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- Author or Editor: Machteld C. Mok x
Cytokinins are widely used in tissue culture and transformation systems; however, little is known of their mode of action or the mechanisms regulating their levels in plant tissues. We are studying enzymes responsible for the metabolism of zeatin in immature seeds of Phaseolus. Selective expression of genes encoding such enzymes may regulate the level of active cytokinins during seed development as well as in in vitro systems. A zeatin O-xylosyltransferase, which mediates the formation of O-xylosylzeatin from trans-zeatin and UDP-xylose, has been isolated and monoclonal antibodies specific to the enzyme have been produced. Tissue print analyses demonstrated that the enzyme is primarily localized in the endosperm. ln situ localization and EM studies indicated that the enzyme is present in the cytoplasm and the nucleus. cDNA libraries were constructed from immature seed mRNA and immunopositive clones were selected. The products of these clones are being analyzed in E. coli and baculovirus expression systems.
Interspecific hybrid embryos resulting from crosses between Phaseolus species generally fail to reach maturity, and embryo rescue techniques are required to recover plants. To determine if ovary (pod) culture could permit interspecific hybrid embryo development, pods of P. vulgaris L. × P. acutifolius A. Gray and P. vulgaris L. (control) were cultured upright, supported by glasswool, in modified Murashige-Skoog liquid medium. The weight of seeds and length of embryos in P. vulgaris pods increased significantly during culture. However, usually only one or occasionally two seeds located at the middle positions of the pods developed to maturity. The same pod culture procedure promoted precocious germination of early cotyledonary-stage P. vulgaris × P. acutifolius embryos without further maturation of the embryos. These findings confirm that developmental arrest of the interspecific hybrid embryos in vivo is due to intrinsic deficiencies.
Quince (Cydonia oblonga) is widely used as a dwarfing rootstock for pear (Pyrus communis). We have devised procedures for Agrobacterium-mediated gene transfer using leaf discs of quince. The following factors were particularly important for recovery of shoots after incubation of leaf discs with Agrobacterium: an efficient regeneration system, the use of an effective antibiotic to eliminate Agrobacterium while maintaining a high regeneration frequency, and the choice of selectable markers. The regeneration frequency of control leaf discs on a medium containing 30 μM thidiazuron and 0.3 μM naphthaleneacetic acid was 100%. The frequency decreased linearly with increasing concentrations of antibiotics. Timentin. which consists of ticarcillin and a 6-lactamase inhibitor, was more effective in eliminating Agrobacterium than cefotaxime and carbenicillin. Vectors with the bar gene (bialaphos resistance) were better than those with the npt 11 gene (kanamycin resistance), since kanamycin bleached the leaf discs, resulting in poor regeneration. Bialaphos-resistant quince shoots which were positive in Southern hybridization have been obtained. The same procedures are being applied for transformation of pear.
Two approaches are used to improve iron utilization of fruit trees under iron-limiting conditions: 1) selection of somaclonal variants; and 2) cloning and incorporation of genes encoding Fe(III) reductases. Two somaclonal variants of quince with tolerance to low iron availability have been selected from 2000+ shoots regenerated from leaf discs. In greenhouse tests, under iron stress conditions, the variant clones continued to show improved iron utilization, having significantly higher chlorophyll concentrations in the new leaves when compared to the quince control. Cloning of Fe(III) reductase genes is based on mutant rescue utilizing a yeast mutant deficient in Fe(III) reduction (Dancis et al., PNAS 89:3869, 1992). A shuttle vector is used to incorporate a tomato root cDNA library into the yeast mutant. Complementing cDNAs, restoring growth to wild-type levels, are selected for further analyses. Cloning of genes involved in iron utilization will eventually lead to transgenic plants with improved iron efficiency.
Abstract
Carotenoid synthesis was studied in tissue cultures derived from different carrot phenotypes of red, dark orange, orange, light orange, yellow and white root colors. Cultures derived from a single genetic source or even from a single root often differed in color. Yellow, pink, orange-red and red cultures were derived from red roots; yellow and orange cultures from dark orange and light orange roots; and yellow cultures from the roots of the other genetic sources. The total carotenoid content in tissue cultures partly reflected that of the parent root, but was generally lower than in the intact root. Cultures derived from white roots were exceptions to this; the carotenoid contents in these cultures sometimes exceeded the contents of the parent roots. Carotenes predominated in cultures and roots of the red and orange sources; xanthophylls were the main components in cultures and roots of the yellow and white sources. Individual cultures from one root differed in quantities of carotenes, but the main carotene was the same as in the root, lycopene for the red sources and B-carotene for the orange sources.
