A Micropropagation Protocol for Ecballium elaterium (L.) A. Rich

. , . .' N 0 0 . (l) ::. > td .~ N ~ (1) ~ o '0 -u • l . . . . .. , " A Micropropagation Protocol for Ecballium elaterium (L.) A. Rich. E. Attard l and H. Attard2 I Institute of Agriculture, University ofMalta, Msida, MSD 06, Malta, and 2Col/ege of Agriculture, Luqa Road, Qormi, QRM08, Malta Introduction: Squirting cucumber, Ecballium elaterium (L.) A. Rich. (Cucurbitaceae), is a wild medicinal plant found abundantly in the Mediterranean region. It has been utilized as a rootstock for many cucurbitaceous crops, mainly attributed to its resistance to abiotic as well as biotic stress (2). Important pharmacological uses (1,9) are attributed to the bitter principles, cucurbitacins (5), which make the crop inedible. Micropropagation was aimed at determining the regeneration potential of this resistant rootstock. Bonferroni post-hoc test for equality of means. Only p::;:0.05 were considered statistically significant. The results were analyzed statistically by the oneway analysis of variance (ANOVA) followed by the Results and Discussion: Effects of PG& on explants. The effects of the different PGRs or additives on the nodal explants are shown in table 1. The best responses for shoot multiplication were with NAAIBAP combination (Figure 1), followed by Ki (p<0.05, v=10). BAP responded synergistically with auxins unlike K1. Nodal explants produced more than 5 shoots within 1 month especially with the NAAIBAP combination. In Gomphrena species, the index was three or more shoots per nodal segment after I month (8). A low auxin (0.1 mgll NAA) and a high cytokinin (5 - 10 mg/l BAP) . combination were optimum. For E. eiaterium, decreasing the auxin concentration decreased the bud multiplication effect. As regards shoot elongation, the best and significant response was observed with Ki, BAP and GA 3• In their absence no elongation took place indicating that the plant in culture does not store or produce any endogenous cytokinins. Also cucurbitacins have anti-gibberellic activity (6) hence intrinsic gibberellins that may be possibly present are inhibited by these secondary metabolites. When NAA was completely omitted from the media, shoot elongation was noted in all treated shoot explants. Callus production was seen with all PGRs or additives except for IAA and charcoal. The 2,4DIKi combination showed significant effects on callus production with no effects on the other parameters. This goes in accordance with the observations made by Esaka (3) on Cucurbita pepo explants. Rooting was a parameter that posed several problems in the regeneration of Ecballium elaterium plantlets. In fact, the whole plantlet was not regenerated in tissue culture. Although IAA induced rooting, the low response might be due to the fact that IAA produces a response in the concentration range between 1 and 30 mg/l (4). Nevertheless, if the auxin had a higher activity, callus induction and _ Cucurbit Genetics Cooperative Report 25:67-70 (2002) 67 Materials and Methods: E. elaterium seeds were obtained from immature fruit collected in the Southern region of Malta. The fruit were washed with tap water for 15 min., surface sterilized with 70 % ethanol for 30 sec, soaked in 10 % hypochlorite solution for 20 min and rinsed in three changes of sterile distilled water. Seeds were carefully removed under aseptic conditions, and placed on Murashige and Skoog (MS) basal medium (7). Two weeks from germination, node explants were taken for tissue culture. The sectioned node explants were inoculated on MS medium containing different plant growth regulators (PGRs) or additives (Table 1), and every 4 weeks the surviving explants were either subcultured on the same medium or transferred to a different medium, in cases of impaired growth. The conditions for growth were 25 ± 1 °C and 3250 ± 250 Ix. Bud multiplication, shoot elongation, root production and callus induction and proliferation were observed. The plantlets were transferred to Jiffyil> pots (Sigma, U.S.A.) and closed in a Phytatray® (Sigma, U.S.A.) to maintain a high percentage of humidity. With the emergence of roots from the pot, the plantlets were transferred to larger pots until flowering. Fig. 1. Emergence of multiple shoots frem nodal explants treated with theNAAIBAP combination Fig. 2. Flowering and fruiting of the micropropagatedE. elaterium plantlets. 