Development of roots on M.26 apple shoots grown in vitro induced by A. rhizogenes was compared with that of roots induced by NAA. Shoots were inoculated with 4-day colonies of A. rhizogenes strain A4 and were sampled at 1, 2, 4, 8 weeks after inoculation. Roots formed on approximately 30% of inoculated shoots. Roots induced by A. rhizogenes typically were agravitropic and branching. The outer layer of cells on these roots, especially on older roots, often resembled callus and sloughed off easily when the plants were transferred. The internal structure of the roots did not differ between the two treatments. Roots induced by NAA always arose endogenously and clear connections to the vascular system of the shoots were apparent. Many roots induced by A. rhizogenes appeared to develop exogenously, arising from anomalous cellular proliferation in the cortex of the apple stems or in callus at the base of the stem. These roots also showed vascular connections to the shoot.
Juvenal Luza and Ellen G. Sutter
Ellen G. Sutter and Hamid Ahmadi
Somatic embryos of Juglans regia transformed with Agrobacterium rhizogenes Rol B gene and non-transgenic lines were proliferated on basal DKW medium. They were then transferred to media containing different concentrations of ABA, IBA and BA to increase the rate of proliferation and maturation. Transgenic embryos required 50 μM ABA and 40 μM IBA whereas non-transgenic embryos required 40μM ABA and 10 μM IBA. Neither kind of embryos required BA. Roots were. induced by drying embryos at 75% for 2-3 weeks until they lost about 30% fresh weight and then transferring them to basal DKW medium for an additional 2 weeks in the dark. Over 50% of the somatic embryos grown on medium containing both ABA and IBA developed well defined root systems compared to less than 15% of embryos grown on basal medium. A combination of 27 μM GA, and 9 μM BA was needed for development of shoot systems and germination of both transgenic and non-transgenic rooted embryos. Anatomical studies followed to characterize the extent of development at each stage.
Bihua Huang and Ellen G. Sutter
Development and maturation of somatic embryos is known to be abnormal in many species, particularly woody species. Precocious germination, abnormal cotyledon formation, and shoot development are three problems, among others, that occur during the growth and germination of walnut somatic embryos. Depending on the cultivar or line being cultured, as much as 50% of the embryos in any given culture may be abnormal. Reports in the literature have shown that ABA is useful in enhancing maturation and producing normal germination of somatic embryos of a variety of plant species. In order to overcome the difficulties of producing plants from somatic embryos in walnut, we have incorporated ABA in the nutrient medium in different concentrations and for different periods of time. Globular and cotyledonary embryos were separated and placed on DKW medium containing four different concentrations of ABA, 30, 60, 80, and 120 μM. Morphology, fresh weight, and germination of embryos grown on these different media were recorded. Embryos grown on ABA had lower fresh weight increases than controls, the actual growth depending on both the concentration of ABA present and the length of time the embryos were grown on ABA-containing media. In addition, the percentage of embryos with normal morphology was considerably higher when embryos were grown on ABA. Other factors that were affected by the presence of ABA included the total number of embryos produced and the amount of senescence in the cultures. Germination of embryos was also improved as a result of their being cultured on ABA-containing media.
Scott J. Nissen and Ellen G. Sutter
The relative stabilities of IAA and IBA under various tissue culture procedures were determined. IBA was significantly more stable than IAA to autoclaving. IBA was also found to be more stable than IAA in liquid Murashige and Skoog medium (MS) under growth chamber conditions. The stabilities of IBA and IAA were similar in agar-solidified MS. Light provided by cool-white fluorescent bulbs promoted degradation of IAA and IBA in both liquid and agar media. Activated charcoal in concentrations as high as 5% was found to adsorb more than 97% of IAA and IBA in liquid MS. These results have important implications for the preparation, storage, and handling of IBA and IAA in plant tissue culture. Chemical names used: indole-3-acetic acid (IAA); indole-3-butyric acid (IBA).
