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Chapter 1
Poplar Shoot Cultures: Their Generation and
Use in Biotechnology1
Brent H. McCown
Introduction
Although most biotechnologists would welcome an opportunity to transform a plant, such as poplar, without resorting to in vitro techniqu es (microcu lture), currently
microculture is central to any genetic engineering protocol commonly used for plants. Functional microculture
methods are more essential to tree biotechnology than to
annual crop biotechnology. Unlike annual crops where
seed-based technologies can be used for various aspects
of biotechnology, many tree selections are don ally propagated. Thus, access to uniform and responsive tissues can
most readily be achieved only through in vitro culture. Although the need for microculture is highest in tree biotechnology, optimizing such procedures for wood y
perennials is among the most challenging of any in vitro
work.
Fortunately, a microculture tool that is relatively easy to
perfect and is useful for various aspects of tree biotechnology is shoot culture (figure 1A). In this chapter, a shoot
culture is defined as an in vitro culture that: 1) is derived
from shoot explants that contain at least 1 preformed meristem (shoot-tip or node); new shoots are derived solely
from preformed meristems (apical or nodal) and adventitious meristem generation is avoided; and 2) remains actively growing through most or all of the culture cycle; the
culture can be maintained indefinitely by subculturing
shoot explants. Since poplar has become the model species for tree biotechnology, detailed aspects of poplar microculture have been extensively reviewed (Ahuja 1987;
Chun 1993; Douglas 1986; Ernst 1993). This chapter examines the various uses of shoot cultures to complement tree
' 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.
biotechnological efforts and summarizes major factors and
challenges involved in establishing shoot cultures of trees,
especially poplar.
Use of Shoot Cultures in
Tree Biotechnology
The responsiveness, stability, and reproducibility of
shoot cultures are particularly suitable for tree biotechnology. However, the complex physiological growth cycles
of woody perennials is a major limiting factor. The phase
change and mid-seasonal growth cycles are of interest. The
vegetative life cycle is exemplified by strong juvenile and
adult phases, the former is typified by rapid vegetative
growth and the latter by flowering (Hackett 1987). As a
woody plant progresses through the juvenile and into the
adult phase, its tissue responsiveness to microculture manipulation usually decreases markedly (Bonga 1987;
Francelot et al. 1987; Hackett 1987). Strong physiological
changes accompany seasonal growth cycles (e.g., spring
flush, bud set, and dormancy) and confound this progressive life cycle change. For microculture, the most responsive tissues are usually obtained from the spring flush
growth. Shoot cultures of trees are a powerful research tool
because, once established, they suspend these progressive
changes in a more or less permanent condition equivalent
to the juvenile phase and the spring flush of growth. Thus,
a well-established shoot culture will provide a tissue source
that typically is more responsive to various biotechnological manipulations than most other sources, excluding embryonic materials that are usually unsuitable for clonal
crops.
Besides providing responsive tissues, shoot cultures stop
progression through life and seasonal cycles. Plant shoot
growth is essentially suspended while plants are maintained as shoot cultures. For example, shoot cultures of a
birch clone (Betula populifolia cv. 'Whitespire') and a poplar clone (Populus tremula cv. 'Erecta') have been maintained
continuously for more than 15 years without noticeable
5
Section 1 In Vitro Culture
Figure 1A. Steps in establishing a shoot culture of a Populus alba hybrid clone. Nodal
and tip explants are taken from actively growing shoots, sterilized, and
placed in culture. New shoot growth is rapidly subcultured until uniform and
continuous growth is obtained.
Figure 1B. Microcuttings (center) are harvested from mature shoot cultures and rooted/
acclimated ex vitro to produce young plants for testing and evaluation.
6
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Poplar Shoot Cultures: Their Generation and Use in Biotechnology
changes in their shoot growth characteristics (Deborah D.
McCown, Knight Hollow Nursery, Middleton, WI, USA,
personal communication). Plants derived from these cultures appear to begin their ex vitro growth at the same lifecycle stage (i.e., as a seedling-like plant). Thus, shoot
cultures can supply tissues of relatively uniform and reproducible physiology throughout the year.
A second aspect of stability and reproducibility is genetics. Because shoot culture depends on growth from preformed meristems (apical or nodal) of subcultured
explants, any single cell mutations and other aberrations
rarely develop into aberrant shoots. Mutated shoots should
appear at a rate approximately equal to the rate that such
aberrant shoots appear in source plants grown ex vitro.
Some plant genotypes, especially those that are chimeric
in character, may be inherently genetically unstable,
whether grown as shoot cultures or in the field. However,
a vast majority of plants are genetically stable when maintained properly (i.e., without adventitious shoot generation) as shoot cultures. For example, no aberrant shoots
have been seen in the long-term shoot cultures of the poplar and birch clones mentioned previously.
