No abstract

Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 g l −1 maltose, 30 mg l −1 hygromycin and 20 mg l −1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD 600 =0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.

Agrobacterium-mediated genetic transformation system was established in Dendrobium Formidible 'Ugusu' by inoculating PLBs with A. tumefaciens strain EHA101 (pIG121Hm) harboring hygromycin phosphotranferase (hpt) and neomycin phosphotranferase II (nptII) genes as selectable marker gene and β-glucuronidase (gus) gene as a reporter gene. For obtaining the optimum conditions for the transformation, several factors such as the stage of PLBs after subculture, bacterial concentrations, kind of inoculation medium, inoculation time and inoculation condition (with or without rotary shaking), were examined. After inoculation, PLBs were cocultivated for 3 days and then transferred for selection onto 2.5 g l −1 gellan gum-solidified ND medium containing 10 g l −1 maltose, 20 mg l −1 hygromycin and 20 mg l −1 meropenem. Hygromycin-resistant plantlets were regenerated from secondary PLBs after 4 months of selection. Transformation of these plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The highest transformation efficiency of 18.5% was obtained when PLBs 3 weeks after subculture were inoculated with 1 : 10 diluted bacteria (OD 600 ≈0.1) with liquid medium containing only 10 g l −1 maltose and 100 µM acetosyringone with shaking for 30 min.

This experiment was conducted to assess the effect of indigo waste on the feed intake, digestibility, rumen fermentation, hematology, immune response and growth performance in growing beef cattle. Twenty crossbred beef cattle with an initial body weight (BW) of 145 ± 11 kg were fed four levels of indigo waste for 90 days in a trial. Additions of indigo waste at 0%, 10%, 20% and 30% in a concentrate diet using a completely randomized design (CRD). Cattle were fed concentrate at 1.8% BW, with rice straw fed ad libitum. The concentrate intake decreased linearly (p = 0.01) with the addition of indigo waste. The supplementation with indigo waste reduced dry matter (DM) and organic matter (OM) digestibility cubically (p = 0.03 and p = 0.02, respectively), while increasing neutral detergent fiber (NDF) digestibility cubically (p = 0.02). The final BW of beef cattle decreased linearly (p = 0.03) with the addition of indigo waste. The inclusion of indigo waste decreased the average daily gain (ADG) and gain-to-feed ratio (G:F) linearly (p < 0.01) from 0 to 90 days. The nutrient digestibility, ADG and G:F of beef cattle fed 10% indigo waste in the diet was similar when compared with the control (0% indigo waste). The ruminal pH, ammonia-nitrogen (NH3-N) and total volatile fatty acid (VFA) concentrations were similar among treatments (p > 0.05). The proportion of acetate increased linearly (p < 0.01) but propionate decreased linearly (p < 0.01), resulting in an increase in the acetate to propionate ratio (p < 0.01) when cattle were fed with indigo waste supplementation. Increasing indigo waste levels did not influence blood urea nitrogen (BUN) levels, hematological parameters or immune responses (IgA, IgM and IgG) (p > 0.05). In conclusion, the inclusion of indigo waste at 10% in a concentrate diet did not have a negative effect on feed intake, nutrient digestibility, rumen fermentation, hematology, immune function or growth performance in growing beef cattle.