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Chapter 3D
Application of Tissue Culture Systems for
Commercial Plant Production 1
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.
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
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.
236
~~, ~·iJ
LABORATORY
ttHJ
\I
I
4w,Ks
-~
\ "----~
.·
4 WEEKS
~
Figure 1. Representation of a traditional shoot-tip/axillary
bud culture method used at Minn vitro, Inc.
Application of Tissue Culture Systems for Commercial Plant Production
solution (10 percent for 10 min), followed by 3 sterile water rinses. Explants are asep tically 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 micros hoots 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 h arves ted 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 axillary 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.
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 initi ates and
develops adventitious buds that form shoots,
followed by rooting. For details of this method,
see Louis and Eils (1994).
237
Section V Biotechnological Applications
tissue cultured plantlets are used. Plantlets 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 characte ris 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 tha t naturally propagate in this manne r (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
wa ter, followed by a bleach trea tment (full-strength for 10
min), and a tap-wate r rinse. Root segments are then buried in sand and maintained unde r moist conditions. Within
2 to 4 weeks, shoots develop from preformed and adventitious buds within the roots. When the shoots a re 4 to 6
em in length, they are removed from the parent root segme nt and trea ted as mini-cuttings (as previous ly described ). Papers by Schier (1974, 1976, 1981) provide more
detail regarding preformed and adventitious root-d e rived
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 ve rsion of this method was d eveloped at
the University of Minnesota (Hanson e t al. 1992; Louis
et al. 1992a, 1992b) in which in vitro g rowing shoots
were treated to induce roots (i.e., BA was rem oved from
the medium). These in vitro roots were the n 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 s uckering
238
has been used to clone some aspen genotypes, to our
knowled ge, the use of in vitro root suckering as a comme rcia 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 pe rcent sucrose, 0.8 mg/1 BA, and
0.01 mg/1a-naphthaleneacetic acid (NAA). Afte r 4 weeks,
adventitious buds begin to form. For microshoot d evelopment, 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 ad ventitious 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 s ubs trate is used . Five "offthe-shelf" subs trates can be used to replace agar: 1) cotton balls; 2) cotton cosmetic rounds; 3) Grod an®rock wool;
4) lsolite®soil amendment; and 5) Sorbarod®cigarette filters. This has led to the envisioned MCS Technology illustrated in fig ure 2. The results of our continued effort to
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Application of Tissue Culture Systems for Commercial Plant Production
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 10times 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.
Acknowledgments
This research was supported in part by the U.S. Department of Agriculture under Grant No. SBIR, Phase II 9233610 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;
239
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.
240
Schier, G.A. 1981. Physiological research on adventitious
shoot development in aspen roots. Gen. Tech. Rep. INT107. 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.