James R. McKenna and Ellen G. Sutter
The use of auxin-impregnated toothpicks stimulated adventitious root formation in genotypes of Juglans `Paradox' that had been backcrossed to J. regia. These genotypes were selected as potential rootstocks because of improved tolerance to cherry leaf roll virus and Phytophthora spp. Other auxin applications including quick dips and talc formulations had little or no effect. The use of toothpicks lowered the concentration of IBA necessary for root initiation compared to previously reported results using quick dips. Toothpicks were inserted transversely into holes drilled 1 to 2 cm above the base of cuttings. Callus and roots always formed at the location of the toothpicks rather than at the base of the cutting. Roots were formed using this method in simple layering, hardwood, and semi-hardwood cuttings. Of all the cuttings that rooted, 90% rooted with toothpicks whereas only 10% rooted using a quick dip. This method may have potential for increasing the efficiency of rooting other difficult-to-root plants.
I-lun Lu, David W. Burger, and Ellen G. Sutter
Long term exposure to high benzyladenine (BA) concentrations inhibited shoot elongation and root growth of Cymbidium cv. Yuh Hwa rhizome explants. Through transfer experiments, it was determined that the commitment to shoot induction occurred between 10 and 14 days of exposure to at least 2.5 μM BA. BA when supplied at 20 μM during the fit 14 days of culture was found to be sufficient to induce shoot formation. Both shoot elongation and root formation were greatly improved by decreasing the BA concentration. By using radiolabelled BA, adenine was found to be a major metabolite in the rhizome tissue. Free IAA levels, quantitated by GC-MS, did not correlate well with the organogenesis of rhizomes, nor did the levels of free IAA correlate well with the activities of peroxidase and IAA oxidase, indicating a complex relationship between hormone concentration and differentiation.
Juan C. Díaz-Pérez, Kenneth A. Shackel, and Ellen G. Sutter
Little is known about the physiological changes that occur during acclimatization and how these changes influence plant survival and growth in the new environment. In particular, it is unclear to what extent in vitro-formed roots are functional in water uptake, particularly when the plantlet is exposed to conditions of increasing evaporative demand. Tissue-cultured shoots and plantlets (shoots with roots) were acclimatized by exposing them to a linear reduction in relative humidity (RH) from 99 % to 75%over 4 days. When conductance was measured at 95% RH (21 C), in vitro shoots and plantlets showed a very high initial conductance, followed by a gradual decline, reaching steady state in 12 hours. Acclimatized shoots and plantlets had a 50% lower initial conductance compared to nonacclimatized ones, and reached steady state in 4 hours. The reduction in conductance as a result of acclimatization most likely contributes to a reduced transpiration under conditions of increased evaporative demand. Roots formed in vitro were associated with a higher plant water status, suggesting that these roots were functional in water uptake. Relative water content of the shoot was positively correlated with leaf conductance and net photosynthesis. We suggest that tissue-cultured plantlets behave as hydraulically integrated units, in which there must be a coordination between control of water loss by the shoot and uptake of water by the root to maintain a favorable plant water balance. Our results also indicate that methods that use excised shoots or leaves to determine transpiration gravimetrically may not accurately represent the stomatal water loss characteristics of tissue-cultured plants.
Mark A. Ritenour, Ellen G. Sutter, David M. Williams, and Mikal E. Saltveit
This study was undertaken to determine if endogenous IAA content and axillary bud development correlate with phenylalanine ammonia-lyase (PAL) induction and russet spotting (RS) susceptibility among RS susceptible and resistant cultivars of Iceberg lettuce (Lactuca sativa L.). Final levels of ethylene-induced PAL activity and RS development were highly correlated among cultivars, field conditions, and harvest dates. Harvested Iceberg lettuce midribs contained relatively low amounts of free IAA (maximum of 5.2 ng·g-1 fresh weight). There was poor correlation between free IAA content in lettuce leaf midribs and final RS development among all cultivars, growing conditions, and harvest dates. Axillary bud development, as measured by the number of visible buds per head, bud weight, or bud length, were not significantly correlated with final RS development or midrib IAA content. Cultivars with higher initial free IAA content lost much of their IAA after 8 days storage at 5C in air ± ethylene.