Because of their responsiveness, stability, and reproducibility, shoot cultures are an excellent tissue source for various in vitro manipulations. For poplar, shoot cultures
provide useful source tissues to establish cell I callus I nodule cultures (McCown et al. 1988), protoplast cultures
(Chun 1985; McCown 1985; Russell and McCown 1986;
Smith and McCown 1983), and genetic engineering. Tissues from shoot cultures were proven adaptable to vector
(Confalonieri et al. 1994; DeBlock 1990; Fillatti et al1987;
Riemenschneider and Haissig 1991) and particle-bombardment transformation of Populus (McCown et al. 1991).
Conducting routine assays can be hampered by complex secondary compounds (e.g., phenolics or tannins)
often present in the tissues of many woody perennials.
Protein isolations, DNA extractions, or common assays of
gene activity (e.g., the reporter gene GUS) (Vainstein et al.
1993) are frequently difficult with woody perennials. Culture-derived shoots usually contain few interfering compounds and often provide the best source tissue for such
assays (Francis 1996). With at least some poplar genotypes,
DNA for molecular analyses is more readily obtained from
shoot cultures than greenhouse- or field-grown leaves
(Francis 1996). However, unlike many other woody plants,
there were no compounds interfering with the GUS reporter gene assays detected in poplar leaf tissues, even in
tissues collected from field plants (Francis 1996).
Shoot cultures also provide the basis for the most widely
used method of micropropaga tion in commerce today.
Shoot cultures provide uniform microcuttings that can be
rooted and acclimated (figure 1B) to produce liners that
are handled like seedlings in container and field plantings
(Douglas 1986). Micropropagation is particularly useful
to provide a multitude of uniform plants in a relatively
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
short time for use in biotechnological tests. Transgene expression in poplar was tested under laboratory conditions
(Confalonieri et al. 1994; Francis 1996), in growth chambers/ greenhouses (McCown et al. 1991; Riemenschneider
and Haissig 1991; Robison et al. 1994), and in the field
(Kleiner et al. 1995) using shoot cultures directly or plants
derived from shoot cultures.
Finally, shoot cultures provide a convenient way to store
selections under evaluation. Cultures can be continuously
subcultured and I or maintained under cold storage conditions (Chun 1993). We have routinely maintained more
than 100 transformants as shoot cultures, which preserves
these unique genotypes and provides a convenient "on
demand" source of new plants for further testing or distribution.
Generation of Shoot Cultures
As previously described (McCown and McCown 1987),
new isolations from most woody perennials pass through
an in vitro stage called stabilization before reproducible
and uniform shoot growth is achieved. Physiological processes of the stabilization phase are not well understood;
however, rejuvenation apparently is involved (reviewed
in McCown 1986; for poplar example, see Whitehead and
Giles 1977). Stabilized shoot cultures are achieved most
rapidly when the explant sources are juvenile materials
such as collar shoots, adventitious shoots, or rejuvenated
plants (Hartmann et al. 1990). However, gradual rejuvenation often can be achieved by repeated subculturing of
new shoots (Mullins 1987).
Once stabilized, the growth and multiplication rate of
the shoot cultures can be optimized in the production phase
(McCown and McCown 1987). Shoots can be multiplied
by either cytokinin stimulation of axillary bud growth,
often in conjunction with the loss of apical dominance
(shoot tip removal), or by manually dividing a shoot explant into its component nodes during subculture. Both
methods are useful with poplars (e.g., Chun 1993; Sellmer
et al. 1989).
With Populus, the optimal growth and multiplication regime usually depends on the specific genotype. Although
many specific differences and variations are observed, 3
general groups of poplars can be discerned from the literature.
1. Many clones, particularly those in the Leuce (currently termed Populus) section can be successfully
cultured as shoot cultures grown on standard MS
medium (Murashige and Skoog 1962) supplemented
with the cytokinin benzyladenine (Sellmer et al.
1989). Auxins are usually not required.
7
Section I In Vitro Culture
2. Some clones, especially those of P. tremula and P.
tremuloides species/hybrids, often perform better as
shoot cultures on media with a lower salt formulation, such as Woody Plant Media (WPM) (Lloyd and
McCown 1980; McCown and Sellmer 1987) or its
derivatives (Ahuja 1987; Sellmer et al. 1989}, supplemented with benzyladenine. Again, auxins are usually not required.
