Chapter 3D

Application of Tissue Culture Systems for Commercial Plant Production1

Kathryn A. Louis and LoriAnn E. Eils

Introduction

Tissue culture propagation or micropropagation in the commercial production of Populus species for large scale clonal plantings has limited use due primarily to the high cost per plantlet, especially when compared to species that are readily cloned using cutting propagation. Howe,·er, two more practical applications of tissue culture propaga­tion of Populus species are: 1) propagation of those species that do not readily propagate via other less costly meth­ods (e.g., aspen and hybrid aspen); and 2) multiplying stock plants of newly developed or released genotypes with lim­ited availability. This chapter discusses methods used to clone Populus species including: 1) traditional shoot-tip/ axillary bud culture; 2) recutting or hedging mini-plants; 3) root suckering in vitro and in situ; and 4) leaf micro-cross section (MCS) technology.

Traditional Shoot-Tip/ Axillary Bud Culture

An excellent bibliography about tissue culture and cell culture of Populus species was compiled by the USDA For­est Service (Ostry and Ward 1991). Readers are encour­aged to review papers listed in that document. A brief review of the method used at Minn vitro, Inc. is described below and illustrated in figure 1.

' Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. Dillon, M.C.; Carman, R.C. ; Eskew, L.G., tech. eds. 1997. Micropropagation, genetic engineering, and molecular biology of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p.

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Stock plants, which provide explants, are obtained as small plants or dormant branches. Plants are grown in­doors under a cool-white, fluorescent (CWF) photoperiod of 16 h until new shoot growth occurs. Dormant branches are stripped of existing leaves, and proximal branch ends are recut and placed in water under a 16 h photoperiod. Branch ends are recut every 2 to 3 days at the time of wa­ter replacement. After 2 to 4 weeks, new forced growth occurs. In Minnesota, dormant branches can be success­fully forced beginning in March through normal bud break (early May) and again in August through early Novem­ber. Explant material is not usually field collected because of difficulties eliminating fungal spores and other contami­nants on field-grown material.

To initiate Populus species cultures, shoot-tip stem cut­tings (approximately 2.5 to 7.5 em long) are collected from actively growing plants or forced branches. Existing leaves are removed and stems with terminal and axillary buds are surface disinfested using a standard commercial bleach

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bud culture method used at Minn vitro, Inc.

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solution (10 percent for 10 min), followed by 3 sterile wa­ter rinses. Explants are aseptically cultured on Murashige and Skoog (MS) (1962) basa l medium supplemented with 3 percent sucrose, 0.1 to 0.5 mgl I benzyladenine (BA), and 5 g/1 agar with a preautoclave pH of 5.75. The culture cycle is 4 weeks with a CWF photoperiod of 16 h. High BAcon­centrations are used for multiple shoot production during the proliferation cycles, followed by reduced BA concen­trations for shoot development before harvest. Although most literature reports the use of Woody Plant Medium (WPM) (Lloyd and McCown 1980) as the basal medium, we have found that MS medium prod uces superior microshoots (data not shown). During the process of har­vesting microshoots from the culture vessel, small microshoots (less than 2.5 em) and proliferating clusters are transferred to fresh medium for later harvest.

For ex vitro rooting, microshoots that are 2.5 em or longer are harvested a nd placed in a standard 288 seed germination plug covered tray (trimmed to fit into a standard 1020 flat). The plug tray and fl at are covered with a clea r dome and placed under a CWF 16 h photo­period. The plug tray is filled with a rooting medium composed of peat:perlite:vermiculite (1:1:1 ). All preformed in vitro roots are removed before ex vitro rooting. (In our experience, in vitro formed roots of Populus species did not survive after transplanting into peat-based rooting me­dium. New adventitious roots developed at a slower rate than from microshoots without p reformed roots.) Visible roots usually form within 10 days. After 3 weeks of root­ing, we recommend tha t weekly fertilization begin at 200 ppm nitrogen, using a 20:20:20 (N:P:K) formulation. At ap­proximately 4 weeks, acclimation is accomplished by prop­ping open the clear dome slightly for the first day, then increasing the opening each day, until day 7 when the dome is completely removed. Plantlets are then transplanted to larger containers and placed in the greenhouse or nursery bed with 70 percent shade for the first few days. The amount of shade is gradually decreased over a 7-day pe­riod, then fertilizer applications can be doubled.

