Plant Tissue and Organ Culture * Walter Tulecke, Boyce Thompsoninstitute, Yonkers, New York

Plant Tissue and Organ Culture * Walter Tulecke, Boyce ThompsonInstitute, Yonkers, New York

The in vitro culture of plant tissues and sterilize the media and glassware. Ordinary organs is a useful technique for studying Pyrex test tubes and flasks can be used for plant cells and plant development. By pro- the cultures. For more details on equipment viding a means for growing a portion of an for plant tissue culture, see White (4) and intact plant on a suitable nutrient medium, Gautheret (1), but bear in mind that simple this method makes it possible to study the equipment and standard procedures for factors which influence differentiation, sterile technique are the primary require- Downloaded from http://online.ucpress.edu/abt/article-pdf/25/2/90/19331/4440218.pdf by guest on 03 October 2021 metabolism, and growth. The physical, ments. chemical, and biological conditions of an Culture media: No single medium can be experiment can be controlled and varied to suggested as suitable for all types of tissue suit a particular requirement. and organ culture. A medium which has been In general, three kinds of excised plant used for many years as a basal nutrient for parts are grown in vitro: 1) organ cultures, plant tissues (4) is now available from Difco 2) initial explants, and 3) tissue cultures. The Laboratories, Detroit 1, Michigan (TC organ cultures include such parts as flowers, Medium-White, codes 0779 to 0784). It fruits, ovules, roots, embryos, leaves, etc. should be pointed out that this medium is The initial explants usually refer to the cul- low in phosphates and other major elements, ture of special tissues such as the pith tissue contains no pantothenic acid or auxin, and of tobacco stems, the secondary phloem of has only glycine as an amino acid source. carrot roots, and the parenchyma of potato Modification of this minimal medium may tubers. The initial explants are removed from be made in various ways to suit a particular the stem, root, or tuber by using sterile tech- experimental requirement. For example, the niques and employing a sterile cork borer to major salts could be increased from 2 to 10 remove the tissue desired. The tissues are cut times, either singly or collectively, to study to uniform size from the cork boring and their effect on plant development. The basal cultured; they are used only for the duration White's medium also serves well when addi- of the experiment and then discarded. Tissue tions of coconut water (1-20%), yeast ex- cultures, by contrast, are continuously cul- tract (0.001-0.1%), casein hydrolyzate tured plant tissues which are derived from (0.001-0.5%), or other nondefined nutrient the cambium, parenchyma, or other tissues supplements are being tested for growth pro- of specific plant parts, such as leaves, stems, motion. Glass distilled or deionized water roots, or cotyledons. are recommended but are not absolutely Equipment: To assure that sterile trans- essential; ordinary distilled water can be used fer procedures may be carried out with a in many procedures. minimum of contamination (5% or less), it Growth substances, such as naphthalene- is necessary that a portion of a laboratory or acetic acid or 2, 4-dichlorophenoxyacetic a room be set aside where air movements are acid, are usually required for continued restricted. The laboratory bench should be growth of normal tissue cultures. The levels made of material which can be cleaned with commonly used are 0.01 to 10.0 ppm. The a disinfectant, such as 70% ethanol, 0.1 % optimum amount for a given tissue should mercuric chloride, or 10%ocommercial Clor- be determined experimentally. ox. A microbunsen burner (Scientific Glass Procedures for sterilizing plant material: Co., Bloomfield, New Jersey, No. B-8872) There are several principles which apply to or alcohol lamp, scalpel and forceps (or most methods of sterilizing plant materials: similar instruments) are necessary for dis- 1) the choice of sterilizing agent and the section and transfer operations. An auto- duration of use must be determined by the clave or pressure cooker is required to material; 2) all portions of the surface to be

90 PLANTTISSUE AND ORGAN CULTURE 91 sterilized must be throughly wetted; 3) the Usually one to two months are required to disinfectant should be removed from the sur- obtain sufficient callus growth for the tissue face of the material by washing with sterile to be subcultured to new media free from distilled water. the original tissue. It is important to bear in Sodium hypochlorite or mercuric chloride mind that any medium proved for tissue is most commonly used for sterilizing. Clor- growth is only an approximation of the ox, a commercial bleach, serves satisfactorily medium provided in the plant or a theoret- for many plant materials. It is usually avail- ically optimum medium. Consequently, able as a 5.25%osodium hypochlorite solu- rapid transfers of tissues would tend to over- tion and may be used at dilutions of 1 to come any medium insufficiency due to low 20%ofor 5 to 30 minutes. Ten percent Clorox levels of nutrients provided. Longer intervals for ten minutes, when used with a very small between transfers, however, have the advan- amount of wetting agent (household deter- tage that the tissue modifies or conditions the Downloaded from http://online.ucpress.edu/abt/article-pdf/25/2/90/19331/4440218.pdf by guest on 03 October 2021 gent), is generally satisfactory. Three to five medium. Until more is known about the washes with distilled water are usually conditioning factors, it is probably best to sufficient to remove the hypochlorite and transfer tissues at three-week intervals after most of the detergent. After surface steriliz- their original growth and isolation. ing the plant part, it is ready for culture or Conclusion: The methods of plant tissue for dissection of the desired tissue. and organ culture may be used for a wide Materials: One of the best materials to range of experimental studies, including begin tissue cultures with is seeds. The seeds cytological, physiological, biochemical, or may be obtained from tomatoes, grapefruit, morphogenetic problems (3). Microdissec- pumpkins, sweet corn, eggplant, or other tion, grafting, the use of radioisotopes, the fruits available at the produce market. When culture of single cells, and other procedures the fruit is opened carefully the seeds are may be applied to the plant tissues. It has also uncontaminated and may be germinated di- been shown that whole plants may be de- rectly on White's medium. The seeds can be rived from original cell cultures of higher used to set up experiments. For example, with plants, such as the carrot (2). These and pumpkin, squash, or cucumber seeds, the other studies indicate that the plant tissue primary roots of seedlings can be removed and organ culture can be used to increase to study adventitious root formation; one or our understanding and control over plant both cotyledons can be removed to study development. the effect of nutritional supplements on seedlings deprived of their natural food source; References or roots may be excised and cultured alone. With a little ingenuity, the development of 1. Gautheret, R. J. 1955. The nutrition of plant other plant organs, such as leaves, stems, or tissue cultures. Ann. Rev. Plant Physiol. 6: young ovules, may be investigated. 433-484. 2. Steward, F. C. For 1958. Growth and organized isolating tissue cultures, bean pods, development of cultured cells. III. Interpreta- rose stems, carrot roots, and potato tubers tions of the growth from free cell to carrot are good sources to start with. A medium plant. Amer. Jour. Bot. 45: 709-713. which will support the growth of tissue cul- 3. Tulecke, Walter. 1961. Recent progress and tures from these plants consists of White's the goals of plant tissue culture. Bull. Torrey Bot. Club. 88: 350-360. basal medium, 10% coconut water and 1.0 4. White, P. R. 1943. A Handbook of Plant Tis- ppm of 2- 4-dichlorophenoxyacetic acid. sue Culture. The Ronald Press Co. New York.