Immature `Redhaven' peach [Prunus persica (L.) Batsch] embryos were infected with a shooty mutant strain of Agrobacterium tumefaciens, tms328::Tn5, which carries an octopine-type Ti plasmid with a functional cytokinin gene and a mutated auxin gene. Shoots were regenerated from embryo-derived callus that was initiated on MS medium lacking phytohormones. Shoots exhibited increased frequency of branching and were more difficult to root than the noninfected. Transcripts of the tms328::Tn5-cytokinin gene were detected using northern analyses of total plant RNA. Polymerase chain reaction of genomic DNA and cDNA resulted in amplification of DNA fragments specific for the cytokinin gene, as determined by restriction enzyme and Southern analyses. The concentrations of the cytokinins zeatin and zeatin riboside in the leaves of regenerated plants were on the average 51-fold higher than in leaves taken from nontransformed plants. None of the shoots or callus tissues were postive for octopine. The expression of the T-DNA encoded cytokinin gene promotes growth of peach cells in the absence of phytohormones, thus serving as a marker for transformation. In addition, this gene appears to promote morphogenesis without an auxin inductive step.
Ann C. Smigocki and Freddi A. Hammerschlag
Ann C. Smigocki
Cytokinins were first recognized as a class of phytohormones for their ability to promote cytokinesis in cultured plant cells and have since been shown to be involved in a wide range of physiological processes. Most recently, the availability of phytohorm one-specifying genes from Agrobacterium tumefaciens has allowed for direct in planta manipulation of cytokinin levels. Overexpression of the isopentenyl transferase (ipt) gene by constitutive promoters led to enhanced ability of plant cells to undergo shoot organogenesis but the high endogenous cytokinin levels almost completely suppressed root development. Transient overproduction of cytokinins using promoters regulated by environmental and/or developmental factors did not inhibit regeneration of rooted plants. Transgenic plants in which cytokinin levels can be modulated are being used to characterize the participation of cytokinins in fundamental regulatory mechanisms of morphogenesis, delayed senescence, disease resistance and directed nutrient transport. The potential for using reconstructed cytokinin biosynthesis genes in economically important crops is of tremendous agronomic significance.
Cary A. Mitchel
Brief, periodic seismic (shaking) or thigmic (contact rubbing) stress treatments applied to plants growing in a wind-protected environment typically reduce but strengthen vegetative growth and often inhibit reproductive development. Cell division and cell enlargement both are affected. Mechanically dwarfed plants accumulate less leaf area than do undisturbed controls and undergo temporary stomatal aperture reduction following an episode of stress, leading to reduced photosynthetic productivity. Vibration or mild shaking may lead to a slight stimulation of plant growth. Most classes of phytohormones have been implicated to mediate different growth responses to mechanical stress. Physical perturbation turns on the transcription of several genes coding for calmodulin-like proteins. Calcium chelators and calmodulin inhibitors partially negate effects of thigmic stress. Growth rate responses of naive seedlings are immediate and dramatic, suggesting turgor collapse, whereas recoveries are slow and sometimes partial, suggesting reduced wall extensibility in the cell enlargement zone. Mechanical stress may be used for height control of intensively cultivated bench crops or to physically toughen bedding plants prior to outdoor transplant. Physiological hardening remains a question. Mechanical height control avoids use of chemicals but increases risk of wounding and pathogen infection.
