Tissue Culture and Cloning of Carnegiea gigantea, Cactaceae Item Type Article Authors Baker, William P.; Hanks, Tyrone Harvard; Marin, Louis Eduardo Publisher University of Arizona (Tucson, AZ) Journal Desert Plants Rights Copyright © Arizona Board of Regents. The University of Arizona. Download date 26/09/2021 08:33:25 Link to Item http://hdl.handle.net/10150/554346 BakerCloning 13 and Tinus 1996; Guibert 1996; O'Dea et al. 1995; Ritchie Tissue Culture and Cloning of 1997). Carnegiea gigantea, Cactaceae Cloning of these plants allows multiple copies to be pro- duced from superior genotypes without the time and expense William P. Baker of traditional methods. Tissue culture and cloning, however, Tyrone Harvard Hanks are not routinely used to cultivate members of the cactus family. According to Havel and Kolar (1983), "one of the Biomedical Sciences reasons can be seen in the morphological structure of cacti Midwestern University making the traditional way of explant isolation and surface 19555 North 59th Avenue sterilization of the plant material difficult." Glendale, Arizona 85308 [email protected] Material and Methods In this study, in vitro clones were produced from phenotypi- cally selected, commercially available saguaro. Eight plants Louis Eduardo Marin measuring 8 -10 cm tall were purchased from a local nurs- Life Science Department ery. Stem portions were excised, dipped in 95% ethanol, and flamed to burn off the needles. The cooled samples were Mesa Community College sterilized by soaking in 10% sodium hypochlorite (10% v/v 1833 West Southern Avenue in dH2O) for 5 minutes. They were rinsed once in distilled Mesa, Arizona 85202 H20, surface sterilized in 95% ethanol, flamed and trans- ferred to a sterile surface. Abstract Cloning has become an established method of supplying Plugs of tissue were then removed with a sterile cork borer. valuable timber trees and other plants for commercial pur- Following Havel and Kolar (1983) these explants were ob- poses. Cloning of these plants allows multiple copies to betained "from the side walls of the ribs so as to intersect the produced from superior phenotypes. In this study, in vitrovascular bundles proceeding from the nearest areola." Ex- clones were produced from phenotypically selected, commer-plant plugs were transferred aseptically to culture dishes con- cially available saguaro (Carnegiea gigantea). The clonestaining 20 ml of solid callus initiation medium consisting of were produced from tissue plugs obtained from surface ster-B -5 salts (Gamborg et al. 1968) supplemented with 10 mg /1 ilized saguaro. The plugs were transferred using standard auxin and 8 g/1 agar. Each transferred explant was pressed aseptic technique to culture dishes containing solid Callusinto the surface of the agar to insure good contact with the Initiation Medium (Gamborg's B -5 medium supplementedmedium (Figure 1A). The cultures were then incubated un- with 10 mg /1 auxin and 8 g/1 agar). The cultures were incu- der cool fluorescent lights at ambient temperature and hu- bated under continuous cool fluorescent lights at 24 C untilmidity until callus formation was observed. The lights were callus formation was observed. Healthy callus were trans-on 24 -hours a day at approximately 1300 lux. ferred to solid Development Medium (Gamborg's B -5 me- dium supplemented with 10 mg /1 auxin, 10.0 mg Kinetin, Of 30 explants prepared, 25 became contaminated and were and 8 g/1 agar) and further incubated. Resulting clones werediscarded. In the remaining 5, callus proliferation was ob- prepared for in vivo conditions by transfer to sterile pottingserved after 8 -12 weeks of incubation (Figure 1B) and found soil and successfully outplanted to the green house. Suchto be green and friable. They contained necrotic areas that clones may supply scarce C. gigantea for future research.were excised, and the healthy tissue sectioned into 2 cm The use of single genotypes for ecological applications shouldsquares. This was done in a laminar flow hood restricted to be avoided since they lack natural population variability. plant cell tissue culture using aseptic technique. The healthy callus squares were then transferred to tissue culture dishes Introduction containing 20 ml of shoot regeneration medium consisting Cloning refers to the in vitro isolation and growth of an ex- of B -5 major salts (Gamborg et al. 1968) supplemented with cised plant tissue plug (explant) into a mass of cells termed10.0 mg kinetin, 10 mg/1 auxin, and 8 g/1 agar. a totipotent callus (McNabola and Kitto 1989). The totipo- tent callus is an undifferentiated mass of cells that can be Transferred callus explants were pressed into the surface of induced to form new plants when stimulated with appropri-the agar to insure good contact with the medium. The cul- ate growth factors. The growth factors used are plant