Organogenic and Embryogenic Potential of Several Commercial Lines of Cucumis melo L.

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Cucurbit Genetics Cooperative Report 14:71-73 (article 27) 1991

M. Bordas, V. Moreno and L.A. Roig
Plant Cell and Tissue Culture Laboratory. Department of Biotechnology. E.T.S.I.A. Universidad Politecnica. Camino de Vera 14, 46922-Valencia. Spain

Introduction. Availability of efficient methods for plant regeneration of melon is required to take advantage of biotechnological alternatives for crop improvement. A reliable protocol for regeneration from primary explants can be useful in somaclonal variation as a way for producing new genetic variability. Furthermore, successful plant regeneration could permit the introduction of desirable traits by the “leaf disc transformation” procedure.

Nowadays, regeneration in tissue culture of cucumis melo from several sources of explants has been successfully achieved (1, 3, 4, 5, 6, 7, 8, 9). However, the frequency of regeneration appears to be genotype dependent (10). Therefore, some study of the morphogenic response of the agronomically important lines or varieties is needed if we aim towards the improvement of this important crop by new technologies.

The purpose of the present study is the selection of suitable genotypes for somaclonal variation programs and genetic transformation studies by screening commercial lines of melon.

Materials and Methods. The genotypes of Cucumis melo L. tested were lines of ‘Pinyonet Piel de Sapo’, ‘Amarillo Canario’ and ‘Amarillo Golda’ (kindly provided by G.Anastasio, Petoseed Iberica S.A.). Seeds were surface-sterilized and explants were excised from cotyledons and hypocotyle of 9 day-old seedlings and from leaves of axenic plants as described previously (7, 8). The only difference was that cotyledonary explants were prepared by cutting in three segments instead of two. For each explant source, six explants were placed into 250-ml jars containing 40 ml of medium and, in all cases, at least 12 replicate jars were included. Incubation conditions were identical to those previously used in other studies (7,8).

The organogenic response of cotyledonary and leaf explants was determined when cultured on 1K 15/60 medium, consisting of MB3 basal medium (8) + 1.5 mg/l indole-3-acetic acid (IAA) + 6.0 mg/l kinetin (K). Recently, the efficiency of this protocol has been significantly enhanced (4). Inspite of this, the present study was focused to evaluate the morphogenetic capacity of lines, cultivars or varieties, and with this idea we considered IK 15/60 medium more suitable in order to reveal the genotype effect. Organogenic response was rated after 30 days of culturing. Explants were scored for growth (fresh weight), shoot-bud and shoot formation (both frequency and index) (see Tables).

Embryogenic response was tested using hypocotyl explants cultured on NP 25/25 medium (MB3 + 2.5 mg/l naphthalenacetic acid NAA + 2.5 mg/l 2-isopentyl adenine2-iP) in order to obtain unorganized calli. After 25 days incubation in the dark, calli were subcultured onto MB3 medium without growth regulators. Once calli showed embryoid-like structures, they were transferred to low intensity light conditions and after an additional 3 days were incubated under the standard intensity light (8). Embryogenic response was rated 25 days after culturing in MB3. Calli were scored for growth and embryoid-like structures formation.

Results and Discussion. The organogenic response of the primary calli from cotyledonary and leaf explants is outlined in Tables 1 and 2.

When cotyledonary explants were used, the highest overall organogenic response was obtained with ‘Pinyonet Piel de Sapo’. It should be noted that the response of this line was similar to or slightly higher than that obtained with the standard line of ‘Cantaloup charentais’ (C.Ch.) used in our previous studies. Although the frequency of shoot-bud formation of C.Ch. in the same medium was 76.8%, this result was obtained when cotyledons were excised in two segments. Later studies have confirmed that organogenic response decreases significantly when smaller explants are used (68% in C. Ch. sec 4).

Table 1. Growth and organogenic response on cotyledonary explants on IK 15/60 medium.

P. PIEL SAPO

A. CANARIO

A. GOLDA
Growthz 2.29 + 0.07 1.34 + 0.06 1.39 + 0.07
Shoot-buds (%)y 77.08 + 3.50 52.78 + 5.88 41.67 + 5.81
Shoots (%)y 37.5 + 4.03 44.44 + 5.86 30.56 + 5.43
Organogenic Indexx 1.39 + 0.06 0.89 + 0.09 0.74 + 0.08
Shoots Indexw 0.66 + 0.09 0.94 + 0.16 0.65 + 0.15

Table 2. Growth and organogenic response in leaf explants on IK 15/60 medium.

