when plants were grown on higher levels of Cd. This indicated that only partial tolerance to Cd was observed in these transgenic lines. Similarly, partial tolerance for Zn was also observed in these transgenic lines, but up to levels of 400 μm ZnCl2. Expression levels of AtPCS1 protein were not related to tolerance responses for either Cd or Zn stresses in transgenic lines.
Ksenija Gasic and Schuyler S. Korban
Douglas G. Bielenberg and Ksenija Gasic
Inexpensive plug-and-play temperature controllers have recently become available. These allow a chest freezer to be programmed easily to hold a desired set point across a range of biologically relevant temperatures. Installation can be completed in a few minutes using consumer-grade chest freezers. We used these temperature controllers to create five temperature-controlled chambers at 12, 14, 16, 18, and 20 °C. We demonstrated the use of these controlled-temperature chambers with two biologic assays: floral budbreak of peach [Prunus persica (L.) Batsch] stem cuttings and germination of sunflower (Helianthus annuus L.) seeds. We used the budbreak and germination rates at multiple temperatures to estimate base temperatures and thermal time requirements for development.
Scott Schaefer, Ksenija Gasic and Schuyler Korban
Peach shoots were grown in vitro for 0, 6, 12, 24, and 48 h on a basal medium containing one of several phytohormones or chemical elicitors, including abscisic acid (ABA), indolebutyric acid (IBA), indoleacetic acid (IAA), kinetin, gibberellic acid (GA3), aminocyclopropane-1-carboxylic acid (ACC), methyl jasmonate (MeJ), and salicylic acid (SA). Northern blot analysis was conducted using the 3' end of a peach-1,3-glucanase gene, PpGns1, used as a probe. Variations in levels of PpGns1 expression patterns were observed for each of the treatments. Shoots treated with ABA displayed high levels of transcripts at 12 and 24 h, followed by a sharp decline at 48 h. Shoots treated with ACC displayed a steady increase in transcripts throughout the 48 h period. The synthetic auxin IBA displayed a steady increase in mRNA accumulation for the first 24 h followed by a sharp decline at 48 h. Shoots treated with kinetin displayed low levels of transcripts after 24 h, while GA3 did not induce any accumulation of PpGns1 transcripts. Both SA and MeJA induced steady mRNA accumulation in peach shoots over the entire 48-h period. Induction of PpGns1 in response to SA, MeJ, and ACC also resulted in observed necrotic lesions on peach shoots, thus suggesting a different defense mechanism response.
Scott C. Schaefer, Ksenija Gasic and Schuyler S. Korban
Several plant defense genes, including an iris ribosomal-inactivating protein (I-RIP) and a maize β-glucanase (M-GLU) as well as a small antimicrobial peptide (5 kd) from Mirabilisjalapa(Mj-AMP) were separately introduced into tomato (Lycopersiconesculentum) cv. Sweet Chelsea using Agrobacteriumtumefaciens-mediated transformation. Transgenic lines carrying each of the transgenes were confirmed for integration into the tomato genome using Southern blot hybridization. Transcription of I-RIP, M-GLU, and Mj-AMP genes in various transgenic lines was determined using Northern blot analysis. Plants of some transgenic lines were inoculated with a 2 × 104 to 3 × 104 conidial spores/mL suspension of the fungal pathogen Alternariasolani, the causal agent of tomato early blight disease. Several transgenic lines carrying either a M-GLU or Mj-AMP transgene showed a higher level of resistance to early blight than control (nontransgenic) plants. The implications of this approach on developing disease resistance in tomato will be discussed.
Ksenija Gasic, John E. Preece and David Karp
Ksenija Gasic and John E. Preece
Chengyan Yue, R. Karina Gallardo, James J. Luby, Alicia L. Rihn, James R. McFerson, Vicki McCracken, Tom Gradziel, Ksenija Gasic, Gregory L. Reighard, John Clark and Amy Iezzoni
We conducted audience surveys at three major peach producer meetings across the United States. We found that the relative importance assigned to fruit quality and tree traits by producers varied across producers’ end markets. Fresh peach producers indicated fruit flavor and size were the most important fruit quality traits, whereas processed peach producers viewed fruit size, fruit firmness, and absence of split pits as being the most important traits for a successful peach cultivar. These results have potential to ensure that peach breeding programs are consonant with fresh and processed peach producers’ needs for fruit and tree traits.
Allan F. Brown, Gad G. Yousef, Ivette Guzman, Kranthi K. Chebrolu, Dennis J. Werner, Mike Parker, Ksenija Gasic and Penelope Perkins-Veazie
The objective of this study was to examine the relative impact of genetics and environment on phenolic and carotenoid profiles in peach (Prunus persica) germplasm. Fully mature, (“ready-to-eat” stage) firm fruit of peach cultivars China Pearl, Contender, and Carolina Gold were collected from established trees at two North Carolina locations in 2009 and 2010. Advanced breeding selections NC Yellow and NC 97-48 were collected from a single location in both years. Using tandem extractions and chromatography analyses, 10 carotenoids and 24 phenolic compounds were quantified separately in the peel and flesh. Statistically significant differences were noted among peach cultivars and advanced selections for β-carotene, cyanidin-3-glucoside, cyanidin-3-rutinoside, cholorogenic acid, quercetin-3-glucoside, and individual procyanidins. Peel anthocyanin (ANC) concentration ranged from 183 mg/100 g in ‘Contender’ to non-detectable levels in NC97-48 and NC Yellow. ‘China Pearl’ and ‘Carolina Gold’ produced ANC levels approximately half of ‘Contender’. Chlorogenic acid concentration also fit a discrete pattern of accumulation but was not related to the accumulation of ANC. ‘China Pearl’, NC 97-48, and NC Yellow contained the highest levels of chlorogenic acid (105 to 136 mg/100 g), ‘Carolina Gold’ contained the lowest (52 mg/100 g), and ‘Contender’ represented an intermediate phenotype (70 mg/100 g). Statistically significant genetic variation was found for almost all compounds identified, whereas location and year effects tended to be compound-specific. For chlorogenic acid, 28% of the phenotypic variance was explained by location (year = nonsignificant), whereas 40% of the phenotypic variation of ANC was explained by differences in years (location = nonsignificant). Analyzing fruit from the same environment over 2 years or from two locations in the same year would not have adequately accounted for the variation associated with environment. The detailed phytochemical profile of peach reported here demonstrates the importance of multiyear, multilocation analysis in revealing accurate measures of phytochemical genetic variation and provides a comprehensive baseline analysis of phytochemicals in commonly grown peach cultivars that can be used to evaluate novel germplasm.