Indian Journal of Experimental Biology
Vol. 37, July 1999, pp. 627-638
Review Paper
Applications of genetic transformation to tree biotechnology
Paramjit Khurana & Jigyasa Khurana
Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India
The developments in the field of biotechnology have advanced to a stage where its benefits can be apparent in the field
of forestry. The ability to manipulate forest tree species at the cellular and molecular levels holds promise for circumventing
inherent limitations in tree improvement programs, e.g., long generation cycles, space for large segregating populations, and
the lack of genetically pure lines. Genetic engineering methods complement plant breeding efforts by increasing the
diversity of genes and gerrnplasm available for incorporation into desirable plant species, and shortening the time period
required for the production of new varieties and hybrids. Future developments in the area!\ of molecular markers and
efficient regeneration methods would help in ameliorating the dilemmas of several existing silvicultural practices. The .
present article focusses on the first generation applications of genetic engineering of trees, i.e., pest resistance, disease
resistance and herbicide tolerance, and discusses the significant progress made in the fields of lignin modification and other
traits currently being prospected for genetic manipulation.
The present day global awareness has led to the
realization of the unique contribution of trees to the
well-being of this planet. Parallel research and
developmental efforts are therefore being undertaken
to increase the forest area worldwide and to replace
plantations being destroyed by industrialization and
urbanization. Trees have thus become increasingly
important to restore the lost forest stands and to meet
the escalating needs of the world economy.
Tree improvement involves not only managing
genetic resources but also includes conservation,
selection, breeding and propagation of select
genotypes'. Traditional breeding methods involve sets
of genes being introduced through sexual
hybridization and is restricted by problems of sexual
incompatibility manifested at the interspecific and
intergeneric level, and related problems like sferility
and apomixis. In addition, these methods have proved
to be extremely time consuming due to the long
generation cycles of trees. Recent developments in
molecular biology have, however, opened new vistas
for broadening gene pools and have provided impetus
to various crop improvement programs. Genetic
engineering methods thus complement traditional
breeding methods and hasten the process of
introduction of the desirable traits into \cultivars/
varieties of commercial interest.
Success with genetic engineering is not easy and is
*Corresponding author:
Fax: 91-11 -6885270, 91-11-6886427
E-mail: [email protected] n
often limited by the n<?n-availability of suitable
regenerating systems. Another major prerequisite for
the application of recombinant DNA technology to
tree improvement is the development of gene transfer
systems. In addition to Agrobacterium-mediated
transfer, the other promising delivery systems
available
are
through
electroporation
and
microprojectile bombardment. Also, the ultimate
regeneration of transgenic plants from transformed
cells, continues to remain a major challenge.
The intensive research in the field of crop
biotechnology has enabled the identification and
isolation of a number of genes responsible for varied
characters. Based on these identified traits, tree
biotechnologists are concentrating on genetic
manipulation of important . tree species. Some of the
target traits which are currently being prospected for
genetic engineering are:
Category
Target Character
I. Hybrid production
Self incompatibility
Male sterility
Structure and architecture
(height, branching, leaves, roots)
Flowers (structure, color, timing)
Herbicide resistance
Improved nutrient uptake
Improved photosynthetic
efficiency
Sugar and starch (composition
and content)
Oils (composition and content)
Storage proteins (composition
and content)
2. Plant growth
3. Altering inputs
4. Products
INDIAN J EXP BIOL, JULY 1999
628
5. Environment
Flavors and fragrances
Pharmaceuticals
Fibres (textiles)
Fruit (ripening and quality)
Biotic factors (pest, bacteria,
viral and fungal resistance)
Abiotic factors (drought, salinity,
temperature, heavy metal
tolerance)
Cell, Tissue and Protoplast Culture
Owing to rapid deforestation, depletion of genetic
stocks and escalating product demand, mass
propagation and production of short duration trees
with rapid turnover of biomass is the need of the
hour. It is therefore, envisaged that in comparison to
the traditional time consuming breeding programs,
tissue culture techniques are competent to meet the
demand for increased biomass production.
During the last two decades, dramatic progress has
been made in developing and refining various tissue
culture techniques. As a result, methods are now
available to culture and regenerate plants from
somatic cells, pollen, and protoplasts of a large
number of plant species. Considerable progress has
been made since plantlets were obtained via
organogenesis from PopuLus tremuLoidei. It is now
possible to regenerate successfully many of the
woody species in vitro which were initially
considered to be recalcitrant3.4. There are a number of
advantages of micropropagation of trees over sexual
propagation. Some of these are:
1. Cloning superior trees, such as hybrids or
selected specimens from field populations.
2. Due to the long breeding phase of trees,
improvement of planting stock by sexual means is
slow, whereas with cloning it is immediate.
3. Quite often the juvenile phase of development
can be bypassed.
4. The genetic uniformity of a clone is generally an
asset.
5. Elite trees, such as hybrids and polyploids, can
be propagated vegetatively.
The availability of protoplast-to-plant technology
for various tree species has initiated the exploitation
of this technology for various aspects of ttee biology.
Biotechnological procedures with direct application
to tree improvement include somatic hybridization
and in vitro selection using both somaclonal and
gametoclonal variation. Somatic hybridization is a
prime objective for protoplast manipulation and
certain intergeneric and interspecific protoplast
fusions have been performed in which hybrid callus
has been produced. Somatic hybrids have been
produced by the fusion of protoplasts of sexually
incompatible rootstock genotypes of Pyrus communis
var. pyraster and Prunus avium x pseudocerasus5 .
The somatic status of the regenerated plantlets was
confirmed through chromosome counts, isozyme
assessments, and morphological markers.
The existence of an inverse relationship between
cell wall regeneration and tolerance to salinity has
been demonstrated 6 • Salt and drought tolerant
plandets have been obtained from colt cherry
protoplasts, Prullus avium x pseudocerasus, as a
result of protoclonal variation and recurrent in vitro
selection strategies? The exploitation of protoplast
technology has thus opened up the possibility of
creating agronomically useful genetic novelties. Plant
regeneration from protoplasts can be a source of
spontaneous somaclonal variatIOn for certain
genotypes and the same is true for in vitro selection
based on protoplasts. By placing particular emphasis
on selection of monogenic and allelogenic traits such
as disease resistance and herbicide tolerance,
extremely desirable phenotypes can be obtained.