P.PIEL SAPO

A. CANARIO

A. GOLDA
Growthz 2.35 + 0.08 1.86 + 0.06 1.68 + 0.05
Shoot-buds (%)y 16.66 + 4.39 63.89 + 5.66 8.33 + 3.26
Shoots (%)y 4.16 + 2.35 41.67 + 5.81 4.17 + 2.36
Organogenic Indexx 0.51 + 0.05 0.95 + 0.08 0.18 + 0.04
Shoots Indexw 0.05 + 0.03 0.87 + 0.16 0.04 + 0.02

zFresh weight (g) expressed as mean + 1 SE.
yFrequencies of calli with shoot-buds or developed shoots √ p(1-p)/n(%), p = number of calli with response/total number of calli (n).
xOrganogenic Index = weighted average obtained from arbitrary vslurd z90 to 3) corresponding to the organogenic response level shown in eah callus + 1 SE.
wShoots Index = average number of shoots per callus + 1 SE.

The results obtained when leaves were used as an explant source indicates that cv. Amarillo canario showed the highest shoot-bud and shoot formation at a frequency of 63.89%, respectively. Increasingly, the highest regenerating capacity from leaf explants was obtained with the above mentioned genotype among all lines screened for response in tissue culture. Colijn-Hooymans et al. (2) found in cucumber that the number of endopolyploid cells pre-existing is larger in cotyledonary than in leaf issues. If this is the case in melon, the explant source will be very important depending on the purpose of the research. Leaves especially could be a useful tissue source for applying “leaf disc transformation.”

Somatic embryogenesis response was very high in all cases. The frequency of calli which showed embryoid-like structures, after 10-15 days of culturing, were 78.47%, 83.33% and 75.00% in ‘Pinyonet Piel de Sapo’,, ‘Amarillo canario’ and ‘Amarillo Golda’ respectively. After 30 days, these values increased up to 98.88%, 98.61% and 88.70%. However, embryoids usually showed abnormal development. Consequently, results indicate that these lines require further optimization for efficient regeneration by way of somatic embryogenesis.

In conclusion, it has been confirmed that the regenerating protocol established can be applied in all genotypes tested in order to obtain calli-derived plants in a somaclonal selection program. In addition, it has been proved that cultivars tested in the present study will be suitable for experiments on the production of transgenic plants using the technology of Agrobacterium-mediated gene transfer by “leaf disc transformation.”

Acknowledgments: The authors express their appreciation to CICYT (Comision Interministerial de Ciencia y Tecnologia, Ministry of Education and Science, Spanish Government) for financial support (Project BIO89-0446). M. Mordas is grateful for her Grant from the same organizaiton (CICYT).

Literature Cited

  1. Branchard, M., M. Chateau, B. Megnegneau and I. Debeaujon. 1988. Somatic embryogenesis and plant regeneration from cotyledon callus culture of Cucumis melo. In: cucurbitaceae 88. Proc. Eucarpia Cucurbit Genetics and Breeding, pp. 133-136. Avignon-Montfavet, France.
  2. Colijn-Hooymans, C.M., R. Bouwer, W. Orczy and J.J.M. Dons. 1988. Plant regeneration from cucumber (Cucumis sativis) protoplasts. Plant Sci. 57:63-71.
  3. Dirks, R. and M. Buggenum. 1989. In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L. Plant Cell Reports 7:626-627.
  4. Garcia-Sogo, B. 1990. Morfogenesis en cultivo de melon: regeneracion de plantas con alta eficacia a partir de celulas y protoplastos. PhD Thesis. Universidad de Valencia (Spain). 337 p. (in Spanish).
  5. Kathal, R., S.P. Bhatnager and S.S. Bhojwani. 1988. Regeneration of plants from leaf explants of Cucumis melo cv. ‘Pusha Sharbati’. Plant Cell Reports 7:449-451.
  6. MacKay, W.A., T.J. Ng and F.A. Hammerschlag. 1989. Direct and indirect regenerationof Cucumis melo L. from cotyledon culture. Cucurbit Genet. Coop. Rpt. 12:55-56.
  7. Moreno, V., and L.A. Roig. Somaclonal Variation in Cucurbits. In: Y.P.S. Bahah (ed.) Somaclonal Variation in Crop Improvement. I. Biotechnology in Agriculture and Forestry SEries, Vol. 11. Springer-Verlag Berlin Heidelberg, pp. 345-464.
  8. Moreno, V., M. Garcia-Sogo, I. Granell, B. Garcia-Sogo and L.A. Roig. 1985. Plant regeneration from calli of melon (Cucumis melo L. cv. Amarillo Oro). Plant Cell Tissue & Organ Culture 5:139-146.
  9. Neidz, R.P., S.S. Smith, K.B. Dunbar, Ch. T. Stephens and H.H. Murakishi. 1989. Factors influencing shoot regeneraiton from cotyledonary explants of Cucumis melo. Plant Cell Tissue & Organ Culture 18:313-319.
  10. Orts,. M.C., B. Garcia-Sogo, M.V. Roche, L.A. Roig and V. Moreno. 1987. Morphogenic response of calli derived from primary explants of diverse cultivars of melon. HortScience 22:666.