RESUMO
The abundance and scattered distribution of simple-sequence repeats (SSR) in eukaryotic genomes prompted us to explore the use of SSR-based oligonucleotide primers in single primer amplification reactions. In a pilot experiment, 23 primers were used across a panel of evolutionarily diverse eukaryotic genomes, including grapes, lettuce, tomato, pine, maize, salmon, chicken, Holstein cows and humans. The primers were 16-20 bases in length and represented SSRs of di-, tri-, tetra-, and pentanucleotide repeats. The results showed that tetranucleotide repeat primers were most effective in amplifying polymorphic patterns. Of 11 such primers tested, 70% produced polymorphic patterns from the DNA of one or more species. Primers representing a combination of two tetranucleotide repeats, or compound microsatellites, were equally effective. The polymorphisms contained in such fingerprints were able to identify individuals of vertebrate species as well as lines or varieties of plants. Inheritance of the polymorphic bands was studied in a maize recombinant inbred population, DE811 x B73. Thirty-two polymorphic bands, derived from two amplification patterns, were mapped as dominant markers on an existing RFLP map of the same population. The bands were distributed across nine of the ten chromosomes.
RESUMO
A successful Agrobacterium-mediated transformation system involving a disarmed Ti plasmid is composed of two stages: transformation of cells and recovery of transformed plants. A tissue transformation system with 34% efficiency was developed using stem segments of the interspecific tomato hybrid Lycopersicon esculentum × L. pennellii. This transformation system emphasizes three factors favoring the recovery of transformed plants: 1) promotion of cell division activity at the inoculation site with kinetin in the incubation medium, 2) promotion of adventitious bud initiation by using organized tissue explants in culture, and 3) application of selection at the shoot development stage of adventitious regeneration.
RESUMO
We have attempted to confirm previous reports of "egg transformation" in seven Nicotiana species, including genetic stocks obtained from the original experimenter. The methods employed in the original experiments were duplicated as closely as possible. In total, 1,622 pollinations were made involving irradiated pollen and mixtures of irradiated and self pollen. Consequently, 995 seedlings from 9,052 seeds were screened for 1,594 potentially detectable transformation events. A very low frequency of unexpected progeny resulted, but these results were not repeatable and appear to have arisen by mechanisms other than transformation. These results are strongly at odds with previous claims, when 50% of offspring were found to be transformed. We conclude that the previous observations of high frequency egg transformation are not reproducible. However, due to the ambiguous nature of the markers employed, it is very difficult to prove that such transformation events do not occur as very rare events.
RESUMO
Experiments were designed and carried out to investigate the possibility of inducing "egg transformation" in tomato, as described by Pandey in Nicotiana L. Pollinations were made, which included the following treatments: irradiated donor pollen, irradiated donor pollen mixed with normal self pollen, irradiated donor pollen followed by delayed self-pollination, and a simple pollen mixture of non-irradiated donor and self pollen. No transformants were found after screening 5,620 seedlings representing 22,300 potential transformation events. If egg transformation occurs, it would appear to be limited to species outside of Lycopersicon esculentum Mill.
RESUMO
Experiments were conducted to determine if "egg transformation" could be achieved in Zea mays L. as described by Pandey in Nicotiana L. Multiple recessive and multiple dominant marker stocks were employed, as well as a tester and a donor line for the "En" transposable element. Recipient tester females were pollinated with dominant donor pollen, which was applied in several treatment combinations. The pollination treatments included: 1) pollen irradiated at 20, 30, 40, 80, and 100 Krad; 2) pollen irradiated with the same doses, mixed with non-irradiated recipient pollen; 3) pollen irradiated at 80 Krad, followed by self pollination delayed 18 h; 4) non-irradiated donor pollen mixed with non-irradiated recipient pollen. Zero seed were produced from 100 pollinations with irradiated pollen. There were 258 pollinations made with irradiated donor plus self pollen mixtures, producing over 21,300 seed. Of these seed, 3 were unexpected. One was clearly from pollen contamination, one was clearly derived from a pre-meiotic mutation, and the third occurred as a mutant sector in the seed's endosperm. There were 56 pollinations with non-irradiated pollen mixtures, producing over 5,000 seed. Among these seed, there were 7 unexpected seed. Three of these were clear-cut cases of heterofertilization. Four progeny were dominant for all seed and seedling markers except one endosperm marker. These cases appear to represent spontaneous recessive endosperm mutations. More than 59,000 potential transformation events were screened producing only 6 apparent mutations. It is concluded that if egg transformation occurs in Zea mays, it is a very rare event, and is not likely to be useful in corn improvement.
RESUMO
Numerous pollination treatments involving heavily irradiated (40-100 krad) pollen of diverse plant species failed to produce any clear cut "egg transformants" of the type reported by Pandey in Nicotiana. Genetic stocks of pea, rapeseed, and apple, bearing multiple Mendelian markers, were employed to detect any possible transformation events. For each plant species, an optimal level of irradiation was determined which would allow normal pollen tube growth leading to fertilization, but which would prevent the formation of normal hybrids due to the "pulverized" condition of the chromosomes contributed by the irradiated pollen. Pollination treatments included selfing, pollination with donor pollen mixed with self pollen, pollination with irradiated donor pollen mixed with self pollen, pollination with irradiated pollen followed by a delayed self pollination, and pollination with irradiated pollen by itself. None of these treatments produced clearly transformed seedlings. The total number of potential transformation events screened was in excess of 6,046 including 2,268 for pea, 3,309 for rapeseed, and 469 for apple. It is concluded that if egg transformation occurs outside of Nicotiana it is a rare event, and its frequent occurrence in Nicotiana must be, at best, an isolated phenomenon.