3. Other clones, especially those belonging to the
Aigeiros and Tacamahaca sections, do not perform
particularly vigorously as shoot cultures on any medium so far defined. Some selections may not be amenable to long-term maintenance as shoot cultures
(SeUmer et al. 1989). For others, acceptable shoot cultures may be generated using more complex cultural
regimes. MS medium supplement~d with cytoki~ins
and auxins may be helpful (Whttehead and Giles
1977). For some selections, benzyladenine may be
phytotoxic, but naturally-occurring cytokinins~ such
as zeatin, may be stimulatory (Ernst 1993). Multistage
protocols separating bud stimulation and elongation,
each with a separate medium requirement, was useful in other work (Chun 1993; Ernst 1993; Whitehead
and Giles 1977). DeBlock (1990) used a buffered medium supplemented with calcium gluconate to overcome shoot-tip necrosis (Sha et al. 1985) problems
associated with some poplar clones.
Conclusion
Although not a comprehensive remedy fo~ the difficu~­
ties associated with working with trees for biotechnological research, shoot cultures are a major and often essential
tool. Shoot cultures provide tissues to begin manipulations
and offer an effective avenue for moving plant materials
from culture to testing or production. For a program beginning work on a selection of poplar, establishing t~e
genotype in shoot culture will sensitiz.e researchers to Its
idiosyncrasies in the microculture environment.
Acknowledgments
This work was supported by the University of Wisconsin-Madison College of Agricultural and Life Sciences,
HATCH, the UW-Graduate School, and the Plant Biotechnology Consortium.
8
Literature Cited
Ahuja, M.R. 1987. In vitro propagation of poplar and aspen. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue
culture in forestry, Vol. 3. Case histories: Gymnosperms,
angiosperms, and palms. Dordrecht, The Netherlands:
Martinus Nijhoff Publishers: 207-223.
Bonga, J.M. 1987. Clonal propagation of mature trees:
· Problems and possible solutions. In: Bonga, J.M.;
Durzan, D.J., eds. Cell and tissue culture in forestry,
Vol. 1. General principles and biotechnology.
Dordrecht, The Netherlands: Martinus Nijhoff Publishers: 249-271.
Chun, Y.W. 1985. Isolation and culture of in vitro cultured
Populus alba x P. grandidentata protoplasts. J. Korean For.
Soc. 71:45-59.
Chun, Y.W. 1993. Clonal propagation in non-aspen poplar
hybrids. In: Ahuja, M.R., ed., Micropropagation of
woody plants. Dordrecht, The Netherlands: Kluwer
Academic Publishers: 209-222.
Confalonieri, M.; Balestrazzi, A.; Bisoffi, S. 1994. Genetic
transformation of Populus nigra by Agrobacterium
tumefaciens. Plant Cell Rep. 13: 256-261.
DeBlock, M. 1990. Factors influencing the tissue culture
and the Agrobacterium tumefaciens-mediated transfo~a­
tion of hybrid aspen and poplar clones. Plant Phystol.
93: 1110-1116.
Douglas, G.C. 1986. Poplar (Populus spp.). In: Bajaj, Y.P.S.,
ed. Biotechnology in agriculture and forestry, Trees II,
Vol. 5. Berlin: Springer-Verlag: 300-323.
Ernst, S.G. 1993. In vitro culture of pure species non-aspen
poplars. In: Ahuja, M.R., ed. Micropropagation of
woody plants. Dordrecht, The Netherlands: Kluwer
Academic Publishers: 195-207.
Fillatti, J.J.; Sellmer, J.; McCown, B.; Haissig, B.; Comai, L.
1987. Agrobacterium mediated transformation and regeneration of Populus. Mol. Gen. Genet. 206: 192-199.
Francelot, A.; Boulay, M.; Bekkaoui, F.; Fouret, Y.;
Verschoore-Martouzet, B.; Walker, N. 1987. Rejuvenation. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue
culture in forestry, Vol. 1. General principles and biotechnology. Dordrecht, The Netherlands: Martinus
Nijhoff Publishers: 232-248.
Francis, K.E. 1996. Genetic transformation and transgene
analysis of hybrid poplar NM6 (Populus.nigr~ x Popul.us
maximowiczii). Madison, WI, U.S.A.: Umverstty of Wisconsin: 80 p. M.S. thesis.
Hackett, W.P. 1987. Juvenility and maturity. In: Bonga,
J.M.; Durzan, D.J., eds. Cell and tissue culture in forestry, Vol. 1. General principles and biotechnology.
Dordrecht, The Netherlands: Martinus Nijhoff Publishers: 216-231.
USDA Forest Service Gen. Tech. Rep. AM-GTA-297. 1997.