The effects of kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D) and gibberellic acid3 (GA3) on growth and pigmentation were tested in 13 selected cultures. Kinetin inhibited and 2,4-D enhanced carotenoid synthesis in several of the cultures. GA had no effect on pigment formation. Kinetin and 2,4-D affected the relative quantities of carotenoid components in one orange-red culture, R 1-1, but little in the orange-red culture R 1-2. Low total carotenoid content was associated with a relatively high xanthophyll content and a low lycopene to B-carotene ratio.
Variation in culture pigmentation due to mutations was not confirmed. Plants regenerated from tissue cultures which had been derived from a single root had the same phenotype even though the colors and carotenoid contents of the callus cultures differed widely. If plantlet regeneration is limited only to cells with a diploid chromosome number, aneuploidy, which is very common in callus cultures, may have contributed to the observed pigmentation differences in the cultures.
Abstract
Plant growth regulator studies and plant tissue culture research have been closely related and mutually supportive. The manipulation of plant cells, tissues, and organs in culture, with important applications in propagation and genetic modification of plants, is highly dependent on the use of appropriate growth regulator regimes. Conversely, tissue culture systems are useful as bioassays to define the growth-regulating activity of many compounds. The discovery of the cytokinin N-(2-furanylmethyl)-1H-purin-6-amine (kinetin) by Miller et al. (17) was particularly relevant in this respect. Whereas the testing of this cytokinin and its structural analogues for biological activity was dependent on callus culture bioassays, the subsequent availability of synthetic cytokinins created many new opportunities in the field of plant tissue culture.
Interspecific hybridization between Phaseolus vulgaris and P. coccineus results in mature seeds or abnormal embryo formation depending on the direction of the cross. In addition, differential fertility and reversion to parental types occur in later progeny populations, accompanied by recurrence of various embryo types. Normal and abnormal embryos exhibited isozyme patterns resembling P. vulgaris and P. coccineus parents respectively, suggesting that developmental abnormalities may be associated with specific combinations of parental genes. RFLP between parental species was examined and probes were selected for analyses of F2 populations. Differential transmission of alleles occurred for some RFLP markers. Statistical analyses were applied to detect possible association between probes and abnormal developmental events. The high incidence of interspecific polymorphism will also facilitate the construction of a linkage map in Phaseolus.
Interspecific hybridization between Phaseolus vulgaris and P. coccineus results in mature seeds or abnormal embryo formation depending on the direction of the cross. In addition, differential fertility and reversion to parental types occur in later progeny populations, accompanied by recurrence of various embryo types. Normal and abnormal embryos exhibited isozyme patterns resembling P. vulgaris and P. coccineus parents respectively, suggesting that developmental abnormalities may be associated with specific combinations of parental genes. RFLP between parental species was examined and probes were selected for analyses of F2 populations. Differential transmission of alleles occurred for some RFLP markers. Statistical analyses were applied to detect possible association between probes and abnormal developmental events. The high incidence of interspecific polymorphism will also facilitate the construction of a linkage map in Phaseolus.
Somaclonal quince (Cydonia oblonga Mill.) variants IE-1 and IE-2 (Dolcet-Sanjuan et al., 1992) were more tolerant to Fe-deficient conditions in vitro than the original clone Quince A. The tolerance was evidenced by higher chlorophyll concentrations in leaves, higher Fe(III) reduction in roots, and increased acidification of the medium. In Summers 1993 and 1994, the two variants and Quince A were compared in the greenhouse, grown in normal potting soil (pH 5) and in soil obtained from an Fe-deficient orchard, with and without the addition of lime and KHCO3 solution to increase the pH (up to pH 8.3) and intensify Fe stress. In both years, the variants had significantly higher leaf chlorophyll concentrations than Quince A when grown in high-pH soils. The Fe+2 concentrations in leaves were also higher in the variants. Under field conditions at IRTA Reus, Spain, IE-1 and IE-2 had higher chlorophyll concentrations than the controls at some, but not all, analysis dates. These results suggest that IE-1 and IE-2, when grown as young plants in the greenhouse, have higher tolerance to Fe deficiency than Quince A, as observed in vitro, but that the tolerance is more variable under uncontrolled conditions in the field.
Development of optimum protocols for micropropagation of C. avellana is particularly important due to the threat of Eastern Filbert Blight and the need for rapid increase of resistant varieties and advanced selections. Therefore, efforts were directed at in vitro establishment, multiplication and rooting, starting with buds from mature trees. The frequency of shoot formation from buds was highest in August but varied with the genotype. Medium containing high Ca levels was more effective in preventing bud necrosis than MS medium. Multiplication rates of 4-7 new shoots/propagule were obtained over a 6-week culture period. Rooting of some genotypes could be accomplished by inclusion of 1 or 3 μM β- indolebutyric acid (IBA) in the medium. Other genotypes responded better to a dip of shoot bases in 1-10 mM IBA for 10 sec., followed by a passage on auxin-free medium. Large numbers of healthy plantlets have been produced for transfer to soil.