C.N.R. - Istituto Biosintesi Vegetali

Recovery of callus lines from transformed protoplasts of endosperm suspension cultures

--S. Faranda and L. A. Manzocchi

Studies on the regulation of endosperm-expressed genes in maize have been hindered by the difficulties in obtaining fertile transformed plants. This has led to a renewed interest in tissue culture from maize endosperms (Lyznik, LA and Tsai, CY, Plant Sci. 63: 105-114, 1989; and Quayle, TJA et al., Plant Cell Rep. 9:544-548, 1991); their ability in tissue-specific synthesis suggests that, while maintaining a population of meristematic cells, endosperm cultures retain characteristics of the differentiated tissue (Felker, FC and Goodwin, JC, Plant Physiol. 88:1235-1239, 1988).

We have obtained stable suspension cultures from endosperm cells of A69Y maize, expressing reserve proteins (zeins), although at reduced quantitative level with respect to developing endosperms (Manzocchi,, LA, Plant Cell Rep. 9:555-558, 1991); we report experiments of protoplast transformation and recovery of callus lines.

Protoplasts were obtained from cultures in log phase with good yields (4-6 x 106 protoplasts per gram of packed cells). They were transformed by PEG-mediated DNA delivery with chimaeric gene constructs kindly provided by Dr. A. Viotti, A and containing the reporter genes GUS (Jefferson, RA et al., EMBO J. 6: 3901-3907, 1987) or NPTII (Beck, E et al., Gene 19:327-336, 1982) driven by CaMV 35S RNA promoter (Pierce, DA et al., in "Plant Gene Systems and Their Biology", A. R Liss Inc., pp. 301-310, 1987).

Different plating media and techniques were tested to obtain callus regeneration from PEG-treated protoplasts. The best results were obtained by including protoplasts in 1.2% agarose in KM8P medium (Kao, KN and Michayluk, MR, Planta 126:105-110, 1975) at 0.5-1 x 106 cells/ml immediately after transformation.

4 days after, division of 20-40% of the cells was observed; visible microcalli were distinguished 20-30 days after. Microcalli of 0.5-2mm could be picked up manually two months after plating and transferred to agar MSE medium routinely used for the growth of endosperm cultures (Manzocchi, LA, Plant Cell Rep. 9:555-558, 1991). Plating efficiency was 0.03-0.05%.

Calli recovered from protoplasts transformed with the GUS gene were screened for GUS expression by histochemical stain two months after transformation. 13% of the calli stained blue with X-gluc reagent (Jefferson, RA et al., EMBO J. 6:3901-3907, 1987), while no GUS expression was detected in untransformed control calli. GUS-expressing calli were successively subcultured and tested histochemically every 20 days; stable transformed phenotypes were observed in 60% of the calli 5 months after transformation.

Difficulties were found in the selection of calli from protoplasts transformed with the NPTII gene, because, as already described for cereals (Vasil, V et al., Biotechnology 9:743-747, 1991) endosperm cells are quite resistant to growth inhibition by kanamycin. Microcalli of 1-2mm diameter were subcultured on agar media containing 100 - 200 - 400mg/l kanamycin for 5 months, with an average survival of 25%. NPTII activity (McDonnell, RE et al., Plant Mol. Biol. Rep. 5:380-386, 1987) was detected in 42% of the kanamycin resistant calli derived from transformed protoplasts, while no activity was found in control cultures.

Experiments are in progress to define a more effective selection procedure for the isolation of NPTII expressing cultures; Southern blot hybridization experiments are being carried out to test for NPTII and GUS coding sequences in our putative transformed callus lines.

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