A Practical Guide to Woody Plant Micropropagation John W Einset

BOTANY: THE STATE OF THE ART

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 acid) can be added operation. separately or, alternatively, in preformulated Of course, the key component of the mixes. At the Arnold Arboretum, we use a medium is the phytohormone; specifically, formulation called "Murashige’s Mmimal when micropropagation mvolves shoot mul- Organics Medium" (actually a misnomer), tiplication, the cytokinin. Although over 200 which contams sucrose, the vitamins thia- different cytokinins are available, they all mine and i-mositol, and MS salts, all in the seem to have similar effects on plants, so it proper proportions. After dissolving this is usually only necessary to test a few com- mixture in the appropriate volume of deion- pounds to find an effective cytokinin. Almost

Figure 3 The Tissue Culture Rooms at Nourse Farm m Whateley, Massachusetts. Nearly 500,000 strawberry and raspberry plants are produced annually by micropropagation at Nourse Farm 40

all plants, for example, respond well to a kinin in these plants. medium containing the basal components Once all nutrients have been incorporated plus 1 milligram per liter (mg/1) to 5 mg/1 of into the medium, the mixture is supple- the cytokinin N6-benzyladenine (abbreviat- mented with 1 percent agar and then heated ed BAP, BA, or, preferably, bzl6Ade). (One mg/ to dissolve the agar, and then the medium is I is equal to one part per million [ppm].) dispensed into the culturing container. Prac- Likewise, N6-isopentenyladenine (2iP or tically any type of container can be used, the i6Ade), kinetin, and thidiazuron are usually only requirements being that it permit light also effective as cytokinins, though bzl6Ade to enter, provide for ventilation, and not be is generally the best choice. A curious excep- destroyed by the heat involved in steriliza- tion to this rule involves ericaceous species tion. At the Arnold Arboretum, we use glass such as rhododendrons, azaleas, and kiwi- test tubes with plastic caps, but have used fruits, which respond poorly, if at all, to baby-food jars, canning jars, and even kitchen bzl~Ade but exhibit extensive shoot prolif- cooking pans. eration with i6Ade. Obviously, there is some- The agar provides an inert, jelly-like sup- thing unique about the biochemistry of cyto- port that prevents the plant tissues from

Figure 4. Subdmdmg Clusters of Shoots and Planting Indmdual Shoots on Fresh Cytokmm-Contammg Medium under Stenle Conditions. This procedure is carned out after each cycle of shoot muluphcation 41

sinking to the bottom of the culture vessel be uniformly distributed throughout the and suffocating from lack of oxygen. To most medium. Next, this very hot, agar-contain- tissue culturists, however, agar (several dif- ing medium needs to be dispensed into the ferent brands are available) is one of the most culture vessels both accurately and quickly, troublesome aspects of micropropagation. before the agar solidifies. If all has gone well As a way of illustrating some of the problems to this point, the culture vessels can now be involved, consider that, first, it is necessary sterilized (for 15 minutes at 120 C in an auto- to dissolve the agar by heating so that it will clave, or for 30 minutes in a pressure cooker) and, after cooling, used for tissue culture. Unfortunately, this usually is not the end of Table 1. Chemical Constituents of a Standard Tis- problems with agar because, after growth is sue-Culture Medium Used at the Arnold Arbor- completed and the plant tissues have been etum for the of Several Micropropagation Woody it becomes to redissolve Species by Shoot Multiplication removed, necessary the agar so that the used medium can be Based largely on research conducted at the Um- discarded-not by pouring it down the sink, Wisconsin m the 1960s F. versity of early by however, as the agar will gel and plug the Skoog, T Murashige, and E. M. Linsmaler-Bednar. drain!

Explants for Shoot Multiplication By far the best starting material for micro- ’ propagation is a growing stem tip from a vigorous, healthy plant. Although seedlings are usually better sources for tips than mature specimens, the disadvantage of using seedlings is that the characteristics of the resulting adult plants are unpredictable. On the other hand, when shoot explants are taken from mature plants, one can be fairly certain that the individuals produced by micropropagation will be identical (that is, "clonal") to the stock plant. Optimally, shoot tips are collected during the early flush of vegetative growth in spring rather than during summer, when growth has ceased, or during fall, when buds have entered their dormant period. The size of the explant depends on the objective of the micropropagation procedure. If the goal is to use micropropagation to obtain virus-free plants, for example, it is usually necessary to excise only the terminal millimeter of the growing pomt, to clean it, and then to plant this tissue onto the nutrient medium. Unfor- tunately, these manipulations require con- 42

