An In Vitro Selection Method for a Melon Variety which Regenerates by Direct Organogenesis

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Cucurbit Genetics Cooperative Report 15:65-66 (article 24) 1992

Victor Gaba and Yeheskel Antignus
Dept. of Virology, Agricultural Research Organisation, Volcani Center, POB 6 Bet Dagan 50250, Israel

The Israeli F1 hybrid melon cv Galia is an important export crop. We wish to engineer this melon variety with coat protein mediated protection against Zucchini Yellow Mosaic Virus (ZYMV). We have cloned ZYMV (5), and identified and isolated the ZYMV coat protein gene (6). In devising a transformation system an important step is to identify a selection agent which is capable of blocking regeneration in non transformed tissues i.e. which lack an introduced resistance gene. In cucurbits the antibiotic kanamycin has been used successfully as a selection agent at concentrations of 25-100 mg/l (1, 3, 4, 9(. Here we report that the responses of cv Galia to kanamycin are atypical of published cucurbit selection methods.

Materials and methods. Seeds of Cucumis melo L. cv Galia were pealed and surface sterilised in 1.2% solution of hypochlorite, 1 drop of Tween 20 per 100 ml. Seeds were washed 4X with sterile water and cut into 4 longitudinally through the embryo, each explant thus being half of a cotyledon with attached embryo fragment. The primary explants were then plated onto autoclaved Murashige and Skoog (8) medium, with 3% sucrose, 8-10 g/l agar, and 1 mg/1 benzyl adenine (MSBA1 medium). Plant material was incubated in a growth room at 26 C in continuous light. Explants were transferred to fresh MSBA1 medium with kanamycin at periods up to 7d after primary explant preparation. In a second set of experiments explants were cut in pieces prior to transfer to kanamycin-containing MSBA1 at 5d old (table 2). Regeneration was scored as the % of explants with shoots or shoot buds after 30d total in culture.

Results and Discussion. The response to kanamycin was independent of age for exposures to kanamycin beginning up to 7d of age, for the concentration range 100-250 mg/l (data not shown). The effect of kanamycin on regeneration also appeared to be independent of concentration within that range (Table 1). Clearly kanamycin has a small effect (17%) in reducing regeneration – statistically significant, but inadequate for a selection system.

However, in a second series of experiments, the antibiotic had a powerful effect when explants were cut into fragments on transfer to MSBA1 with kanamycin (Table 2). This technique is the basis of our selection system for genetic transformation of melon cv. Galia.

The recalcitrance of”Galia” melon to kanamycin is probably due to anatomy of regeneration in this variety. The published cucurbit transformation systems (1, 3, 4, 9) all feature organogenesis or embryogenesis via a callus phase or on the edge of the explant, all directly exposed to the selection agent. “Galia”, however, regenerates by direct organogenesis across the surface of the explant (2; Gaba, unpublished). The developing buds are therefore protected from the antibiotic by a mass of cotyledon tissue. Such protection is obviated by reducing the size of the explant (Table 2). This technique will be useful for a selection method for the transformation of other cucurbit varieties which regenerate directly on the cotyledon surface, such as some cucumber cultivars (7).

Table 1. Kanamycin has little effect on regeneration of explants of melon cv Calia. All treatments for transfer to kanamycin at ages 0-7d have been bulked. Data from 7 separate experiments.

Kanamycin concentration (mg/l)

% regeneration

Standard error of mean

Number experiments n
0 87 2 7
100 75 3 6
15 77 8 5
200 73 6 3
250 65 11 3

Table 2. Kanamycin has a strong effect when explant size is reduced. Primary explants were cut into fragments on transfer to MSBA1 with 250 mg/l kanamycin, after 5d on MSBA1. Minimum of 30 explants per treatment.

Primary explant

& of pieces regenerating

kanamycin

control
uncut 65 87
cut in 2 0 65
cut in 4 0 48
cut in 8 0 27

Literature Cited

  1. Chee, P.P. 1990. Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants. Plant Cell Rep. 9:245-248.
  2. Dirks, R. and MN. van Buggenhum. 1989. In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L. Plant Cell Rep. 7:626-627.
  3. Dong, J. -Z., M.-Z. Yang, S. -R. Jia, and N. -M. Chua. 1991. Transformation of melon (Cucumis melo L.) and expression from the cauliflower mosaic virus 35S promoter in transgenic melon plants. Bio/Technology 9:857-863.
  4. Fang, G. and R. Grumet. 1990. Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant Cell rep. 9:160-164.
  5. Gal-On, A., Y. Antignus, A. Rosner, and B. Raccah. 1991/2. Infectious in vitro RNS transcripts derived from cloned cDNA of the cucurbit potyvirus, zucchini yellow mosaic virus. J. Gen. Vir. (In press).
  6. Gal-On, A., Y. Antignus, A. Rosner, and B. Raccah. 1990. Nucleotide sequence of the zucchini yellow mosaic virus capsid-encoding protein and its expression in Escherchia coli Gene 87:273-277
  7. Gambley, R.L. and W.A. Dodd. 1990. An in vitro technique for the producion of multiple shoots in cotyledon explants of cucumber (Cucumis sativus L.). Plant Cell Tissue Organ Culture 20:177-183.
  8. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-498.
  9. Trulson, A.J., R.B. Simpson, and E.A. Shahin. 1986. Transformation of cucumber (Cucumis sativus L.) plants with Agrobacterium rhizogenes. Theor. Appl. Genet. 73:11-15.

Contribution of the Agricultural Research Organisation, No. 3432-E, 1991 Series.