This traditional shoot-tip I axillary bud culture procedure is based on a plant biology that allows existing shoot tips and axillary buds to develop and continue initiation of new shoot tips and axi llary buds. These shoot tips and axillary buds then elonga te to produce microshoots. This cycle can be repeated indefinitely, provided that transfers occur in a timely manner. Reducing the detrimental effects of sys­temic bacteria that can become evident over time can be accomplished by a more rapid transfer cycle (every 2 to 3 weeks), using only shoot tips (1 to 2 em), and I or the addi­tion of an tibiotics to the medium. (Based on research by Young et a!. (1984) and our in-house research, we found that a combination of 25 mg/1 cefotaxime, 25 mgl l tetra­cycline, and 6 mg/1 rifampicin will successfully suppress bacterial growth in mos t Populus species cultures.) Other problems are low tooting rates and poor acclimation for

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

Application of Tissue Culture Systems for Commercial Plant Production

some genotypes. Our overall success rate has been that ap­proximately one-third of the genotypes are readily cloned, one-third of the genotypes are cloned with difficulty, and one-third of the genotypes are not amenable to this method.

Recutting or Hedging of Plantlets

Although this method is not an in vitro method, it is worth mentioning because it is often very successful when

MEDIA 'L-------------'-------' EXCHANGE

c

• .

. .

MICROSECTION BUDS SHOOTS ROOTS

Figure 2. Schematic representation of Micro-Cross Section (MCS) Technology (courtesy of Minn vitro, Inc.). A) Leaf tissue is placed on a tape carrier and sliced into strips. B) The leaf tape strip is placed on a support substrate. The culture medium is liquid (no agar) and the culture vessel has flow-through medium ex­change as required. No physical transfer of the explant is required. C) Leaf explant initiates and develops adventitious buds that form shoots, followed by rooting. For details of this method, see Louis and Eils (1994).

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Section V Biotechnological Applications

tissue cultured plantlets are used. Plantle ts that arc pro­duced as previously described can be grown in a green­house or growth chamber until their main shoots are 8 to 10 em in height. Cuttings are then collected from these mini-stock plants and rooted as previously d escribed. The remaining portions of the mini-stock plants continue to grow via axillary shoots, which can also be used as mini­cuttings once the axillary shoots are 6 to 8 em in length.

Mini-cuttings produced in this manner usually root at a higher percentage than those from tissue culture (data not shown). A likely reason for this rooting increase is that mini-cuttings have a more developed cuticle. Thus, al­though mini-cuttings are less fragile, they apparently main­tain the juve nile characteris tics of tiss ue-c uI tu red microcuttings. This hedging me thod can be successfully performed for several flushes.

Root Suckering

Cloning Populus species using root suckers has been successful for species that naturally propagate in this man­ner (e.g., P. tremuloides). The field I greenhouse method be­gins with digging up roots (1 to 3 em in diameter and 15 to 20 em in length). These roots are cleaned with soa p and water, followed by a bleach trea tment (full-strength for 10 min), and a tap-water rinse. Root segments are then bur­ied in sand and maintained under moist conditions. Within 2 to 4 weeks, shoots develop from preformed and adven­titious buds within the roots. When the shoots are 4 to 6 em in length, they are removed from the parent root seg­ment and trea ted as mini-cuttings (as previous ly de­scribed ). Papers by Schier (1974, 1976, 1981) provide more detai l regarding preformed and adventitious root-derived buds. In our work, this method seems most successful in the spring and is apparently limited by the inherent suckering ability of the parent plant. A major problem is correctly identifying which roots belong to what tree. This p roblem can be alleviated by cloning the identified plant with the usual methods (e.g., cuttings, tissue culture, etc.) then growing the cloned plants in large containers unti l roots of sufficient size are obtained.

