Istituto Sperimentale per la Cerealicoltura
Our approach to transformation is essentially based on the use, as acceptor system, of highly embryogenic callus tissue of type 1 (compact and nodular), obtained from a particular F2 segregating population, which we refer to as WA1. This callus type is also characteristically produced in other monocots such as Pennisetum, Panicum and Sorghum, and its peculiarity relies on the continuous proliferation through embryogenesis at the upper surface. When 2,4-D is removed from the culture medium and the embryogenic tissue fragmented into 1 mm2 pieces, in the first 10 days it develops further into embryogenic structures. Subsequently, these structures can be divided again in single germinating somatic embryos and regenerated into complete plantlets. Although the efficiency of this last step varies greatly depending on various factors of "maturation" of the tissues during transfer, it is possible to raise the efficiency of regeneration up to 30-60% of the isolated somatic embryos by selecting an appropriate timing of the regeneration step. The embryogenic structures in an early phase of development can be desiccated until 20-25% of their initial fresh weight and, when rehydrated and cultured again, they can be induced to proliferate and regenerate. WA1 plantlets are recovered with the same efficiency as reported for fresh non-desiccated tissue.
By using the materials previously described we have developed a possible method of transformation, herein referred to as DUT (desiccation-uptake technique of transformation). Although various plasmids carrying different reporter genes were used, most of the work was done with the plasmid pCGN778, kindly provided by Calgene, Davis, USA, containing a NPT-II chimaeric (neomycin phosphotransferase-II) reporter gene. The expression of the NPT-II gene was tested in callus and plant tissues either after SDS-PAGE electrophoresis of the extracts assayed according to Reiss (Reiss et al., Gene 30:211-218, 1984), or through the protein dot-blot assay performed according to McDonnell (McDonnell et al., Plant Mol. Biol. Rep. 5:380-386, 1987). In the case of dot-blot assays, we included as an additional step an incubation in 0.1% Proteinase-K, 1% SDS, at 65 C for 45 min for a better cleaning of the false positive signals. DUT was performed in a series of seventeen independent experiments each one consisting of 150-200 mg initial fresh weight of tissues to be treated, with 30-50 ug plasmid DNA in 50 mM NaCl, 50 mM Tris HCl pH 7.8. DUT was performed by a desiccation (about 4 hrs depending on the amount of tissue at 27 ± 2 C, constant flow rate 0.45 m . sec-1) followed by uptake (2 hrs at 27 ± 2 C). Rehydrated calli were incubated for complete recovery 48 hrs in non-selective medium and subsequently transferred to selective and regenerative conditions. At this level, concentrations as low as 20 ug/ml of kanamycin were effective. In about 15 days complete regeneration of plantlets was obtained; the leaves of these were white (NPT-II negative), green (NPT-II positive) and striped, thus showing a chimaeric composition of the tissues (NPT-II positive or negative respectively in green and white sectors till the 6th leaf stage). Callus tissue after DUT, if propagated on 2,4-D medium in the presence of kanamycin, was NPT-II positive for at least one month. Resistance, detected as NPT-II activity as well as capacity of healthy growth on kanamycin, was then lost, thus revealing the transient nature of the transformation at the callus level. Some of the plants putatively transformed (Rt0) have been grown to maturity, selfed or crossed. Rt1 and Rt2 progenies were screened for NPT-II activity. Green tissue of the Rt0 plants was NPT-II positive. Four Rt0 plants were considered and among these plants two (RK4 and RK7) were green and two (RK5A and RK5B) were variegated at various levels. The first two plants were barren with vital pollen which was used to pollinate B79 ears. RK5A was a plant not bearing a tassel and was pollinated with normal A69Y pollen, while the RK5B plant was a fertile complete plant and was selfed. Table 1 reviews the situation observed. Various seed sets were obtained from the crosses. For each Rt0 some Rt1 seeds were considered and seeded for the screening of Rt1 plants. The strongest signal was given by the two plants from RK5A. Eighteen out of 28 plants of RK5B were positive, and none from the other three progenies screened. When Rt2 progenies of the NPT-II positive Rt1 plants were tested in vitro for germination on kanamycin (50 and 100 ug/ml) on MS medium as isolated mature embryos, they showed different degrees of resistance at the seedling level which, however, ended in a complete discoloration of the seedlings in vitro of the next leaves when potted into soil. These results are indicative of the transient expression of the gene which might have been inserted and expressed in a first period and then excised and lost, or even replicated as plasmid DNA molecules independently from the genomic DNA and subsequently lost. Although several parameters have still to be optimized and molecular analysis on the genomic DNA of the plants performed, these results might be indicative of the possible use of this approach for maize transformation.
Table 1. Putatively transformed
Rt0 plants and their progenies. Situation. (Table unavailable
- see hard copy.)
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