68 Cucurbit Genetics C()()perative Report 25:67-70 (2002) Table 1. The over all effects of different medias on the different parameters studied. Percent!ige for each Stimulus (%) Rooting Multiplication Elongation Callus 8.80 26.39 x 23.09 x 0.00 0.00 14.66 0.00 0.00 0.00 16.50 x 10.56 16.70 x 7.31 19.27x 0.00 0.00 8.56 0.00 0.00 19.27x 9.63 19.27x 5.76 13.89 x 3.60 14.39 x 14.39 x 14.39 x 14.39 x 0.00 14.39 x 0.00 2.88 IAAlKi NA.AlBAP Ki 2,4-D lKi lBA NAAfB AP (Y2)Y MS IAA BAP Charcoal GA3 8.73 0.90 6.55 0.00 26.19 x 0.00 26.19 x 3.49 26.19 x 0.00 5.24 SThe media contained MS medium and 1 mg/L of each PGR or additive listed: indole acetic acid (IAA) , kinetin (Ki), naphthalene acetic acid (NAA) , benzylamino purine (BAP), 2,4-dichlorophenoxyacetic acid (2,4 -D), indole butyric acid (IBA) and gibberellic acid (GA3) . YNAAIBAP (y2) contains 0.5 mg/L ofNAA and 1 mglL ofBAP. xp < 0.05 (v=10). The experim ent was repeated three times with 15 replicates. Table 2. Time (day s) for rooting and rep otting for the four treatments. IAA Rooting in Jiffy* pots Repotting GA-i +RH.:l - RH. +RH. - RH. 10 25 23 46 58 63 37 72 ~oting hormone powder (1 % NAA and thiram, Secto, UK) . The experiment was repeated three times with 10 replicates. Cucurbit Genetics Cooperative Report 25:67-70 (2002) 69 proliferation might have set in and hence posing a problem to the rooting process. Transfer of explants. Based on the above findings, the shoot explant grown on GA3 and IAA media were selected for pot trials, with the use of a rooting hormone (1 % NAA and thiram, Secto, UK). The best treatment was IAA cultures treated with rooting hormone (Table 2). For the IAA with rooting hormone treatment, flowering took place at approximately day 62 from transfer to Jiffyil> pot. This was eventually followed by fruiting (Figure 2) In conclusion, an ideal protocol would involve the germination of seeds on MS medium followed by the inoculation of node explants on NAAIBAP for three consecutive subcultures at 4-week intervals, elongation on GA3 medium for another 4 week and an auxin (IAA) shock for 1 week, in tissue culture. Subsequently shoots should be treated with rooting hormone (NAA), transferred to Jiffy pots and after 3 to 4 weeks the p1antlets should be repotted and acclimatized for another 4 to 5 weeks. Our results give a clear protocol for the regeneration of the squirting cucumber for possible use as a resistant rootstock for the edible cucurbitaceous crops. Literature cited 1. Attard, E., A. Scicluna-Spiteri, M. Grixti and A. Cuschieri. 1996. The cytotoxic activity of Cucurbitacin E and Busulphan on ovarian and stomach cancer cells in vitro: A comparative study. Xjenza 1:29-34. 2. Bar-Nun, N. , and A.M. Mayer. 1990. Cucurbitacins protect cucumber tissue against infection by Botrytis cinerea. Phytochem., 29:787-791. 3. Esaka, M. 1993. Cucurbita pepo (Pumpkin): In vitro production of ascorbate oxidase. In: Bajaj, Y.P.S., ed. Biotechnology in Agriculture and Forestry: Medicinal and Aromatic Plants IV . Volume 21 , Berlin: Springer-Verlag. pp. 115 - 13l. 4. Finnie, J.F. and J. Van Staden. 1994. Gloriosa superba L. (Flame Lily). Micropropagation and in vitro production of colchicines. In: Bajaj, Y.P.S., ed. Biotechnology in Agriculture and Forestry: Medicinal and Aromatic Plants VI. Volume 26, Berlin: Springer-Verlag. pp. 146166. 5. Lavie, D. and E. G lotter. 1971. The Cucurbitanes; a Group of Tetracyclic 70 Triterpenes; Progress in the Chemistry of Organic Natural Products XXIX; New York; U.S.A. - English Translation, pp. 352-354. 6. Morgan, E.D. and LD. Wilson. 1999. Insect Hormones and Insect Chemical Ecology. In: Barton, D.; Nakanishi, K. , ed. Comprehensive Natural Products Chemistry, Volume 8, UK: . Elsevier, pp. 320 - 321 . 7. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473497. 8. Vieira, C.C.J., H. Mercier, E.P. Chu and R.c.L. Figueiredo-Ribeiro. 1994. Gomphrena species (globe amaranth): In vitro culture and production of secondary metabolites. In: Bajaj, Y.P.S., ed. Biotechnology in Agriculture and Forestry: Medicinal and Aromatic Plants VII. Volume 28, Berlin: Springer-Verlag. pp. 257- 270. 9. Yesilada, E., S. Tanaka, E. Sezik and M. Tabata. 1988. Isolation of an anti-inflammatory principle from the juice of Ecballium elaterium. J. Nat. Prod. 51 :504-508. Cucurbit Genetics Cooperative Report 25:67-70 (2002)