Homozygous plants obtained
by
somatic
hybridization (homofusion) of protoplasts of haploid
genotypes will prove helpful in the study of
inheritance of horticultural traits. Heterofusions
between different haploid clones might serve to create
genetic novelties of great value for rootstock breeding
and might be of interest as novel scion cultivars.
Finally, protoplasts are an ideal system for the
production of novel transgenic trees and genetic
transformation of protoplasts is the ultimate goal of
various tree breeders.
Genetic Engineering and Tree Biotechnology
Genetic engineering methods complement plant
breeding efforts by not only increasing the diversity
of genes and germplasm available for incorporation
into crops but also by shortening the time period
required for the production of new varieties and
·hybrids. The ability to manipulate forest tree species
at the cellular and molecular level also holds promise
for ~i,rcumventing inhere?t limitations in tree
improiement programs, e.g., long generation cycles,
space for large segregating populations, and the lack
of genetically pure lines.
KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY
Since the production of the first transgenic plants
of tobacc0 8, the use of genetic engineering methods
has increased by leaps and bounds. Although a
number of transgenic crop species have been
produced, success with tree species has been limited
primarily due to lack of efficient in vitro regeneration
protocols and suitable gene transfer mechanisms.
However, with the growing focus on regeneration and
transformation of tree species, it is likely that within
few years, the economically important tree species
will be amenable to genetic manipulation.
!
Methods for Foreign Gene Introduction
The Natural Gene Vector-Agrobacterium
Recognition of the ability of the soil bacterium
Agrobacterium tumefaciens to transfer a portion of its
DNA to plants was perhaps the most important
milestone in plant biotechnolog/,IO. Major advances
contributing to the popularity of Agrobacteriumbased
transformation
systems
include
the
development of disarmed strains where the oncogenes
which result in tumorigenesis are deleted from the
plasmid; the development of binary vectors, in which
the T -DNA borders are located on a wide host range
plasmid and the virulence genes of the Ti plasmid are
located on an independent plasmid and act in trans;
were crucial in expanding the use of Agrobacteriumbased vectors in plant transformation studies.
The ability to transform tissues and to regenerate
transgenic woody trees successfully using the natural
gene transfer system of Agrobacterium is dependent
primarily on three factors:
1.
2.
3.
Virulence of Agrobacterium cells,
Efficiency of selection that would allow
growth of transformed cells, and
Frequency of regeneration among the
transformed cell population.
Besides the above, quite a number of other
biological and physical parameters influence the
transformation efficiency of Agrobacterium, the
foremost being the susceptibility of the host to
infection by Agrobacterium. The host range of
Agrobacterium is quite extensive. Populus is known
as a natural host for A. tumefaciens ll and various
species of Populus have been investigated for their
susceptibility to Agrobacterium. A wide variety of
trees have also been shown to be transformed by
various wild type strains of A. tumefaciens -and A.
rhizogenes I2. 15 • Of the several recombinant strains of
629
A. tumefaciens that have been evaluated for their
ability to transform different tree species, LBA 4404
has proved to be most successfuI12.16.19. Besides
Agrobacterium cell densitlo.21 and the period of cocultivation22.27 , the transfer of T-DNA is significantly
influenced by the plant phenolics secreted in response
to wounding and high levels of acetosyringone have
resulted in increased foreign gene expression l5 .
Trees often show unusual sensitivity to kanamycin
and low concentrations of 25 mgIL have proved to be .
sufficient for selection, although a high of 100 mgIL
in Malus has also been reported 27 . Preculturing of
explants before co-cultivation, so as to enhance the
susceptibility of cells to infection and T-DNA
transfer, has also been reportedI5.28.3o. Preculturing of
Malus for 1-8 days significantly increased the
transient expression of the gus marker gene, although
it was found to be detrimental for stable gene
29
expression and subsequent regeneration .
Electroporation
Electroporation of protoplasts could be an efficient
means of introducing DNA into plant cells, especially
in those species not susceptible to Agrobacterium
infection. However, for electroporation to be
successful, one of the major requirements is a
functional protoplast-to-plant regeneration system.
With the few exceptions that have been published so
far this approach is certainly not easy. Some of the
major problems with transformation of protoplasts
and subsequent plant regeneration are the possibilities
of somaclonal variation among the regenerants, the
presence of multiple copies of inserted DNA,
rearrangements, instability of inserted DNA, etc . It is
thus not surprising that although transient gene
expression via electroporation has been reported in
Alnus incana 31 , Eucalyptus citriodora 32 and E.
gunnii 33 , no report of stable transformation via this
technique has been published so far.
Particle bombardment
Particle bombardment, which uses explosive, high
speed acceleration to deliver biologically active DNA
into a large range of target tissues and cells has
become the second most widely used technique in
plant genetic engineering. This potential to transfer
foreign DNA in regenerable cells, tissues or organs
provides the best method for genotype-independent
transformation, surpassing Agrobacterium host
specificity and tissue culture related regeneration
/
630
INDIAN J EXP BIOL, JULY 1999
difficulties. The first report on the production of a
transgenic Allium plant using high velocity
microprojectiles 34, stimulated the use of this versatile
technique for the production of transgenics from
recalcitrant tree species. It took three years before the
first transgenic trees of a hybrid poplar, Populus alba
x P. grandidentata 35 and Carica papaya 36 could be
produced. Since then transgenics have been produced
in Liriodendron tulipijera 37 and Citrus reticulata x C.
paradisP s, and transient expression has been reported
9
in Elaeis guineensii , Hevea brasiliensis40 and Ulmus
procera41 and many others.
