Embryogenic cultures of interior spruce derived from 12 full-sib families were subjected to cryopreservation, with a 97 % success rate for 357 genotypes. Analyses suggested that cryotolerance was not related to family ranking (height increment), embryogenic potential or culture dispersability in suspension, and long-term storage in or above liquid nitrogen did not affect regenerative potential. By contrast, differences in cryotolerance among cell lines appeared to be prevalent in certain families. Analysis with a DNA fingerprinting probe used for clonal identification demonstrated no evidence of somaclonal variation as a result of cryopreservation. The results of this work indicate the applicability of cryopreservation as a long-term storage strategy for spruce embryogenic cultures from a wide genetic background.
Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0-80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.
Strategies for the selection of transformed cells and the counterselection (clearing) of Agrobacterium in genetic engineering requires a balance between the selection pressures and the normal morphogenetic response of transformed tissue. In this paper, we report the effect of antibiotics used for plant transformation studies on the elongation and induction of buds from embryos and the induction of callus from seedling tissue of Piceaglauca. Carbenicillin, ampicillin (500 and 750 μg/mL), and cefotaxime (250 and 500 μg/mL) are used to clear the invitro environment of bacteria and have little effect on the elongation or bud formation from P. glauca embryos. Concentrations of hygromycin greater than 1 μg/mL and kanamycin greater than 10 μg/mL inhibited elongation, whereas concentrations of kanamycin greater than 5 μg/mL totally inhibited adventitious bud formation, callus formation, and caused seedling mortality. At concentrations as low as 1 μg/mL, kanamycin decreased adventitious bud formation by greater than 50%. These results suggest that effective selection of transformants expressing marker genes can be performed using relatively low concentrations of kanamycin or hygromycin.
The quality of shoots in cultures of the apple rootstock, M4, was used as a criterion for the selection of an optimum medium. The frequency of shoots in defined shoot clases was monitored for each of five media, which differed in the type and concentration of phytohormone. Media containing BA (1.15 mgl-~) and IBA (either 0.15 or 0.20mgl -t) produced the maximum number of shoots that were desirable for transplantation and acclimatization.Abbreviations used in the text BA = Benzyl adenine (benzyl amino purine) IBA = Indole butyric acid
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