An in vitro version of this method was developed at the University of Minnesota (Hanson e t al. 1992; Louis et al. 1992a, 1992b) in which in vitro growing shoots were treated to induce roots (i.e., BA was removed from the medium). These in vitro roots were then cut into 0.5 em or longer segments and returned to culture. Adventi­tious shoots were initiated and developed from the root explants cultured on MS medium supplemented with 0.01 to 1.0 mg /1 thidiazuron (TDZ). Our attempts to produce large qualities of roots by g rowing roots w ithout s tem tis­sue were unsuccessful. Although in situ root suckering

238

has been used to clone some aspen genotypes, to our knowled ge, the use of in vitro root suckering as a com­mercia l method to clone plants has not yet occurred.

Micro-cross Section Technology

Micro-cross section (MCS) Technology is a method used to clone plants based on adventitious bud initia­tion and d evelopment from leaf tissue. This method was initia lly d eveloped by research~rs at the Univers ity of Minnesota (Lee-Stadelmann et al. 1989). Very small (400 J.lm to 3 mm) leaf segments are placed onto MS medium supplemented with 3 percent sucrose, 0.8 mg/1 BA, and 0.01 mg/1 a-naphthaleneacetic acid (NAA). After 4 weeks, adventitious buds begin to form. For microshoot develop­ment, the explant is transferred to medium supplemented with a reduced BA concentration (0.1 mg / 1) and without

AA. As microshoots develop, they are harvested and rooted as previously described . Because very small pieces of lea f tissue can be used, a large number of explants can be generated for adventitious shoot production. Thus, a proliferation stage is hardly needed and a large number of plants can be produced in a short time.

The uniform size and shape of the explants makes this process amenable to robotics. Continued refinement of this technique along with development of automated handling equipment would probably close the cos t gap be tween tissue culture propagation and seedling propagation. Ad­ditionally, the semi-solid medium can be replaced with a. liquid medium if a support subs trate is used . Five "off­the-shelf" subs trates can be used to replace agar: 1) cot­ton balls; 2) cotton cosmetic rounds; 3) Grodan® rock wool; 4) lsolite® soil amendment; and 5) Sorbarod® cigarette fil­ters. This has led to the envisioned MCS Technology il­lustrated in figure 2. The results of our continued effort to

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

develop MCS Technology are summarized in a TAPPI Bio­logical Symposium paper (Louis and Eils 1994). Forty-five different Populus genotypes were tested, of which P. tremuloides x P. tremula (hybrid aspen) genotypes were the most responsive. These results are most promising because hybrid aspen plants do not readily propagate using other asexual methods.

The first commercial trial of MCS Technology was a 10-times scale-up of our research size (i.e., 10 times our stan­dard 100 micro-cross sections), which was replicated for a total of 2,000 micro-cross sections. This scale-up resulted in reduced microshoot numbers per leaf section and ex­tensive fungal growth during acclimation (data not shown). This reduced production was likely caused by using leaves of a less than optimal developmental stage. Fungal growth during acclimation was apparently caused by the residual medium in the liquid support substrates. Even though the substrates were flushed with water be­fore acclimation, adequate sucrose/nutrients apparently remained to support fungal growth.

Discussion

Populus species tissue culture propagation can be clas­sified as either preformed (terminal or axillary) or ad ven­titious meristem culture methods. Preformed meristem culture is the traditional method in which shoot tips with existing meristems are grown in an environment that en­hances the production of more meristems at a much faster rate than occurs on a whole-plant basis.

Adventitious bud initiation and subsequent development of microshoots captures the plant's totipotent biology. For example, using in vitro root suckering and MCS Technology, a cell, or a small group of cells, divides and differentiates into a new meristem that subsequently develops into a microshoot. Adventitious bud initiation has been used only on a research basis at our laboratory. MCS Technology has shown the greatest potential for producing hybrid aspen at a lower cost than traditional shoot-tip culture.

The most commercially successful method is the tradi­tional shoot-tip I axillary bud method. We have used this method to mass produce 45,000 hybrid aspen for a pilot field planting and 10,000 aspen for horticultural use. We have also used traditional.shoot-tip culture to clone unique or rare plants to increase stock plant numbers for cutting propagation.

Generally, regardless of the tissue culture propagation method used, differences occur among Populus species and genotypes within a species. Our results have followed the one-third:one-third:one-third "rule" of success; approxi­mately one-third worked very well, one-third worked moderately well, and one-third did not work at all.