Like any other transformation method aimed at
obtaining high frequency stable transformation,
particle bombardment also demands the optimization
of DNA delivery conditions. Various biological (type
of target tissue, osmotic treatment) and physical
(target distance, rupture disc pressure, type of
microparticles,
DNA
and
relative
particle
concentration, chamber vaccum) parameters · of
bombardrpent, availability of suitable promoters,
select~ble markers and a sensitive selection agent
significantly influence gene transfer. Embryos and
embryogenic cell lines are the most preferred target
tissues. The enrichment of the bombardment medium
with sucrose considerably increases the transformation efficiency of zygotic embryos42. The gun
powder system has been reported to be as reliable as
the helium device for DNA transfer into zygotic
embryos of Eucalyptus globulus, and a relatively
higher efficiency of the gold particles as compared to
42
the tungsten projectiles is also reported .
The first transformation of a tree species (Carica
papaya) via particle bombardment with an
agronomically useful gene (prv cp - coat protein gene
of papaya ringspot virus) resulted in virus resistant
plants 36 • Herbicide tolerant papaya transgenic plants
have also been produced following the delivery of bar
gene, which confers resistance to phosphinothricin43 .
The BT gene confering insect resistance has also been
35
introduced in Populus alba x P.grandidentata .
In addition to producing agronomically useful
transgenic trees, particle bombardment has also been
used for studying gene expression and regulation, and
identification of plant promoters to ensure strong and
constitutive expression of the foreign gene.
Transformation of Coffea arabica by the
~-glucuronidase gene using different promoter
sequences resulted in significant variations 44 • EFla..
At promoter of Arabidposis thaliana was found to be
most effective. In a similar study, on the bombarded
embryogenic callus of ELaeis guineensis, Emu
promoter was found to be most efficient as compared
39
tt four other promoters .
#~tications to Tree Improvement
For the application of genetic engineering methods
to tree improvement, initial research had focused on
engineering of traits that relate directly to the
traditional roles of industry in farming, such as pest
resistance, disease resistance and herbicide tolerance
(also see, Tzfira et al. 45 ). Progress is now being
observed in the areas of drought and cold tolerance,
altering lignin composition, improving fruit quality,
reducing juvenility phases and alteration of tree form
and architecture.
Herbicide tolerance
Engineering herbicide tolerance into crop plants
represents a new alternative for conferring selectivity
and enhancing crop safety. Work has been largely
concentrated on herbicides with properties of high
unit activity, low toxicity, low soil mobility, rapid
biodegradability and with a broad spectrum activity
against various weeds. The development of crop
plants that are tolerant to such herbicides would
provide more effective, less costly and environment
friendly weed control.
Two general approaches have been employed in
engineering herbicide tolerance:
1.
2.
Altering the level and sensitivity of the target
enzyme for the herbicide, and
Incorporating a gene that will detoxify the
herbicide.
An example of the first approach is glyphosate, the
active ingredient of "Roundup" herbicide, which acts
by' specifically inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP). Some level
of tolerance has been engineered into Populus (P.
alba x P. grandidentata) by the introduction of the
mutant aroA gene, which encodes for EPSP less
21
sensitive to glyphosate by Fillati et al. • Transgenic
trees of hybrid aspen (Populus alba x P. tremula) and
poplar (Populus trichocarpa x P. deltoides), resistant
to phosphinothricin, have been produced by
Agrobacterium-mediated transfer of the bar gene46 .
Employing
particle
bombardment,
herbicide
(phosphinothricin) resistant plants of Carica papaya
KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY
have also been produced, which were able to
withstand 3-5 times higher concentration of pJ>"r3.
Insect resistance
Trees are susceptible to many insect pests and
breeding for resistance is a difficult task in long lived
trees. The production of insect resistant plants is
another application of genetic engineering with
important implications for crop improvement. In
plants, resistance to insect pests has been achieved by
employing two types of gene products:
1.
Protease inhibitors, and
2.
Insecticidal crystalline proteins (lCP from
Bacillus thuringiensis).
Protease inhibitors, first characterized by Ryan 47 ,
are synthesized in a variety of plants in response to
insect predation and have been found to be toxic to
diverse groups of insect species. Many plant species
possess the genes coding for these protease inhibitors.
The promoter for one of these genes, pin2, is
inducible. Klopfenstein et a1. 48 , introduced the
chloroamphenicol acetyl transferase (cat) gene fused
to the pin2 promoter into hybrid poplar using
Agrobacterium. In another instance, the introduction
of a gene encoding the cysteine protenase inhibitor,
OCI, from rice into a poplar hybrid clone (Populus
tremula x P. tremuloides) has resulted in tolerance
towards Chrysomela tremulae, a coleoptera49 . I nsects
feeding on these transgenic poplars exhibit reduced
growth, altered development and increased mortality.
Progress in engineering insect resistance in
transgenic trees has also been achieved through the
use of insect toxin protein genes of Bacillus
thuringiensis (BT). B. thuringiensis is an entomocidal
bacterium that produces a crystalline protein which is
lethal to select pests. Most strains of Bacillus
thuringiensis are toxic to lepidopteran (moth and
butterfly) larvae, although some strains with toxicity
to coleopteran (beetle) or dipteran (fly) larvae have
been described. The insect toxicity of B. thurillgiensis
resides in a large protein and has no toxicity to
beneficial insects, other animals or humans. The
mode of action of the BT crystalline protein is
thought to be exerted at the level of disruption of ion
transport across brush border membranes of
susceptible insects.
A lepidopteran specific BT gene has been
introduced into hybrid pioneer elm employing particle
gun and Agrobacterium-mediated gene transfer
631
methods 50 . Transgenic poplars (Populus alba x P.
grandidentata) exhibiting insect resistance via BT
gene expression have been produced 35 . The transgenic
poplars obtained were highly resistant to feeding of
lepidopteran
insects.