siderable manual skill and, therefore, the cation. On the average, one can expect probability of success when small explants approximately a fivefold increase in shoot are used is quite low. Because of this, if the number every six weeks, a rate that theoret- goal is solely clonal multiplication, it is eas- ically would produce more than a million ier to begin with shoot tips that are 0.5 to 1 shoots, starting from a smgle tip, withm 12 centimeter long. months. After they have been collected from the stock plant, the tips need to be disinfected Rooting and Hardening thoroughly, to remove all traces of microbial contamination. At the Arnold Arboretum, Probably because they develop in the humid we normally wash explants in detergent and environment of the culture vessel and there- then rinse them under tap water. Since these fore have leaves that lack a protective cuticle, steps effectively clean the shoot tips of nearly shoots produced through micropropagation all bacteria and fungal spores, the few are particularly sensitive to desiccation. It is remaining microorganisms can be killed essential, therefore, that they be maintained simply by a soak in 1/10-strength household under moist conditions during Stage III. Sev- bleach (the active ingredient being sodium eral strategies have been used to accomplish hypochlorite) for 2 to 15 minutes. Tips from this. In our laboratory, for instance, after dip- most plants can withstand a 10-minute ping the bases of micropropagated shoots in bleach treatment, although some tissues are a rooting powder, we transfer them to a very tender and will brown and die under humid plastic box containing moist vermi- these conditions. For this reason, it is best to culite and a transparent cover. On the other experiment with different times for the hand, at Weston Nurseries in Hopkinton, hypochlorite treatment when a new plant is Massachusetts, tissue-culture shoots of being used for micropropagation. mountain laurels, rhododendrons, and aza- After they have been treated with hypo- leas are planted in the greenhouse, in beds of chlorite, tips are transferred to sterile petri moist peat moss covered with polyethylene plates and a fresh cut is made at the base of tents to maintain a humid environment. At each explant. The tips are then planted in Nourse Farms in Whately, Massachusetts, nutrient medium with sterile forceps, and strawberry shoots are planted in a moist peat the cultures are incubated under light and moss-soil mix, in a greenhouse equipped temperature conditions that promote the with a fogger-type humidifier. optimal multiplication of shoots. We use If the appropriate treatments are used, artificial lighting recommended for house- roots usually form on tissue-cultured cut- plants and normal room temperature (75 F, tings within about two weeks. Once rooting or 24 C). has occurred, the resulting plantlets begin to Growth normally becomes apparent after grow vigorously, and the gradual process of one to two weeks. Within about six weeks, hardening them to lower humidities and it is usually necessary to subdivide the higher light intensities can take place. With resulting shoot cluster and to use individual lilacs, for example, we incubate our covered, branches for further shoot multiplication on plastic boxes in the culture room for two fresh medium or for plantlet production fol- weeks while roots are being initiated, and lowing a rooting treatment. Of course, the then we remove the covers from the boxes. frequency of subculturing varies from spe- Two weeks later, we transfer the boxes from cies to species, as does the rate of multipli- the culture room to the greenhouse, where 43

they are kept initially under shade for four plants are common. For some purposes this weeks and then in full sun. By this time, the is desirable and, in fact, the variability that micropropagated plantlets are hardened can be produced in tissue cultures is already enough to be handled as any other plant being exploited commercially to obtain dis- would be. ease-resistant potato cultivars. Often, plants produced by micropropagation are considerably more than vigorous The Economics of Micropropagation conventionally propagated plants. This is hardly surprising in view of the optimal con- For about $250, one can purchase practically ditions of nutrient supply, moisture, and all of the supplies needed to set up a micro- lighting under which they are grown. Geneti- propagation laboratory. This price includes cally, micropropagated plants are identical to enough Murashige’s Minimal Organics their stock plants, at least as long as shoot Medium, vitamins, cytokinins, and agar for multiplication is used to produce them. If, 2,500 cultures ($100), 500 sterile plastic petri however, the micropropagation method dishes plus covers ($50), 250 culture tubes involves either regeneration from callus or with plastic caps ($50), flasks and beakers for somatic embryogenesis, then variant (mutant) media preparation, and stainless-steel for-

Figure 5 The Rapid Mulriphcation of Raspberry Shoots by Micropropagation 44

ceps, scalpels, and blades. In addition to Commercial tissue-culture laboratories these items, one needs to be able to heat a estimate that they spend approximately $0.50 medium (on a hot plate, for example) so that for each plant they produce beyond Stage III all of its components will be dissolved before of the micropropagation process. This esti- it is dispensed to the culture tubes, and to mate is calculated based on a minimum level sterilize a medium (with a pressure cooker, of production (about 250,000 plants per year), for example). Forceps and scalpels can be at which several economies of scale become sterilized simply by dipping them in 95 per- significant. In addition, the estimate fails to cent alcohol and then burning the alcohol take into account expenses involved in devel- with a flame. A balance is also needed, unless oping refined technology for a new plant. one purchases chemicals already preweighed Because of this, a newcomer to micropropa- into lots. Of course, a transfer bench is gation will probably find that the costs per required free from drafts, as is an artificially plant are much higher. lighted incubation room equipped with racks Research in universities and private firms for cultures. during the last few years has resulted in a rapidly expanding catalog of information about the precise conditions of Stages I, II, and III for over 200 different plant species. Unfortunately, this information is not always presented in a style that is comprehensible to the beginner. Even more serious is the ten- dency of some commercial laboratories to explain micropropagation in an almost mystical, surrealistic fashion when, in fact, the technology involves a principle (phytohormone control of development) that is both simple and straightforward. If you are genuinely interested in micropropagation, remember that patience, flexibility, and con- fidence in the scientific basis of the methodology are the most important requirements success. for _

fohn W Emset, associate professor of biology m Harvard University, is a member of the staff of the Arnold Arboretum.

Figure 6 A Micropropagator at Nourse Farm Subdmd- mg Shoots before ’Iransfernng Them to Soil m the Greenhouse (Shoots produced by tissue culture can either be used for further shoot multiplication, or they can be rootedJ