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

Application of Tissue Culture Systems for Commercial Plant Production

Acknowledgments

This research was supported in part by the U.S. Depart­ment of Agriculture under Grant No. SBIR, Phase II 92-33610 and by funding approved by the Minnesota Legislature ML 1991, Chapter 254, Art. 1, Sec. 14, Subd. 7g as recommended by the Legislative Commission on Min­nesota Resources from the Minnesota Future Resources Fund.

The authors thank M. Ostry, D. Skilling, K. Ward, and B. Bucciarelli (USDA Forest Service); N. Anderson, W. Hackett, W. Johnson, and C. Mohn (University of Minne­sota); G. Wyckoff and B. Li (University of Minnesota, Aspen-Larch Cooperative); B. Pajala (Minnesota Dept. of Natural Resources); R. Schantz-Hansen, R. Settergren, L. Hubbel, M. Fasteland, and J. Eaton (Potlatch Corporation); J. McCoy and L.C. Peterson (Blandin Paper Company); K. Wearstler and C. Wierman (Boise Cascade); D. Ostlie (En­ergy Performance Systems); and G. Larson and D. Langseth

. (WesMin RC&D) for their generosity in supplying plant material for our studies, and G. Betts and B. Berguson (NRRI) for technical and engineering assistance.

Literature Cited

Hanson, C. V.; Hackett, W.P.; Mohn, C.A.; Louis, K.A. 1992. Growth of attached and isolated roots of aspen. In: Pro­ceedings, 12th North American forest biology workshop; 1992 August 17-20; Sault Ste. Marie, Ontario, Canada: 79.

Lee-Stadelmann, O.Y.; Lee, S.W.; Hackett, W.P.; Read, P.E. 1989. The formation of adventitious buds in vitro on micro-cross sections of hybrid Populus leaf midveins. Plant Science. 61: 263-272.

Lloyd, G.; McCown, B. 1980. Commercially feasible micropropagation of mountain laurel, Kalmia latitulia, by use of shoot-tip culture. Comb. Proc. Int. Plant Prop. Soc. 30: 421-427.

Louis, K.A.; Hanson, C. V.; Hackett, W.P.; Mohn, C. A. 1992a. In vitro root suckering of aspen (Populus tremuloides ). Proceedings, International Plant Propagators' Society: 42: 472-475.

Louis, K.A.; Mohn, C.A.; Hackett, W.P.; Hanson, C.V.1992b. In vitro adventitious shoot initiation and development from roots of two aspen (Populus tremuloides) clones. In: Proceedings, 12th North American forest biology work­shop; 1992 August 17-20; Sault Ste. Marie, Ontario, Canada: 78.

Louis, K.A.; Eils, L. 1994. Propagation of Populus tremuloides x P. tremula via leaf micro-cross section technology. In: Proceedings, TAPPI Biological sciences symposium;

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Section V Biotechnological Applications

1994 October 3-6; Minneapolis, MN, U.S.A. Atlanta, GA, U.S.A.: TAPPI Press: 41-45.

Murashige, T.; Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.

Ostry, M.E.; Ward, K.T. 1991. Bibliography of Populus spe­cies cell and tissue culture. Gen. Tech. Rep. NC-146. St. Paul, MN, U.S.A.: U.S. Dept. of Agriculture, Forest Ser­vice, North Central Forest Experiment Station. 26 p.

Schier, G.A.1974. Vegetative propagation of aspen: clonal variation in suckering from root cuttings and in root­ing of sucker cuttings. Can. J. For. Res. 3: 459-461.

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Schier, G.A. 1981. Physiological research on adventitious shoot development in aspen roots. Gen. Tech. Rep. INT-107. Logan, UT, U.S.A.: U.S. Dept. of Agriculture, For­est Service, Intermountain Forest and Range Experiment Station. 12 p.

Schier, G.A.; Campbell, R.B. 1976. Differences among Populus species in ability to form adventitious shoots and roots. Can. J. For. Res. 6: 253-261.

Young, P.M.; Hutchins, A.S.; Canfield, M.L. 1984. Use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters. 34: 203-209.

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.