Transformed
sweetgum
(Liquidambar styraciflua) was generated which
contained a chimeric BT toxic gene and conferred
resistance to the two known defoliators of sweetgum,
?'i
.
i.e., fall web worm and the gypsy moth--. Transgenic
poplar (Populus tremuLa x P. tremuLoides) expressing
B. thuringiensis endotoxin cry ilIA, known to be
49
active against coleopteran has also been produced _
Insects feeding on this transgenic poplar exhibited
increased survival mortality.
Viral disease resistance
The family of potyviruses, the largest and most
widely distributed group of plant viruses, is known
for its ability to severely damage many important
crop species 51 • This is also the case for Plum Pox
Virus (PPV) associated with plums, apricots and
peaches. Originally described in Bulgaria, the Sharka
disease has spread over great parts of Central and
Southern Europe as well as over many Mediterranean
countries. In crop plants, disease resistance has been
approached through viral cross protection using
chimeric genes specifying viral coat protein as a way
of protecting plants from viral diseases 52 . This
approach has been successfully used against a number
of different viruses, for example, Tobacco Mosaic i
Virus (TMV), Alfalfa Mosaic Virus (AMV), etc. This
approach has resulted in the successful production of
.
5l
transgenic Prunus armemaca- an de'
arlca papaya 36.54
resistant to attacks of Plum Pox virus and Papaya
Ringspot virus, respecti vel y.
Fungal disease resistance
A number of tree species serve as useful hosts for
the fungal pathogens. Although no major progress has
been made in the genetic engineering against fungal
diseases in trees, efforts are underway to characterize
the fungal disease resistance genes. Chitinase genes
from hybrid poplars, win6 and Will8, have been
20
characterized • Two major fungal di~eases requiring
immediate attention are: Chestnut blight and Dutch
elm disease. These diseases have devastated both
natural populations and planted specimens of
chestnuts and elms.
A novel method to control chestnut blight caused
by Cryphonectria parasitica has been described by
632
INDIAN J EXP BIOL, JULY 1999
Choi and NUSS 55 . The introduction of a cloned dsRNA
genome from a less virulent strain into virulent strains
converts the virulent strain into a less virulent strain.
Symptoms of the disease were significantly reduced
by the introduction of the genetically altered strains
into chestnut trees. The effect of the cloned dsRNA
genes in making other fungal pathogens less virulent
is under investigation.
;'
/'
Stress tolerance
When plants are subjected to stress, biotic or
abiotic, an increased production of potentially
dangerous active oxygen species is favoured 56 .
Photochemical oxidants, including ozone, are
important sources of stress for trees 57 • Ozone toxicity
has been implicated in decreasing tree growth 58 ,
increasing tree mortalitl9 and causing intraspecific
and interspecific changes in forest stands. Ozone
usually overwhelms the plant's antioxidant defense
mechanism, resulting in the degradation of
chlorophyll and the destruction of cell membranes60 .
Collectively, this results in reduced photosynthesis,
premature foliar aging and increased susceptibility to
other stress and/or pests. There is strong evidence for
the protective role of various antioxidants such as
superoxide dismutases. Superoxide dismutases have
been implicated in ozone tolerance in plants,
including hybrid poplars61 •62 • Another antioxidant,
glutathione, may also have a role in scavenging
superoxide radicals. In an attempt to produce stress
tolerant transgenic poplars, Foyer et al. introduced
genes for glutathione reductase (got)63. Overexpression of glutathione reductase (GR) in the
chloroplast results in increased antioxidant capacity
of leaves and thereby improves the capacity to
withstand oxidative stress.
Efforts to improve growth and productivity of
forest trees under stress conditions could benefit from
an understanding of the expression of specific
drought induced proteins which may contribute to
drought tolerance. Evidence for the accumulation of a
66 kDa water stress responsive protein (BspA) in
shoots of Populus tremula has been presented64 •
Furthermore, a good correlation between the
expression of dehydrin like proteins (DSP 16) and
sucrose synthase, and the degree of tolerance and ion
leakage in Populus clones was found. The Nterminal amino acid sequence of BspA has been
determined and it exhibited high homology to wheat
germins GF-2.8 and GF_3.8 65 • Further analysis of the
expression of these proteins and their mode of action
will allow a better understanding of their role in
drought tolerance.
Lignin modification
Lignins are complex cell wall phenolic
heteropolymers which represent the second most
abundant organic compound on earth after cellulose.
In general, for tree species, lignin makes up about 1535% of the dry wood weight. During paper pulping,
lignin must be eliminated from wood . The
engineering of lignin biosynthesis appears to be a
promising aspect for improving trees used in the
paper/pulp industry. Genetically reducing the quantity
or altering the quality of lignin in pulpwood species
could improve the efficiency of these pulping
procedures. Sense and antisense expression of
sequences encoding CAD (cinamyl
alcohol
dehydrogenase) and COMT (methyl transferases)
enzymes involved in lignin biosynthesis have resulted
in the production of altered lignin in poplar66•67, the
most important source of pulp. Lignin is altered both
in composition and statistics, although the content is
similar to that in the control. Contrasting results with
two enzymes in terms of feasibility of delignification
have been reported. In the case of down regulated
COMT transgenics there is a decrease in S/G ratio
(syringyllguaiacyl) and a consequent difficulty in
chemical extraction 66 • Transformed lines with a down
regulated CAD activity show an increased
accumulation of cinnamaldehydes leading to
improved pulp properties and easier delignification 67 ,
Coexpression of COMT alongwith ferulate-5-hydroxylase, such as was cloned from Arabidopsis, will
lead to the conversion of softwood lignin to the
hardword type68 •69 .
Another enzyme of prime importance involved in
the last step of lignin biosynthesis. is the oxidative
polymerization by peroxidases, Poplars have a
number of different peroxidase isozymes whose
patterns of expression are tissue specific,
developmentally regulated and influenced by
environmental factors 7o . Kajita et al. altered the
expression of a peroxidase isozyme by introducing a
genomic clone for peroxidase (prxA 1) under the
control of the CaMV 35S promoter in a hybrid
poplar, Populus kitakamiensis 17 , Transgenic poplars
obtained by introducing the chimeric peroxidase gene
have been shown to have an increase in total
peroxidase activity that has been accounted for by the .
KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY
633
Table I- Foreign gene expression in tissue or cells of angiosperrnous trees-Coll/d
Species
Target Ce1Vfissue
Mode of gene
Foreign geneh
TGE"
SGEd
Reference
transfe~
Uquidambar styraeijlua
Leaf
A.t.
anionic peroxidase
+
25
Uriodentir(m tulipifera
Embryogenic suspension
Pb
gus, nptl/
+
37
Malus x domestiea
Leaf
Leaf
Shoot
Leaf
A.t.
At.
A.t.
A.t.
IIptll, gus
nptll, gus
nptl/, gus
nptll, gus
+
27
29
94
95
M. pumila
Leaf
A.t.
nptll, nos
Malus
Leaf
Leaf
A.t.
A.t.
nptll, gus
gfp
Mangifera indica
Somatic embryo
At.
gus, nptll
Populus deltoides
Leaf disc
A.t.
gU.f ,
nptll
98
P. euroamerieana
Leaf disc
A.t.
gus, nptll
98
P. nigra
Leaf
A.t.
gus, nptll, hpt
+
18
P. tremula
Stem
A.t.
gus, nptll
+
99
P. tremuloides
Leaf
Leaf
A.t.
At.
npt II, gus
COMT
+
+
30
100
P. alba x P. grandidenta
Leaf
Leaf suspension culture
Stem
Stem
Leaf
Leaf
A.t.
A.t.
A.t.
Ep
A.t.
A.t.
nptll, aro A
np II-ae, hpt, BT
bar, nptll
nptll, gus, BT
cat, np II
aroA, nptll
+
+
+
+
+
+
21
24
46
35
48
101
P. sieboldii x P.
grandidentata
(P. kitakamiensis)
Leaf
A.t.
gus, nptl/, pr XAI
+
17
P. tremula x P. alba
Shoot
Stem
Stem
Stem
Stem
Stem
A.t.
A.t.
At.
A.t.
A.t.
A.t.
nptl/, gus
nptll, gus
COMT, nptll
CAD, nptll, gus
gor, gshll, nptll
FeSOD, nptll
+
+
+
+
Stem
A.t.
Leaf
A.t.
+
+
+
+
19
+
28
96
+
97
\
P. tremula x P.
tremuloides
Bt, cry iliA,
OCI, npt II
rolC, Ae-rol C,
nptll
+
+
+
102
II
66
67
63
103
49
+
+
104
Prunus amygdalus
Leaf
A.t.
lip/II, gus
+
16
P.ameriealla
Embryogenic callus
A.t.
gus, nptl/
+
105
P. armeniaea
Immature embryo
A.t.
gus, ppv ep
+
53
P. domestiea
Hypocotyl
Immature embryo
A.t.
A.t.
nptll, gus
gus, pPV cp
+
+
106
53
P. persiea
St~m
A.t.
oes
+
107
COlltd
634
INDIAN J EXP mOL. AlLY 10519
KHURANA & KHURANA: GENETIC TR;\ NSFORMATION IN TREE BIOTECHNOLOGY
Table I-Foreign gene expression in
ti s~l.1e
635
or cells of angiospermous trees-Collld
l~plll
:n
P. subhinella aulumno
rosa
Embryogenic callus
A.r.
gus,
Pyrus communis
Leaf
A.I.
IIplll, gus
+
26
Robinia pseudoacacia
Hypocotyl
Cotyledon
A.I.
A.I.
nptll
nplll
+
+
108
13
Theobroma cacao
Leaf
A.I.
nptll
+
109
Ulmus procera
Leaf
Shoot
Pb
A.I.
gus
gus
+
41
+
+
110
ABBREVIATIONS
a
A.r.
Agmbaclerium rhil.ogenes
COMT
caffeic acid-O-methyl transferase
A.I.
Agrobaclerium lumefaciens
CAD
cinnamyl alcohol dehydrogenase
Ep
Electroporation
gor
glutathione reductase
Polyethylene glycol
gshll
glutathione synthetase
Ph
Particle bombardment
aroA
agr
agropine
5-enolpyruvyl shikimate 3-phosphate
synthase
PEG
b
nplll
(ICS
Cry III A
octopine synthase
bar
nos
nopaline synthase
gus
~-glucuronidase
kall
kanamycin phosphotransferase
transfera.~e
cal
chloroamphenicol acetyl
chs
chalcone synthase
BT
Bacillu.f Ihuringiensis toxin
hpl
hygromycin phosphotransferase
OCI
cysteine proteinase inhibitor
peroxidase
prXAI
PPVcp
neomycin phosphotransferase
Plum pox virus coat protein
Bacillus Ihurillgiellsis 5-endotoxin gene
phosphinothricin
PRVep
Papaya ring spot virus coat protein
Imsllmr
Tumor genes
nop
nopaline
man
mannopine
FeSOD
iron superoxide dislllutase
gfp
green flourescent protein
c
TGE
Transient Gene Expression
d
SGE
Stable Gene Expression
leading to the specific ablation of floral organs or
using antisense or promoter suppression of specific
homeotic reproductive development genes 73.
While little research has been done on the production
of sterile trees, it is likely that it will be relatively
easy to transfer sterility genes being isolated from
Reproductive sterility and early flowering
agricultural crops into trees . Male sterility would
minimize gene flow via pollen dispersion . Complete
Before genetically engineered trees can be planted
sterility would be desirable for species such as
in the field for commercial application, the trans genes
Populus and Salix, which have small wind blown
introduced into them need to be contained. This is
seeds.
because native and wild populations of trees could
suffer a high degree of contamination from the
In the last few years there has been considerable
transgenic plants71 , or the transgenes could escape
progress towards cloning flower specific genes and
and become wild72.
these could potentially be used to regulate flowering.
