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- Author or Editor: Ellen Sutter x
The relative contributions of stomatal and cuticular water loss to desiccation of tissue-cultured apple (Malus domestica Borkh), cherry (Prunus avium × P. pseudocerasus), and sweetgum (Liquidambar styraciflua L.) leaves after removal from culture were studied. Conductance from both abaxial and adaxial leaf surfaces was measured on plants immediately after removal from culture, after acclimatization, and after being subjected to water stress. In all species, stomatal conductance decreased significantly after acclimatization. Cuticular conductance was significant under some conditions and may have been due to damage resulting from tissue dehydration. The three species had quite different responses to imposed water stress. Responses appeared to be related to differences in stomatal functioning, cuticular integrity, and degree of acclimatization.
Lateral buds excised from unrooted cuttings of Maranta leuconeura E. Moor. ‘Kerchoviana’, prayer plant, were cultured on Linsmaier and Skoog (LS) medium supplemented with various combinations of N6-benzylaminopurine (BA), kinetin, α-anaphthaleneacetic acid (NAA), and 3-indoleacetic acid (IAA) for the production of multiple shoots and complete plants. Of 48 combinations of growth regulators, 4 produced the most vigorous, well-formed shoots after a culture period of 6 weeks: 2.0 mg·liter-1 kinetin, 0.2 mg·liter-1 BA, 0.2 mg·liter-1 BA plus 0.1 mg·liter-1 NAA, and 2.0 mg·liter-1 kinetin plus 1.0 mg·liter-1 IAA. Enhanced shoot production occurred when shoots were transferred at 12-week intervals and when they were maintained on 0.2 mg liter-1 BA. Complete plants with strong, fibrous root systems were produced after the shoots were transferred to basal medium lacking growth regulators for 3-4 weeks.
Research on morphogenesis in vitro began in the early 1900s when Haberlandt theorized that entire plants could be produced from single living cells (2). Early studies on tobacco callus culture by Miller and Skoog (3, 6) indicated that auxins and cytokinins could be used to manipulate morphogenesis in vitro. In subsequent studies, critical factors were determined to be the types, concentrations, and ratios of various plant growth regulators used. However, scientists soon realized that a great many plants would not undergo morphogenesis under conditions being used at the time. Pioneering work by Steward et al. (7) and Reinert (4) on induction of embryogenesis in carrot suspension cultures introduced more questions than answers concerning the role of plant growth regulators and other controlling factors in morphogenesis.
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.
‘Manzanillo’ olive (Olea europaea L.) seeds were subjected to chemical scarification with NaOH and H2SO4 for various periods of time to determine the most appropriate treatment for improving the germination of the seeds. A critical balance of concentration and time was necessary to achieve high germination percentages without loss of viability of the seeds. H2SO4 was more effective than NaOH in increasing germination percentages. Germination percentages as high as 98% were obtained on stratified seed using H2SO4, compared to 0% without chemical scarification.
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.
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.
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).
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.
Epicuticular wax fine structure on leaves from carnation (Dianthus caryophyllus L.) grown in the greenhouse or regenerated from shoot tip culture was compared by scanning electron microscopy. Wax rods densely covered the glaucous leaf surface of greenhouse-grown plants. Irregularly-shaped wax plates were observed on leaves of glaucous plantlets, but no structured wax was seen on non-glaucous plantlets, which comprised the majority of plantlets. During 2½ weeks in the greenhouse, increasing amounts of structured epicuticular wax developed on leaves of glaucous plantlets but not of non-glaucous plantlets. The low survival rate of carnation plantlets regenerated in vitro may be explained by their lack of epicuticular wax structure which results in excess desiccation when they are transferred from in vitro conditions to the greenhouse.