BOTANY: THE STATE OF THE ART A Practical Guide to Woody Plant Micropropagation John W Einset The spinoff from basic research on the physiology of plants, plant micropropagation is a simple, straightforward-and commercially profitable- technique Although its commercial use as a method for plant-tissue-culture technology in the world multiplying plants is still fairly new, tissue- today. culture propagation (micropropagation) has Micropropagation is also becoming very already had a significant impact on the way important for woody plants, although the people think about and handle plants. The scale of this enterprise is minor compared to example of pyrethrum (Chrysanthemum the pyrethrum industry. At the present time cinerariaefolium) is especially striking. Dur- micropropagation is utilized commonly for ing the last 15 years this plant has been species in two families, the Rosaceae (roses, exploited extensively as a source of chemi- apples, raspberries, and strawberries) and the cals, known as pyrethrins, that are used as Ericaceae (rhododendrons, azaleas, and "natural" insecticides. In fact, it is estimated mountain laurels). While it is not yet clear that over 150 million pyrethrum flowers are that the technology will be feasible with all harvested every day of the year in East Africa woody species, the prospects are very prom- or Ecuador for the production of pyrethrin ising for several of them. Because of this, the insecticides. Given the size of the industry most spectacular applications for micropro- (50 billion flowers per year), it is obvious why pagation are undoubtedly still in the future. micropropagation is being used for pyreth- As a technique, micropropagation repre- rum. After all, the technology enables grow- sents a direct, practical extension of scien- ers to obtain rapid, clonal mulitiplication of tific methodology devised over 30 years ago plants that produce exceptionally high con- to study fundamental aspects of plant phys- centrations of pyrethrins. With this capabil- iology, especially the role of phytohormones ity, yearly increases in superior plants equiv- in growth and development. Essentially, alent to one million-fold multiplication are micropropagation takes advantage of the obtained, and the total level of pyrethrin pro- control of plant development that can be duction is increased significantly. As a mat- exerted by phytohormone treatments. Thus, ter of fact, the pyrethrum example probably although tissue-culture media contain over represents the single most important use of 20 different chemical constituents, and in 37 spite of the fact that environmental factors added to the medium, especially the relative such as light intensity and temperature need levels of cytokinin and auxin. Thus, high to be carefully monitored, the crucial varia- cytokinin-to-auxin concentrations result in ble in micropropagation is the phytohor- shoot formation from callus, low ratios result mone content of the medium. (An article on m root formation, while intermediate ratios "Chemicals That Regulate Plants," which result in continued callus proliferation. appeared in the Spring 1985 issue of Arnol- Spectacular as this classic demonstration of dia, discusses other practical uses of phyto- plant developmental control is, there are sur- hormones.) prismgly few plant species that respond in tissue cultures as tobacco does. Even though callus can be from The Three Methods of Micropropagation produced practically any plant, the ability of these calluses to form Depending on the plant, micropropagation shoots and roots in response to phytohor- involves one of three possible strategies: (1)/ mone treatments is rare. regeneration from callus, (2) somatic ~ By contrast, somatic embryogenesis has embryogenesis (embryo formation from veg- already been utilized for species in over 25 etative cells), or (3) shoot multiplication. different families. Like regeneration from ~ Regeneration from callus was demon- callus, somatic embryogenesis involves an strated first in the early 1950s by Professors initial stage of callus formation, in this case F. K. Skoog and C. O. Miller, codiscoverers of using a medium containing auxin as the only the cytokinin class of phytohormones, both phytohormone. The callus is then recultured of whom were working at that time at the on a medium lacking phytohormone or on University of Wisconsin. These investigators medium with cytokinin. Often, several suc- showed that stem segments taken from tobacco plants will proliferate an unorga- nized mass of tissue, known as callus, when placed on a nutrient medium containing cytokinin and auxin. If the callus is then subdivided into smaller pieces and these are placed on fresh media, growth will continue. Significantly, the type of growth depends on the kinds and quantities of phytohormones Figure 2. The Chemical Structures of Some Cytokinins. Figure1 Steps m the Micropropagation of Woody Chemically, naturally occurnng cytokmms are consid- Plants Woody-plant micropropagation mvolves a shoot- ered to be denvatmes of adenme, a basic bmldmg block muluphcation cycle usmg controlled cytokmm treat- of several important plant consutuents Thidiazuron, a ments and a senes of treatments to cause the rootmg of synthetic cytokmm that has been shown to be effectme cuttings and the hardenmg of plantlets m micropropagation, is a phenylurea cytokmm. 