Flower sterility can be achieved through flower " Recent work on meristem identity genes with the
specific expression of cytotoxic structural genes,
model plant, Arabidopsis, has led to precocious
specific overproduction of the peroxidase isozyme
(Prx Ai). The anionic peroxidase isozyme has been
found to have a pI of 4.4 and the tissue specificity and
UV inducibility of this isozyme have been
characterized.
636
INDIAN J EXP BIOL, JULY 1999
flowering in aspen. Hybrid aspen transformation with
the same Arabidopsis LEAFY gene (LFY) under
control of Cauliflower Mosaic Virus 35S promoter
produces flowering after only five months of growth
in the greenhouse74 • Needless to mention, such
investigations have tremendous potential to
revolutionize the entire field of tree breeding.
Future Prospects
The demand has never been greater for improved
forest trees. Several new methodologies are being
used to address problems in forest biology. Besides
addressing problems inherent to the current use of
trees, genetic manipulation of trees brings with it the
potential to create new industries based on novel
characteristics, e.g., trees with trans genes to detoxify
specific pollutants and for remediation of
contaminated and hazardous wastes. The demand for
fibers and wood will continue and advances in wood
chemistry and biotechnology may also make the trees
an ideal feedstock for 'biorefineries' as alternative
sources of petrochemical products. With the
continuing demand for wood and wood related
products, new silvicultural practices will certainly be
needed to speed up the growth of trees and
consequently, biotechnological practices would be
relied upon.
It might be possible to increase biomass production
by altering cellulose biosynthesis in transgenic plants.
One way to increase fiber yield in trees is to create a
sink for photosynthate in xylem cells. Cloning of the
UDP-glucose pyrophosphorylase, responsible for the
synthesis of UDPG, a high energy substrate for
cellulose biosynthesis in both bacteria and plants,
would serve the purpose. It has been found that this
gene has xylem specific expression and that its
overexpression in transgenic tobacco resulted in
increased UDPG-Ppase activity, elevated levels of
cellulose and increased plant biomass 75.
A major breakthrough in genetic engineering of
trees would be the total loss of ability to form
reproductive structures in trees. Such trees will
obviously be sterile, thereby providing gene
containment for environment and commercial
protection, but there is a secondary benefit; avoidance
of the reproductive burden is estimated to provide
vegetative growth increases of up to 15% .. To achieve
this goal, recently a tissue ablation approach has been
proposed76 , whereby a promoter of an early flower
specific gene is used to express a lethal gene such as
Rnase, protease or nuclease, antisense to a central
function, a cytokinin etc.
Fundamental to these efforts would be the
integration of genetic engineering with marker aided
selection. Molecular marker methods will help in
identifying superior genotypes as well as in
monitoring the products of improved breeding
schemes. Markers have significant associations with
height and wood volume, indicating a strong
influence of specific quantitative trait loci. AFLP
markers have been used to study the genetic
architecture of rooting ability in eucalyptus. Maps
have now been constructed for 4 species of
eucalyptus77 • Thus by a judicious amalgamation of
traditional breeding practices and the present day
techniques of biotechnology, the forest industry is
bound to witness a revolution in the practices and
products of forestry.
References
I
2
3
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
Cheliak W M & Rogers D L, Call J For Rcs, 20 (1990) 452.
Winton L L, Am J Bot, 57 (1970) 904.
Haissig B E, Nelson N D & Kidd G H, BiolTcclZllology, 5
(1987) 52.
Zhao Y X, Yoa D Y & Harris P J C, NitrogclI Fixillg Tree
Res Rep, 8 (1990) 113.
Ochatt S J, Patat-Ochatt E M, Rech E L, Davey M R &
Power J B, Theor Appl Gellet, 78 (1989) 35.
Ochatt S J & Power J B, Tree Physiol, 5 (1989) 259.
Ochatt S J & Power J B, Plallt Cell Reports, 8 (1989) 365.
Horsch A, Fraley R T, Rogers S G, Sanders P R, Lloyd A &
Hoffman N, Science, 223 (1984) 496.
Barton K A, Binns A N, Mattzke A J M & Chilton M D,
Cell, 32 (1983) 1033.
Caplan A, Herrera-Estrella L, Inze D, Van Haute E, Van
Montagu M, Schell J & Zambryski P, Scicllce, 220 (1983)
815.
Leple J C, Miranda Brasileiro A C, Michel M F, Delmotte F
& Jouanin L, Plant Cell Reports, II (1992) 137.
Machado A da C, Katinger H & Machado M L da C,
Euphytica, 77 (1994) 129.
Davis J M & Keathley D E, Call J For Rcs, 19 (1989) 1118.
Pang S Z & Sanford J C, J Allier Soc Hart Sci, 113 (1988)
287.
ConfaIonieri M, Balestrazzi A, Bisofli S & Cella R, Plant
Cell Tissue Organ Cult, 43 (1995) 215.
Archilleti T, Lauri P & Damiano C, Plant Cell Reports, 14
(1995) 267.
Kajita S, Osakabe K, Katayama Y, Kawai S. Matsumoto Y,
Hata K & Morohoshi N, Plant Sciellcc, 103 (1994) 231 .
Confalonieri M, Balestrazzi A & Bisofti S, Plant Cell
Reports, 13 (1994) 256.
James D J, Passey A J, Barbara D J & Bevan M, Plallt Ccll
Reports, 7 (1989) 658.
Davis J M, Clarke H R G, Bradshaw H A & Gordon M P,
Plant Mol Bio, 17 (1991) 631.
I
21
Fillati J J, Sell mer J, McCown B. Haissig B & Comai L.
Mol Gen Genet, 206 (1987) 192.
KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Fitch M M M, Manshardt, R M, Gonsalves, D & Slightom J
L, Plant Cell Reports, 12 (1993) 245.
Machado A da C, Puschmann M, Piihringer H, Kremen R,
Katinger H & Machado M L da C, Plant Cell Reports, 14
(1995) 335.
Howe G T, Goldfarb B & Strauss S H, Plant Cell Tissue
Organ Cult, 36 (1994) 59.