Eva Bacaicoa, Ángel María Zamarreño, Diane Leménager, Roberto Baigorri, and José María García-Mina
Some studies suggest that iron (Fe) stress root responses are regulated by variations in specific plant hormones. However, this question remains unclear. A time-course experiment dealing with the relationship between the expression of the Fe-stress root responses at transcriptional (CsFRO1, CsIRT1, CsHA1, and CsHA2) and enzymatic levels [root Fe(III)-chelate reductase and plasma membrane H+-ATPase], and the variation of phytohormone concentrations in the shoot and root of Fe-starved plants have been studied in a Fe-efficient cucumber cultivar (Cucumis sativus L. cv. Ashley). The results indicate that the expression over time of the physiological Fe-stress root responses at transcriptional and enzyme activity levels are consistent with significant increases in indole-3-acetic acid root (transient) and shoot (sustained) concentrations. Fe-starvation also caused transient changes in the root concentration of abscisic acid and nitric oxide. Finally, an increase in root ethylene production and a decrease in the root concentration of some cytokinins were observed under Fe starvation, but they were not clearly timely coordinated with the expression of Fe physiological root responses.
Jinq-Tian Ling, N. Nito, and M. Iwamasa
Protoplasts were isolated from embryogenic calli of Citrus reticulata cv. Ponkan and Citropsis gabunensis, and fused in electric fields. The maximal fusion efficiency was obtained by application of AC at 75 V/cm (1.0 MHz) and DC square-wave pulse at 1.125 KV/cm for 40 usec. Fusion-treated protoplasts were cultured on MT medium without phytohormone, solidified with 0.6% agar. Colonies from the protoplasts were proliferated on MT medium with zeatin 1 mg/l and 0.9% agar. Selection of somatic hybrid callus was based on chromosome count and isoenzyme analysis. The somatic hybrids were tetraploid (2n=36). C. reticulata and C. gabunensis were both homozygous at Got-1 locus, but distinguishable easily because band of the latter migrated faster than that of the former. In zymogram of somatic hybrid, both parent bands were retained and a new hybrid band was also evident between them. Embryos from somatic-hybrid callus regenerated intact plant. The hybrid plants showed intermediate morphological characteristics of the parents.
William R. Woodson, Ky Young Park, Paul Larsen, and Hong Wang
The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased synthesis of the phytohormone ethylene. This ethylene serves to initiate and regulate the processes of programmed cell death. We are using molecular approaches to study the regulation of ethylene biosynthesis in various floral organs during development and senescence of flowers. We have isolated and cloned mRNAs which encode the ethylene biosynthetic pathway enzymes s-adenosylmethionine (SAM) synthetase, 1-aminocyclopropane-1-carboxylate (ACC) synthase and the ethylene forming enzyme (EFE) from carnation flower petals. These cDNAs have been used as molecular probes to determine the steady-state mRNA levels of these transcripts in senescing flowers. The increase in ethylene associated with petal senescence is accompanied by a dramatic increase in the abundance of transcripts for both ACC synthase and EFE. In striking contrast, the level of SAM synthetase mRNA decreases significantly with the onset of petal senescence. Genomic DNA Southern blots reveal both ACC synthase and EFE are encoded by multigene families. We have recently isolated several genomic clones from carnation which represent different ACC synthase genes. The structure and organization of these gene will be presented.
Richard L. Bell
Discs of cambial tissue were excised from actively growing shoots of `Bartlett' pear, and explanted directly on regeneration induction media. The basal medium was 1/2 strength MS macro-nutrients, MS micro-nutrients and organics, 8 g/l agar, and 30 g/l sucrose. Phytohormone treatments consisted of a factorial design of NAA (0 and 5μM) and TDZ (1, 2, 3, 4, and 5μM). After 4 weeks incubation in the dark, the explants were transferred to auxin-free media with identical concentrations of TDZ. There was an absolute requirement for auxin in the induction medium, as all discs on auxin-free initial media died without callusing. Maximum shoot regeneration 4 weeks after transfer to expression media was obtained with an initial medium containing 5μM NAA and 3μM TDZ, from which 30% of the explants produced one or more adventitious shoots. This rate of regeneration is similar to that obtained in some experiments with in vitro leaf explants, and provides an alternative system for regeneration of pear.