38 cessive passages are required before true that ultimately can give rise to plants. Often embryos are formed. The technique, there- in shoot multiplication, explants respond fore, depends in large part on the finesse of almost immediately to the high cytokinin the tissue culturist, a skill demonstrated first concentration of the medium by proliferat- by Professor F. C. Steward of Cornell Univer- ing new shoots. In these cases, Stage II shoot sity, who was able to obtain somatic embryos multiplication has the potential of producing from carrot tissue cultures during the late one million shoots in a year, starting from a 1950s. single growing tip. O The third technique, shoot multiplica- ~ Stage III involves all the manipulations tion, can almost be considered as the "stan- required for establishment of tissue-culture- dard methodology" as far as woody plant derived plants in soil. If, for instance, shoot micropropagation is concerned. Exploited multiplication is used for Stage II, then Stage especially by Professor T. Murashige of the III technology consists of a rooting treatment University of California at Riverside, who that produces plantlets and then a gradual was involved in the early development of this process of acclimation (hardening) of these technology for propagation, the method starts plantlets to the lower humidity and increased with a growing shoot tip and uses media with light intensity of the greenhouse or outdoor high cytokinin concentrations to promote environment. Depending on the tenderness growth and to overcome apical dominance. of the plantlets obtained from tissue culture, The result of this treatment is the produc- hardening may last two to eight weeks. tion of a branched shoot system, which is Individual shoots are then used - subdivided. The Medium for further shoot multiplication, or they are rooted. A surpnsingly large number of nutrients are needed by tissue cultures, at least in com- parison to the requirements of whole plants. The Stages of Micropropagation Thus, in addition to the expected inorganic According to Professor Murashige, all meth- (mineral) nutrients, media for tissue cultures ods of plant micropropagation involve three need to contain sugar (e.g., sucrose, or cane basic types of manipulations, designated as sugar), at least two vitamins, and one or more Stage I, Stage II, and Stage III. phytohormones. Presumably, whole plants LJ During Stage I, establishment of the generate all of these additional nutrients aseptic culture, an "explant" (part of a stock internally, although their production must plant) is cleaned, disinfected, and placed on be restricted to specific tissues. In fact, it is a tissue-culture medium. The objective of likely that localized vitamin and phytohor- Stage I is to obtain a living and growing plant mone synthesis is an important mechanism tissue free from microbial contamination. coordinating growth and function within Surprising as it may seem, this goal is usually plants. the most difficult thing to achieve in micro- Usually, inorganic nutrients are added to propagation. media as a standard mixture of salts. The so- ~ Stage II, also known as the stage of pro- called "Murashige and Skoog salts" ("MS pagule multiplication, sometimes coincides salts"), for example, contain about 15 differ- with Stage I, especially when shoot multi- ent salts, carefully formulated into a mixture plication is used. The aim of Stage II is the that furnishes all of the inorganic require- rapid increase in shoots or other structures ments of tissue cultures, e.g., nitrogen (N), 39 phosphorus (P), potassium (K), and sulfur (S). ized water, we add pyridoxine and nicotinic Even though the MS salts mixture was origi- acid to complete the basal medium. Most nally devised for tobacco tissue cultures, commercial nurseries, on the other hand, experience has shown that it is adequate for prefer to add every component, mcluding most other plants, at least during initial each of the MS salts, separately. As so often attempts at micropropagation. happens, the scale at which one is working Sucrose and vitamms (thiamine, i-mositol, determines the most economical method of pyndoxine, and nicotinic