Sullivan J & Lagrimini L M, Plant Cell Reports, 12 (1993)
303.
Mourages F, Chevreau E, Lambert C & De Bondt A, Plant
Cell Reports, 16 (1996) 245.
Sriskandarajah S, Goodwin P B & Speirs J, Plant Cell
Tissue Organ Cult, 36 (1994) 317.
Schaart J G, Puite K J, Kolova L & Progerbnyak N,
Euphytica, 85 (1995) 131.
De Bondt A, Eggermont K, Druart P, De Vii M, Goderis I,
Vanderleyden J & Broekaert W F, Plant Cell Reports, 13
(1994) 587.
Tsai C-J, Podila G K & Chiang V L, Plant Cell Reports, 14
(1994) 94.
Seguin A & Lalonde M, Plant Cell Reports, 7 (1988) 367.
Manders G, Dos Santos A V P, Utra Vaz F B, Davey M R
& Power J B, Plant Cell Tissue Organ Cult, 30 (1992) 69.
Teulieres C, Grima-Pettenati J, Curie C, Tossie J & Boudet
A-M, Plant Cell Tissue Organ Cult, 25 (1991) 125.
Klein T M, Wolf E D, Wu R & Sanford J L, Nature, 327
(1987) 70.
McCown B H, McCabe D E, Russell D R, Robison D J,
Barton K A & Raffa K F, Plant Cell Reports, 9 (1991) 590.
Fitch M M M, Manshardt R M, Gonsalves D, Slightom J L
& Sanford J C, Plant Cell Reports, 9 (1990) 189.
Wilde H D, Meagher R B & Merkle S A, Plant Physiol, 98
(1992) 114.
Yao J-L, Wu J-H, Gleave A P, Morris BAM, Plant Sci,
1\3 (1996) 175.
Chowdhury M K U, Parvez G K A & Saleh N M, Plant Cell
Reports, 16 (1997) 277.
Arokiaraj p, Jones H, Cheong K F, Coomber S &
CharI wood B V, Plant Cell Reports, 13 (1993) 425.
Gartland J S, Gartland K M A, Main G D, Brasier C M &
Fenning T M, Biologia Plantarum, 36 (1994) 331 .
Rochange F, Serrano L, Marque C, Teulieres C & Boudet A
M, Plant Cell Reports, 14 (1995) 674.
Cabrera-Ponce J L, Vegas-Garcia A & Herrera-Estrella L,
Plant Cell Reports, 15 (1995) 1.
Van Boxtel J, Berthouly M, Carasco C, Dufour M & Eskes
A, Plant Cell Reports, 14 (1995) 748.
Tzfira T, Zuker A & Altman A, TibTech, 16 (1998) 439.
De Block M, Plant Physiol, 93 (1990) II.
\
Ryan C A, Ann Rev Phytopathol, 28 (1990) 425.
Klopfenstein N B, Shi N Q, Kernan A, McNabO'H S, Hall R
B, Hart E R & Thornburg R W, Can J For Res, 21 (1991)
1321.
Cornu D, Leple J C, Bonade-Bottino M, Ross A, Augustin
S, Delplanque A, Jouanin L & Pilate G, In: M. R. Ahuja et
ai, (eds.) Somatic Cell Genetics and Molecular Genetics of
Trees (Kluwer Academic Publishers) 1996, 131.
Bolyard M G, Hajela R K & Sticklen M B, J Arboric, 17
(1991) 34.
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
637
Hollings M L & Brunt A A, In: Kustak E, (ed.) Handbook
of plant virus infections: Comparative diagnosis (Elseiver)
1981, p 731.
Powell A P, Nelson R S, De B, Hoffman N, Rogers S G,
Fraley R T & Beachy R N, Science, 232 (1986) 738.
Machado M L da C, Machado A da C, Hanzer V, Weiss H,
Regner F, Steinkellner H, Mattanovich D, Plail R, Knapp E,
Kalthoff B & Katinger H, Plant Cell Reports, II (1992) 25 .
Fitch M M M, Manshardt R M, Gonsalves D, Slightom J L
& Sanford J C, Biolfechnology, 10 (1992) 1466.
Choi G H & Nuss D L, Science, 257 (1992) 800.
Asada K & Takahashi M, In : D T Kyle et ai, (eds.)
Photoinhibition (Elsevier) 1987, P 227 .
Bernard J E et ai, Changes in forest health and productivity
in the United States and Canada, NAPAP State of
Scienceffechnology Report 16, 1991.
Wang D, Karnosky D F & Bormann F H, Call J For Res, 16
(1986) 47.
Karnosky D F, Bioscience, I (1981) 14.
Podila G K & Karnosky D, Chem & Industry, Dec (1996)
976.
Tanaka K & Sugahara K, Plant Cell Physiol, 21 (1980) 60 I.
Jager H J, Bender J & Grunhage L, Environ Pull, 26 (1985)
1425.
Foyer C H, Souriau N, Perret S, Lelandais M, Kunert K-J,
pruvost C & Jouanin L, Plant Physiol, 109 (1995) 1047.
Altman A, Pelan D, Yarnitsky 0, Tzfira T, Yaari A, Wang
W-X, Shoseyov 0, Vainstein A, Huttermann A & Wang S,
In: M. R. Ahuja et ai, (eds.) Somatic cell genetics alld
molecular genetics of trees (Kluwer Academic Publishers)
1996,47.
Altman A, Alegrand T, Pelah D, Tztira T. Vinocur B, Wang
W & Yarnitzky 0, Dendrome, 3 (1996) 5.
Van Doorsselaere J, Baucher M, Chognot M, Chabbert B,
Tollier M-T, Petit-Conil M, Leple J C, Pilate G, Cornu D,
Monties B, Van Montagu M, Inze D, Boerjan W & Jouanin
I, Plant J, 8(6) (1995) 855.
Baucher M, Chabbert B, Van Doorsselaere J, Pilate G.
Cornu D, Petit-Conil M, Montis B, Van Montagu M, Inze
D, Jouanin L & Boerjan W, In: M. R. Ahuja et ai, (eds.)
