As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high efficiency shoot regeneration from leaf explants of in vitro-propagated shoot cultures. The effect of either thidiazuron at 1 or 5 μM, or zeatin riboside at 20 μM, and two lit levels (18 ± 5 or 55 ± 5 μmol·m-2·s-1) on shoot organogenesis were investigated. With the exception of `Bluecrop', which did not regenerate shoots, maximum shoot regeneration of 13, 12.7, 12.6 and 4.6 shoots per explant for cultivars Duke, Georgiagem, Sierra, and Jersey, respectively, occured on regeneration medium with zeatin riboside and under a light intensity of 55 μmol·m-2·s-1. Whereas `Duke' regenerated equally well on regeneration medium with either zeatin riboside or 5 μM thidiazuron, regeneration frequencies for `Georgiagem' and `Sierra' were significantly higher on zeatin riboside. A light intensity of 55 μmol·m-2·s-1 significantly increased regeneration of cultivars Duke, Jersey, and Sierra on zeatin riboside, but inhibited regeneration of Duke on 5 μM thidazuron.
Xiaoling Cao and F.A. Hammerschlag
Xiaoling Cao, Freddi A. Hammerschlag and Larry Douglass
As part of a program to improve highbush blueberry (Vaccinium corymbosum L.) cultivars via tissue culture and genetic engineering, studies were conducted to determine optimum conditions for organogenesis from leaf explants of the previously recalcitrant cv. Bluecrop. The effects of a pretreatment, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% explants regenerated shoots with a mean of 11 shoots per leaf explant after 62 days when explants of 2-week-old shoot cultures were incubated on the following regime: pretreatment medium #1 containing 5 μm TDZ and 2.6 μm NAA for 4 days, pretreatment medium #2 containing 7 μm zeatin riboside and 2.6 μm NAA for 3 days, regeneration medium containing 1 μm TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatment prior to incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on regeneration medium containing 1 μm TDZ was about three times that on 0.5 μm TDZ or 20 μm zeatin riboside, and nine times that on 5 μm TDZ. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(β-D-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).