Growth responses from whole fruit and fruit halves of lemon cultured in vitro

A comparative growth study was conducted on juice vesicles cultured in the form of various fruit explant types (equatorially bissected fruit halves, longitudinally bissected fruit halves, oneeighth sections of fruit, one-quarter sections of fruit, whole carpel segments, 2 or 3 mm thick equatorial slices of fruit, and 1 cm2 fruit endocarp pieces) from 15 mm diam Citrus limon (L.) Burm. f. cv. Eureka lemons. Juice vesicles within equatorial fruit halves produced the least amount of callus. Furthermore, these juice vesicles grew similarly to juice vesicles occurring in the tree grown fruit. A study of cultured equatorial fruit halves using 10-45 mm diam lemons was then conducted. Fruit half cultures containing juice vesicles could be readily established from 15-45 mm diam lemons. Vesicles from 10 mm diam fruit halves, however, invariably produced callus. Vesicles cultured within fruit halves produced proportionately less callus as their fruit diam increased. Juice vesicles cultured in 15-30 mm diam fruits lost their original green color and turned opaque as they matured (i.e., after 3-6 months in culture). A method is also presented, whereby whole lemon fruits can be established and maintained in vitro. Lemons, 35-45 mm in diam, were the best explant sources for establishing whole fruit cultures. Juice vesicles in whole fruit cultures may remain viable for up to 8 months in culture. CHEMICAL TREATMENTS, environment, husbandry practices, genetic characters, and general plant health influence citrus fruit development and physiology (Sinclair and Bartholomew, 1944; Sauer, 1953; Reuther et al., 1969; Jahn and Young, 1972; El-Zeftawi, 1973; Boswell, Nauer, and Atkin, 1982). In the field, these factors are difficult to control and may obscure the influence of test environments, and of exogenous growth regulators and chemicals on citrus fruit development. Therefore, we sought to develop in vitro systems to study citrus fruit metabolism and growth in a controlled environment. Citrus fruit is a unique complex berry (also called a hesperidium) having 6-20 united carpels clustered around and joined to the floral axis to form locules into which seeds and juice sacs grow (Schneider, 1968). Phylogenetically, carpels are modified leaves. The economically important juice vesicles are unique to the subfamily Aurantioideae of the Rutaceae. Juice vesicles are appendages of the endocarp which ' Received for publication 25 January 1988; revision accepted 16 June 1988. Mention of a trademark, proprietary product or vendor does not constitute approval or guarantee of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. We would like to thank Drs. J. B. Carpenter and L. M. Blakely for critically reading the manuscript and Dr. M. Roose for providing plant material. through various morphogenetic and chemical stages give rise to the juice filled sacs. Over the last few decades several investigators have used various explants from citrus fruit to conduct growth and metabolism studies in vitro (Kordan, 1959, 1974, 1975; Murashige and Tucker, 1969; Schroeder, 1969; Chauhan and Roberts, 1978; Einset, 1978; Unger and Feng, 1978; Kato, 1980; Gulsen, Altman, and Goren, 1981; Altman, Gulsen, and Goren, 1982; Khan, Chauhan, and Roberts, 1986; Tisserat and Galletta, 1987). In these studies, citrus fruit tissue explants readily gave rise to callus. The production of callus from fruit tissue irreversibly alters both the normal morphogenetic and metabolic processes associated with fruit tissues. We report on the development of fruit culture systems to study juice vesicle growth where the preponderance of cultured vesicles remains callus-free (e.g., over 90%). Cultured juice vesicles, in our sterile fruit culture systems, developed similarly to vesicles within fruits grown on the tree. MATERIALS AND METHODS Fruits, 10-4 5 mm diam, of Citrus limon (L.) Burm. f. cv. Eureka (lemon) were obtained from 6-year old trees grown at the University of California, Riverside. Fruits were surface sterilized in a 2.63% sodium hypochlorite solution (containing 2 drops of Tween-20 emulsifier per 100 ml solution) for 30 min and then rinsed 3 times with sterile distilled water. The extreme stylar