Evaluation of selectable markers for the identification of transformation events in regenerating cultures

--F. Locatelli, A. Rossini, M. C. Lusardi1 and E. Lupotto

1Inst. Pflanzenwiss., Zürich

Kanamycin resistance, encoded for by the APH(3') (or NPT II) gene, driven by the 35S CaMV or other promoters, is currently used as a selectable marker in many dicot systems for transformation. Kanamycin resistance has also been applied with success to Gramineous species, such as Zea mays, Triticum monococcum and Oryza sativa. However, tissue cultures of cereals, supported by 2,4-D containing medium, are generally hard to select by using kanamycin because of the relative tolerance of the callus at high levels of the aminoglycoside.

Although widely used, little is known about the effect of other antibiotics related to kanamycin:--neomycin, G418, paromomycin and hygromycin B--on the conditions needed for selection in embryogenic systems, such as embryogenic monocot cultures. This information assumes particular relevance because at least two techniques of transformation, namely microinjection and biolistic method, utilize embryogenic cultures as target tissues. The aim of this study was to screen the effect of these four antibiotics (kanamycin, geneticin, paromomycin and hygromycin B) on the development of embryogenic cultures of maize when selection was applied in regenerative conditions.

Embryogenic tissue cultures of various genotypes were initiated from immature embryos on N6 basic medium, 2% sucrose, 100 mg/l m-inositol; 100 mg/l casein hydrolysate, 2 mg/l 2,4-D (N6I medium), and propagated on the same medium with 1 mg/l 2,4-D (N6P). Regeneration medium was MS basic medium, 2% sucrose, 100 mg/l m-inositol, hormone-free (MShf). The following genotypes, which established type 2, long term, embryogenic cultures were considered: B79 inbred line, LH126 x B79, Lo907 x B79, Lo976 x B79 F2 selections. Regeneration was achieved through a three-step procedure, devised for enhancing the final yield of plantlets. Briefly, 50 mg fresh weight tissue pieces from a 15 day old subculture were transferred from N6P to MShf medium, 16 per 90 mm petri dish, incubated in the light with a 16/8 hours day/night regime. After 5 days, germinating somatic embryos, with enlarged opaque white scutellum arising at the surface, were distinctively detached and transferred into the same conditions. After an additional 7-10 days, regenerated plantlets were separated and secondary somatic embryos, originated from the scutellum of the previous ones, were singularly transferred in the same conditions and induced to regenerate. The whole procedure yields, on the average, from 15 to 25 plantlets, depending on the genotype, per gram fresh weight tissue of the original inoculum. This procedure is virtually more efficient for the final yield of plant regeneration than a simple transfer of calli into hormone free medium as single step. The efficiency of the system makes reliable the recovery of transformed individuals in regenerative conditions where the interference of 2,4-D on the effect of the selective agent is avoided. Therefore, the influence of the various antibiotics during regeneration was investigated. The interference of antibiotics in regenerative conditions was evaluated by recording: i) somatic embryo germination, ii) coleoptile emergence, iii) greening of the coleoptile, iv) shoot emergence (white/green), v) rooting of the shoot, and vi) plantlet development. Each experiment has a minimum size of 16 calli/geno-type/treatment/dosage, and was repeated at least twice. Antibiotics were tested at concentrations of 10, 20, 50 and 100 µg/ml. The results obtained indicated that although kanamycin, geneticin and hygromycin B all act by inhibiting ribosomal protein synthesis, they, however, has a differential effect on the developmental pathway from the early pro-embryogenic structures until complete plantlet formation. Selection applied in regenerative conditions was markedly more stringent than the counterpart applied in propagative conditions in the presence of 2,4-D. For example, a significant inhibition of callus growth was obtained in 21 day subculture in the presence of 100-120 µg/ml kanamycin, whereas 10-15 µg/ml blocked greening of the coleoptile in germinating somatic embryos on MS hormone free medium, in 5-7 days.

Propagation of the embryogenic tissues in a 21 day subculture on 10 µg/ml kanamycin, followed by transfer on 10 µg/ml kan in MShf medium, resulted in somatic embryo germination, swelling of the scutellum and further occasional development of a white coleoptile. No rooting occurred and the general tendency of the tissues was a complete block of development. When occasionally plantlets were produced, they were totally white. Neomycin acted in the same manner. Genotypic differences in response were detected, depending on the genotype of the cultures. Inbred B79 derived cultures were definitively more sensitive than F2 selections from crosses of B79 with other inbred lines; however, no cultures of these genotypes gave escapes when selective agent was applied during regeneration. G418 was less effective than kanamycin. Complete block of the embryo germination occurred at 50 µg/ml G418. Plantlet development and greening occurred at levels as high as 20 µg/ml. Higher selective pressure (50 µg/l) led to embryo swelling and abortive coleoptiles. Among this class of antibiotics, paromomycin was the least effective. Green plantlets could be recovered through germination of the somatic embryos at dosages as high as 50 µg/ml paromomycin. Only at 100 µg/ml a complete block of somatic embryogenesis was recorded. Escapes were detected in the less sensitive genotypes. Hygromycin B was the most effective selection agent in propagative conditions, where 50 µg/ml hygromycin B completely stopped callus growth and no further recovery was achieved upon release of the selection. In regenerative conditions hygromycin B was effective at 10 µg/ml like kanamycin and acted similarly. The sensitivity to hygromycin B was not genotype dependent, selection hampered development in all the genotypes tested, and no escapes were recovered. Selection procedures developed on regenerating embryogenic monocot cultures may help in the recovery of unambiguous events of transformation.

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