Plant Regeneration from Callus Cultures of Citrullius colocynthis

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Cucurbit Genetics Cooperative Report 14:107-108 (article 37) 1991

T.W. Frazier and J.O. Kuti
Horticulture Research Laboratory, College of Agriculture, Texas A&I University, Kingsville, TX 78363.

Citrullus colocynthis (L.) Schrad. belongs to the Cucurbitaceae family and produces edible seeds rich in oil and protein (4). The plant is a long-lived perennial, indegenous to Africa and the Middle East and is probably an ancestral type of watermelon (5). Preliminary studies in our laboratory indicated that C. colocynthis may posess resistance to a number of watermelon diseases including Fusarium wilt. Tissue culture is a powerful technique in the selection of resistance to pathogens and in the creation of genetic variability from cell cultures. (1). There have been few reports on tissue culture of C. colocynthis (3). In this report, we describe regeneration of plantlets from callus cultures of C. colocynthis.

Callus Induction: Seeds of C. colocynthis were obtained from University of Port-Harcourt, Niger,a West Africa. The seeds were surface-sterilized by immersion in 2.5% (v/v) sodium hypochlorite for 15 min, rinsed with sterile distilled waer, germinated aseptically and grown in potting soil in the greenhouse. Cotyledons and leaf petioles of 14-day-old seedlings were used as explants. Explants were surface-disinfected in 2.5% sodium hypochlorits for 10 min, rinsed three times with sterile distilled waer, sliced into 3 mm sections and transferred onto a callus induction medium. The callus induction medium consisted of a basal medium of Murashige and Skoog (2), MS salts plus sucrose (30 gl-1), thiamine HCl (0.8mg 1-1), nicotinic acid (2 mg 1-1 ), pyroxidine HCl (0.8 mg 1-1 ) and Difco Bacto agar (0.8gl-1 ) supplemented with 4 mg l-1 , 2,4-D and 0.1 mg 1-1kinetin. The pH was adjusted to 5.7 prior to autoclaving for 20 minutes at 121 ˚ C, 124 kPa for 15 minutes. Thirty explants from each source were used in the callus induction and the experiment was repeated three times. All cultures were grown under 16 hr photoperiod at 25 ˚ C.

Plant Regeneration: Callus cultures were transferred to a maintenance medium consisting of basal medium supplemented with 1.5 mg 1-1 2,4,-D. Shoots were induced on MS basal medium containing 1,0 mg 1-1kinetin and p.05 mg 1-1 1-napthaleneacetic acid (NAA). At the end of 4 weeks, shoots were excised and placed on the rooting medium which consisted of basal medium supplemented with 0.5 mg 1-1 NAA and 0.05 mg 1-1 6 benzyladenine (BAP). Rooted shoots were transfered to sterile peat and sand soil mixture. When shoot and roots began active growth, regenerated plants were evaluated for somaclonal variation.

Results and Discussion: In all cases, cotyledon-derived callus grew faster and had greater morphogenetic potential than petiole-derived callus. Although the present study focused on the effect of explant source, a previous pilot study on the growth regulator combinations and concentrations required for optimum callus growth or shoot and root regeneration of C. colocynthis indicated similar requirements for other explant sources (data not shown). Studies are under way to use highly regenerative C. colocynthis callus in vitro selectioon for disease resistance.

Table 1. Effect of explant source on callus induction and plant regeneration of Citrullus colocynthis.

Explant source

% Explant with callus

Number of shoots obtained

% shoot with roots

Number of plantlets
cotyledon
Exp. #1 : 38 25 96 24
#2: 34 32 72 23
#3: 41 26 77 20
leaf petiole
Exp. #1: 16 7 24 1
#2: 10 3 0 0
#3: 23 7 29 2

Literature Cited

  1. Daub, M.E. 1986. Tissue culture and the selection of resistance to pathogens. Ann. Rev. Phytopath. 24:159-186.
  2. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15:473-497.
  3. Nag. T.N., M.C. Marsh and D. Singh. 1982. Secondarymetabilites from Indian arid zone plants in vivo and in vitro. In: Plant Tissue Culture, Japanese ASsociation of Plant Tissue Culture, p.293-294.
  4. Sawaya, W.N., N.J. Daghir and P. Khan. 1983. Chemical characterization and edibility of oil extracted from Citrullus colocynthis seeds. J. Food Sci. 48:104-107.
  5. Shimotsuma, M. and Y. Ogawa. 11960. Cytogenetics studies in the genus Citrullus. II. Bitter substances in fruit of C. colocynthis Schrad. Japan J. Genet. 35:143-152.