Somatic cell genetics and molecular genetics of trees
(Kluwer Academic Publishers) 1996. 153.
68
Chapple C C S, Plant Cell, 4 (1992) 1413 .
69
Chapple C C S, Plant Physiol, 108 (1995) 875.
70
Imberty A, Goldberg R & Catesson A M, Planta, 164
(1985) 221.
71
Dale P J, Plant Physiol, 100 (1992) 13 .
72
Keeler K, Biolfechnology, 7 (1989) 1134.
73
Strauss S H, Rottmann W H, Brunner A M & Sheppard L
A, Mol Breeding , 5 (1995) 26.
Weigel D & Nilsson 0 , Nature, 377 (1995) 495.
74
75
76
77
78
Bao X, In: Meilan R & Strauss S H, Dendml11e, 4 (1997) 5.
Teasdale R D, in M. R. Ahuja el ai, (eds.) Somatic cell
genetics and molecular genetics of trees (Kluwer Academic
Publishers) 1996, 69.
Sederoff R R & McCord S, Dendrollle, 4( I) (1997) 3.
Rugini E, Pellegrineschi A, Mencuccini M & Mariotti D,
Plant Cell Reports, 10 (199 J) 290.
638
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
INDIAN J EXP BIOL, JULY 1999
Phelep M, Petit A, Martin L, Duhoux E & Tempe J,
Bioffechnology, 9 (1991) 461.
Franche C, Diouf D, Le Q V, Bogusz D, N'Diaye A, Gherbi
H, Gobe C & Duhoux E, Plant J, II (1997) 897.
Mackay J, Seguin A & Lalonde M , Plant Cell Reports, 7
(1988) 229.
Naina N S, Gupta P K & Mascarenhas A F, Curr Sci, 58
(1989) 184.
Keinonen-Mettala K, Pappinen A & Von Weissenberg K,
Plant Cell Reports, 17 (1998) 356.
Yang J-S, Yu T-A, Chong Y-H, Yeh S-D, Plant Cell
Reports, 15 (1995) 459.
Seabra R C & Pais M S, Plant Cell Reports, 17 (1998) 177.
Le, Q V, Bogusz D, Gherbi H, Lappartient A, Duhoux E, &
Franche C, Plant Science, 118 (1996) 57.
Perez-Molphe-Balche & Ochoa-Alejo N, Plant Cell
Reports, 17 (1998) 591.
Vardi A, Bleichman S & Aviv D, Plant Science, 69 (1990)
199.
Ho C-K, Chang S-H, Tsay J-Y, Tsai C-J, Chiang V L &
Chen Z-Z, Plant Cell Reports, 17 (1998) 675.
Machado L de 0 R, de Andrade G M, Cid L P B, Penchel R
M & Brasileiro A C M, Plant Cell Reports, 16 (1997) 299.
Arokiaraj P, Yeang H Y, Cheong K F, Hamzah S, Jones H,
Coomber S & Chari wood B V, Plant Cell Reports, 17
(1998) 621.
Dandekar A M & Martin L A, J Amer Soc Hort Sci, 113
(1988) 945.
Jouanin L, EIEuch C, Pastuglia M, Capelli P, Doumas P &
Jay-Allemand C, in M. R. Ahuja et al, (eds.) Somatic cell
genetics and molecular genetics of trees (Kluwer Academic
Publishers) 1996, 147.
Sriskandarajah S & Goodwin P, Plant Cell Tissue Organ
Cult, 53 (1998) I.
Norelli J L & Aldwinckle H S, J Amer Soc Hort Sci, 118
(1993)311.
96
97
98
99
100
101
102
103
104
Maximova S N, Dandekar A M & Guiltinan M J, Plallt Mol
Bioi, 37 (1998) 549.
Mathews H, Litz R E, Wilde H D, Merkle S A & Wetzstein
H Y, III Vitro Cell Dev Bio, 28 (1992) 172.
Confalonieri M, Balestrazzi A & Cella R, Plant Cell Tissue
Organ Cult, 48 (1997) 53.
Tzfira T, Ben-Meir H, Vainstein A & Altman A, Plant Cell
Reports, 15 (1996) 566.
Tsai C-J, Popko J L, Mielke M R, Hu W-J, Podila G K &
Chiang V L, Plant Physiol, I 17 (1998) 101.
Donahue R A, Davis T D, Michler C H, Riemenschneider D
E, Carter D R, Marquardt P E, Sankhla N, Sankhl a D,
Haissig B E & Isebrands J G, Can J For Res, 24 ( 1994)
2377.
Brasileiro A C M, Leple J-C, Muzzin J, Ounnoughi D,
Michel M-F & Jouanin L, Plallt Mol BioI, 17 (1991) 441 .
Arisi A-C M, Comic G, Jouanin L & Foyer C H, Plant
Physiol, 117 (1998) 565.
Ahuja M R & Fladung M, in M. R. Ahuja et al, (eds.)
Somatic cell genetics and molecular genetics of trees
(Kluwer Academic Publishers) 1996,89.
105 Cruz-Hernandez A, Witjaksono, Litz R E & Gomez Lim M,
Plant Cell Reports, 17 (1998) 497 .
106 Mante S, Morgens P H, Scorza R, Cordts J M & Callahan A
M, Bioffechnology, 9 (1991) 853.
107 Hammerschlag F A, Owens L D, & Simogocki A C, J Alii
Soc Hort Sci, 114 (1991) 508.
108 Han K-H, Keathley D E, Davis J M & Gordon M P, Plant
Science, 88 (1993) 149.
109 Sain S L, Oduro K K & Furtek D B, Plant Cell Tissue
Organ Cult, 37 (1994) 243.
110 Fenning T M, Tymens S S, Brasier C M, Gartland J S &
Gartland K M A, in M. R. Ahuja et ai, (eds.) Somatic cell
genetics and molecular gelletics of trees (Kluwer Academic
Publishers) 1996, 105.