Tissue Culture Propagation of Field-Grown Cucumber Selections

Cucurbit Genetics Cooperative Report 4:20-22 (article 10) 1981

T. C. Wehner and R. D. Locy
North Carolina State University, Raleigh, NC 27650

One potential application of tissue culture techniques to cucumber breeding is in the propagation of selections made in the field. This would replace vegetative propagation by rooted cuttings (4). Callus has been induced to form from all vegetative and reproductive parts of the cucumber plant: root, stem, leaf blade, petiole, tendril, flower parts, and seedling hypocotyls and cotyledons (1). Callus has also been produced from leaf protoplasts (2).

One method of propagation would involve producing callus from the selected plants, multiplying the callus, and then regenerating plants from the callus. The best source of tissue for use in propagating selections is leaf tissue, since it is available throughout the life of the plant, is available in large quantities, and, if taken near the growing point, represents younger and more rapidly growing tissue than most other plant parts. The major problem in vegetatively propagating cucumber selections from the field is eliminating external and internal contamination without killing the tissue. Our objective was to evaluate a tissue culture approach to propagation of genetically diverse cucumbers grown in the field under normal summer disease load. In addition, we evaluated sodium hypochlorite (chlorine bleach) for use in sterilizing tissues to be cultured in vitro.

Thirty diverse Cucumis plant introduction (PI) lines were planted at Raleigh, NC on June 27. All but two of the PI lines used were Cucumis sativus accessions (Table 1). The exceptions are PI 299570 which is C. africanus, and PI 292190 which is C. metuliferus. After 41 days, the youngest fully-opened leaf was collected from 3 plants of each PI line. Leaves were then sterilized in a 17% solution of laundry bleach (1 bleach:5 water) for 15 to 42 min (depending upon the sterilizing treatment), and then 3 mm square pieces of blade or petiole tissue were cut and placed on the tissue culture medium with 5 tissue pieces per petri plate. The medium contained Murashige-Skoog (3) salts and vitamins with 0.1 mg/l naphthalene acetic acid, 1 mg/l N6-benzylamino purine, and 3% sucrose. A completely random experimental design was used with 30 lines, 2 tissues (leaf blade and leaf petiole), and 2 replications (petri plates). The plates were evaluated for contamination and callus production after 34 days in culture.

Tissue from leaf blades produced callus with a higher rate of success than that from leaf petioles (Table 2). Uncontaminated callus was obtained from 25 of the 30 lines used, with the loss of callus being due to either internal contamination of the tissue or to killing of the tissue for callus during sterilization. Tissue from leaf petioles is not an efficient source of tissue for callus production because of its high rate of internal contamination (Table 3), and its low rate of clean callus production (Table 2). Leaf blades should be sterilized in 17% laundry bleach for about 20 min to eliminate as much contamination as possible without killing the tissue (Table 3). Better than 60% of the petiole and blade pieces of the C. africanus and C. metuliferus PI lines produced clean callus.

It appears that, with further refinements, the system outlined here could be used to reliably establish callus cultures of selections made from field-grown cucumber plants.

Table 1. Plant introduction (PI) lines, and their origins, used in this study.

PI	Origin	PI	Origin	PI	Origin
92806	China	222099	Afghanistan	299570z	South Africa
165499	India	223841	Philippines	342950	Denmark
167050	Turkey	224668	Korea	344442	Iran
177363	Syria	228808	Canada	355052	Israel
181756	Lebanon	249561	Thailand	356809	U. S. S. R.
183224	Egypt	257286	Spain	357837	Yugoslavia
205995	Sweden	264226	France	358813	Malaysia
206043	Puerto Rico	267745	Brazil	360939	Netherlands
212059	Greece	283899	Czechoslovakia	369717	Poland
212233	Japan	288990	Hungary	292190y	Transvaal

^z Cucumis africanus
^y Cucumis metuliferus

Table 2. Callus production from leaf tissue collected from 30 field-grown Cucumis lines.^z

Tissue source	Clean callus (%)	Contaminated callus (%)	Dead tissue (%)
Petiole	38	32	30
Blade	58	7	35

^z Youngest fully-opened leaf harvested from 41-day-old plants growing at Raleigh, NC.

Table 3. Percent loss of leaf tissue pieces to contamination or to killing by sterilization.

	Tissue source z
	Leaf petiole		Leaf blade
Time in sodium hypochlorite (min.)	Dead	Contaminated	Dead	Contaminated
15	0	77	13	33
18	10	33	7	7
21	47	20	0	7
24	30	67	33	0
27	43	10	47	0
30	47	17	40	0
33	30	17	13	0
36	43	23	73	7
39	23	17	53	7
42	33	37	60	13

^z Youngest fully-opened leaf harvested from 41-day-old plants growing at Raleigh, NC.

Literature Cited

1. Alsop, W. R., W. W. Cure, G. F. Evans, and R. L. Mott. 1978. Preliminary report on in vitro propagation of cucumber. Cucurbit Genetics Coop. Rpt. 1:1-2.
2. Coutts, R. H. A. and K. R. Wood. 1977. Improved isolation and culture methods for cucumber mesophyll protoplasts. Plt. Sci. Lett. 9:45-57.
3. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
4. Shehatta, M. A., D. W. Davis, and P. E. Reed. 1974. Vegetative propagation of cucumber. HortScience. 9:575-576.