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Peach [Prunus persica (L.) Batsch] plants #94-1, #99-1, and #40-1, carrying a cytokinin biosynthesis (ipt) gene following transformation with the shooty mutant strain of Agrobacterium tumefaciens, were evaluated for altered growth habit and axillary shoot formation, both in vitro and in the greenhouse. After 9 weeks of in vitro propagation on four different levels of 6-benzyladenine (BA), only transformant #99-1 exhibited significantly greater axillary shoot formation (on 10 μm BA), and significantly greater fresh mass (on 3,10, and 30 μm BA) than the control #RG-3. Tolerance to a supra-optimal (30 μm) concentration of BA was indicated by fresh mass increases for #99-1 shoot cultures. Delayed senescence on 0 μm BA was exhibited by 87% of the transformants, but by only 12% of the control plants. Greenhouse-grown #99-1 and #40-1 were significantly shorter than #RG-3 plants at 6 weeks and at 1 year, but only #40-1 exhibited significantly greater branching than the controls. Chemical names used: 6-benzyladenine (BA); isopentenyl transferase (ipt).
Transgenic plants containing introduced phytohorm one genes have been shown to display altered growth and morphogenetic potential. Peach plants transformed with the ipt gene from Agrobacterium tumefaciens strain tms 328::T n5 and containing elevated levels of cytokinins were screened in vitro for compact growth habit on four different levels of 6-benzyladenine (BA). After nine weeks in vitro, the average number of axillary shoots per plant foe two of the transformants, 99-1 and 40-1, ranged from 1.5 to 6.6 times that for the controls on 0-30 uM of BA, whereas average fresh weight ranged from 1.1 to 3.6 times that for the controls. One of the transformants, 94-1, produced a greater number of axillary shoots only on 30 μM BA. Rooted plants derived through micropropagation from the original transformants were monitored for 30 months under greenhouse conditions. The average height of transformants 94-1 and 99-1 after six months in the greenhouee was 88 and 77% of controls, respectively and after 30 months was 90 and 75% of controls, respectively. In comparison to controls, transformants exhibited a greater number of branches per meter per plant after six weeks, but a lesser number after 30 months. These results suggest that the introduction of a cytokinin gene may be a useful approach to obtaining peach trees with a compact growth habit.
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.
`McIntosh' apple shoots were inoculated in vitro with Agrobacterium tumefaciens strain tms328::Tn5 (tms) carrying a functional cytokinin gene. Callus tissue, removed from the infected stems, produced shoots on shoot proliferation medium. After three subcultures, axillary shoot production from a tms-infected putative transformant was eight times that of controls. Subsequent shoot production on three different levels of BA (3, 6 and 10 uM) was significantly greater than from controls on all levels of BA. PCR analysis of putative transformants revealed an expected 503 bp DNA fragment corresponding to the amplified portion of the cytokinin gene. After 6 months of in vitro propagation, proliferation rates of shoots obtained from the original transformants were similar to the controls and the expected PCR fragment of 503 bp could only be detected by Southern analysis. Even though the T-DNA appears to be lost from the apple genome, the data suggest that the tms strain may be useful in co-infection experiments to induce shoot formation, thus avoiding difficult regeneration procedures.
Abstract
Stem explants of in vitro-propagated peach (Prunus persica L. Batsch.) cultivars Compact Redhaven, Jerseyqueen, Rio Oso Gem, and Suncrest and peach rootstock Nemaguard were infected with several strains of Agrobacterium tumefaciens. Tumor tissue was produced on all cultivars infected with strain tms328::Tn5, which carries a mutated octopine Ti plasmid pTiA6. The tumor tissues were cytokinin-independent and all tissues, except those on ‘Rio Oso Gem’, produced octopine. A DNA probe specific for the T-DNA region of the Ti plasmid hybridized to the DNA extracted from these tumors. No tumor tissue was produced in response to inoculation of the stem explants with the Ti-plasmidless strain A136 or with uninoculated controls. These results demonstrate the transformation of peach cells derived from mature plants and the potential for using A. tumefaciens to transfer economically important genes to peach.
The ipt gene of Agrobacterium tumefaciens T-DNA encodes for isopentenyl transferase, which is an enzyme active in cytokinin biosynthesis. While it is known that cytokinins are associated with in vitro promotion of cell division and stimulation of shoot production, little is known about their mode of action. As the first step in localizing cytokinin synthesis, we present a cloning and expression strategy for the ipt gene. The source of the ipt gene was Agrobacterium tumefaciens octopine Ti plasmid 15955. The ipt gene was amplified by the polymerase chain reaction (PCR) and cloned into pMal-c2 (New England Biolabs, Beverly, MA). This construct was transformed into E. coli and the ipt gene was expressed as a fusion protein. The protein was purified by affinity chromatography to serve as an antigen for polyclonal antibody production. These antibodies will be used to localize isopentenyl transferase in plant tissue.