Poplar Shoot Cultures: Their Generation and Use in Biotechnology
Hartmann, H.T.; Kester, D.E.; Davies, F.T. 1990. Plant propagation: Principles and practices. Englewood Cliffs, NJ,
U.S.A.: Prentice Hall. 647 p.
Kleiner, K.W.; Ellis, D.O.; McCown, B.H.; Raffa, K.F. 1995.
Field evaluation of transgenic poplar expressing a Bacillus thuringiensis cryiA(a) d-endotoxin gene against forest tent caterpillar (Lepidoptera: Lasiocampidae) and
gypsy moth (Lepidoptera: Lymantriidae) following winter dormancy. Environ. Entom. 24: 129-135.
Lloyd, G.; McCown, B. 1980. Commercially-feasible
micropropagation of mountain laurel, Kalmia latifolia,
by use of shoot-tip culture. Proc. Intern. Plant Prop. Soc.
30:421-426.
McCown, B.H. 1985. From gene manipulation to forest
establishment: Shoot cultures of woody plants can be a
central tool. TAPPI J. 68: 116-119.
McCown, B.H. 1986. Adventitious rooting of tissue cultured plants. In: Jackson, M.B., ed. New root formation
in plants and cuttings. Boston: Martinus Nijhoff Publishers: 289-302.
McCown, B.H.; Zeldin, E.L.; Pinkalla, A.H.; Dedolph, R.R.
1988. Nodule culture: A developmental pathway with
high potential for regeneration, au tom a ted
micropropagation and plant metabolite production
form woody plants. In: Hanover, J.; Keathley, D.E., eds.
Genetic manipulation of woody plants. New York: Plenum Press: 149-166.
McCown, D.O.; McCown, B.H. 1987. North American hardwoods. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue culture in forestry,. Vol. 3. Case histories:
Gymnosperms, angiosperms, and palms. Dordrecht,
The Netherlands: Martinus Nijhoff Publishers: 247-260.
McCown, B.H.; Sellmer, J.C. 1987. General media and vessels suitable for woody plant microculture. In: Bonga,
J.M.; Durzan, D.J., eds. Cell and tissue culture in forestry, Vol. 1. General principles and biotechnology.
Dordrecht, The Netherlands: Martin us Nijhoff Publishers: 4-14.
McCown, B.H.; McCabe, D.E.; Russell, D.R.; Robison, D.J.;
Barton, K.A.; Raffa, K.R. 1991. Stable transformation of
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Populus and incorporation of pest resistance by electrical discharge particle acceleration. Plant Cell Reports.
9:590-594.
Mullins, M.G. 1987. Propagation and genetic improvement
of temperate fruits: the role of tissue culture. In: Somers,
D.A., ed. Proc. VI Congress of Plant Tissue and Cell
Culture. Minneapolis, MN, U.S.A.: Univ. of Minnesota:
395-406.
Murashige, T.; Skoog, F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiol. Plant. 15: 473-479.
Riemenschneider, D.E.; Haissig, B.E. 1991. Producing herbicide tolerant Populus using genetic transformation
mediated by Agrobacterium tumefaciens C58: A summary
of recent research. In: Ahuja, M.R., ed. Biotechnology of
woody plants. New York: Plenum Press: 247-263.
Robison, D.J.; McCown, B.H.; Raffa, K.F. 1994. Responses
of gypsy moth (Lepidoptera: Lymantriidae) and forest
tent caterpillar (Lepidoptera: Lasiocampidae) to
transgenic poplar, Populus spp., containing a Bacillus
thuringiensis d-endotoxin gene. Environ. Entomol. 23:
1030-1041.
Russell, J.A.; McCown, B.H. 1986. Techniques for enhanced
release of leaf protoplasts in Populus. Plant Cell Reports.
5:284-287.
Sellmer, J.C.; McCown, B.H.; Haissig, B.E. 1989. Shoot culture dynamics of six Populus clones. Tree Physiol. 5:219227.
Sha, L.; McCown, B.H.; Peterson, L.A. 1985. The occurrence
and cause of shoot-tip necrosis in shoot cultures. J. Amer.
Soc. Hort. Sci. 110: 631-634.
Smith, M.A.L.; McCown, B.H. 1983. A comparison of source
tissue for protoplast isolation from three woody plant
species. Plant Sci. Lett. 28: 149-156.
Whitehead, H.C.M.; Giles, K.L. 1977. Rapid propagation
of poplar by tissue culture methods. N.Z. J. For. Sci. 7:
40-43.
Vainstein, A.; Fischer, M.; Ziv, M. 1993. Application of reporter genes to carnation transformation. HortScience.
28: 1122-1124.
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