hor- tures were incubated at ambient temperature and humidity mones that stimulate cell division (Pietraface 1988). Thisunder cool fluorescent lights (24 -hour photoperiod at ap- differs from rooting in that one callus may be used to giveproximately 1300 lux) until adventitious root proliferation rise to many individuals having identical genomes. Cloningwas observed to occur from the callus (Figure 1C). After has become an established method of supplying valuable tim-approximately 6 weeks of incubation, adventitious roots were ber trees and other plants for commercial purposes (Burr evident in three clones. These clones were prepared for in 14 Desert Plants 2001 Figure 1. Stages in tissue culture and cloning of Carnegiea gigantea, Cactaceae (The Giant Saguaro): a. Explant plug in Callus Initiation Medium b. Healthy proliferating callus in Development Medium c. Clone with adventi- tious roots d. cloned plantlet in greenhouse. BakerCloning 15 vivo transplanting by passage to sterile potting soil in 10 -cmEditor's note: Midwestern University is an upper- division diameter pots. The cloned plantlets were incubated under undergraduate and graduate degree- granting institution spe- continuous cool fluorescent light (approximately 1300 lux cializing in the health sciences with five colleges - the Ari- at ambient temperature and humidity) for 3 weeks and thenzona College of Osteopathic Medicine, the College of Phar- outplanted to a 30 %- shaded greenhouse (Figure 1D). macy, Glendale, the College of Health Sciences, the Chi- cago college of Osteopathic Medicine, and the Chicago Results and Discussion College of Pharmacy. The Glendale Arizona campus is home Contamination rates with this procedure were high, but into 1,079 students, and offers degree programs in osteopathic keeping with those previously reported for other cactus spe- medicine, pharmacy, physician assistant studies, occupa- cies (Weiss et al. 1993; Weng and Xiong 1994). Results in-tional therapy, biomedical sciences, bioethics, cardiovascu- dicate that cloning and outplanting are a viable alternativelar science, and health professions education. The Univer- to traditional propagation for the saguaro. Cloned saguaro sity is accredited by the Commission on Institutions of Higher may supply scarce Carnegiea gigantea for future researchEducation of the North Central Association (NCA) of Col- and re- establishment. As noted in McNabola (1989), "an leges and Schools. advantage of tissue culture is rapid multiplication of plants in a short period of time with minimal maintenance," andThe Bachelor of Biomedical Sciences degree program is "tissue culture also enables you to screen for new geneticdesigned as a baccalaureate completion program for stu- variation within regenerated plants." However, the use ofdents who plan to apply to medical school, dentistry, phar- single genotypes for ecological applications should be avoided macy, or allied health science programs, or for students seek- for field plantings since they lack natural population vari- ing employment in bio- industry research. For more infor- ability. mation, please contact Perry Baker, Ph.D., Associate Pro- fessor and biomedical Sciences Program Coordinator, in the Literature Cited MWU Biomedical Sciences office at (623) 572 -3660. Benson L (1982) The cacti of the United States and Canada. Stanford, University Press. Burr K, Tinus R (1996) Use of clones increases the power of physiological experiments on coastal Douglas fir. Physiologia Plantarum 96(3):458 -466. Gamborg OL, Miller RA, Ojima K (1968) Nutritional re- quirements of suspension cultures of soybean root cells. Experimental Cell Research 50:151 -158. Gibson A, Nobel P (1986) The cactus primer. Harvard Uni- versity Press. Guibert M (1996) Cost of selected varieties produced in two seed orchards of Douglas fir and spruce clones. Re- vue Forestiere Francaise 48(5):431 -445. Havel L, Kolar, Z (1983) Microexplant isolation from Cactaceae. Plant Cell Tissue Culture 2:349 -353. McNabola S, Kitto S (1989) Tissue culture of carrot roots. The American Biology Teacher 51(3):165 -167. O'Dea M, Zasada J, Tappeiner J (1995) Vine maple clone growth and reproduction in managed and unmanaged coastal Oregon Douglas fir forests. Eco- logical Applications 5(1):63 -73. Pietraface W (1988). Plant regeneration. The American Bi- ology Teacher 50(4):234 -235. Ritchie G (1997) Operational use of vegetative propagation in forestry: World overview of cloning and bulking. U S Forest Service General Technical Report PNW 0(389):192 -197. Weiss J, Nerd A, Mizrahi Y (1993) Vegetative parthenocarpy in the cactus pear Opuntia ficus -indica. Annals of Botany 72(6):521 -526. Weng Z, Xiong Z (1994) Molecular cloning and genome or- ganization of saguaro cactus carmovirus. Phytopa- thology 84(10): 1105-1106. Spirit Bird Prays for Rain Sylvester Mubayi.