Plant growth-promoting rhizobacteria (PGPR) enhance plant development by many mechanisms. Indirect growth effects result from PGPR activities that displace soilborne pathogens and thereby reduce disease. Direct effects include improved nutrition, reduced disease due to activation of host defenses, and bacterial production of phytohormones. An understanding of the mode of action is essential for exploitation of PGPR for field use. For instance, bacteria that act as biological control agents can only be of benefit at locations where disease occurs. PGPR that stimulate plant growth directly will likely have more universal uses and greater impacts. Thus, we have been developing model systems for identifying PGPR with such traits. In this presentation, the effects of bacterization of tissue culture-grown plants, plug transplants, and seed with a growth-promoting Pseudomonas sp. (PsJN) will be described. Potential uses for this and other PGPR will also be identified. The talk will consider the advantages and limitations of: a) screening methods used for selection of PGPR, b) model systems available for studying the mechanisms of action, and c) why transplants offer an ideal delivery system for rhizobacteria. Results from field trials with PGPR with different modes of action will be presented and their future role in agriculture considered.
S. Guzman, H. Alejandro, J. Farias, A. Michel, and G. Lopez
Watermelon (Citrullus vulgaris Schrad.) is a widely grown crop throughout the tropics and subtropics. In Mexico, it is an economically important crop. In vitro adventitious shoot regeneration of watermelon has been reported from shoot tip culture, leaf, hypocotyl, and cotyledons. Hence, the objective of this study was to evaluate in vitro plant regeneration from axillary buds of triploid watermelon. Axillary buds explants were prepared from shoot of commercial cultivar in field of 60 old day plants. Explants of 2 to 3 mm were incubated 2 weeks on Murashige and Skoog (MS) shoot regeneration medium containing 2.5 mg/L kinetin (KT) or indole-3-butyric acid (IBA), or gibberellic acid (GA3), followed by 3 weeks on shoot elongation medium supplemented with different combinations of the same phytohormones. The percentage of explants (83% to 90%) that produced shoots, expansion in size of explant (0.81–1 cm) and shoot length (6 mm) were highest in MS medium containing KT or IBA. In the shoot elongation step, shoot length (0.9–1 cm) and leaves number (6–7) were highest in MS medium supplemented with 2.5 mg/L of KT or GA3 and 0.2 mg/L IBA, but the better induction of roots in elongated shoot occurred on MS medium with 2.5 mg/L KT and 0.2 mg/L IBA. The results show that axillary buds from watermelon is an alternative for the micropropagation of this crop.
James D. Spiers, Fred T. Davies, Scott A. Finlayson, Chuanjiu He, Kevin M. Heinz, and Terri W. Starman
This research focused on the effects of nitrogen fertilization on jasmonic acid accumulation and total phenolic concentrations in gerbera. The phytohormone jasmonic acid is known to regulate many plant responses, including inducible defenses against insect herbivory. Phenolics are constitutive secondary metabolites that have been shown to negatively affect insect feeding. Gerbera jamesonii `Festival Salmon Rose' plants were grown in a growth chamber and subjected to either low fertilization (only supplied with initial fertilizer charge present in professional growing media) or high fertilization (recommended rate = 200 mg·L-1 N). Plants were fertilized with 200 mL of a 15N–7P–14K fertilizer at 0 or 200 mg·L-1 N at each watering (as needed). Treatments consisted of ±mechanical wounding with a hemostat to one physiologically mature leaf and the subsequent harvest of that leaf at specified time intervals for jasmonic acid quantification. Total phenolics were measured in physiologically mature and young leaves harvested 0 and 10 hours after ±mechanical wounding. Low-fertility plants had reduced aboveground dry mass, were deficient in nitrogen and phosphorus, and had about a 10× higher concentration of total phenolics when compared to high fertility plants. In low-fertility plants, young leaves had greater concentrations of phenolics compared to physiologically mature leaves. There were no differences in total phenolics due to wounding. The effect of nitrogen fertilization on jasmonic acid accumulation will also be discussed.