CURSO DE MEJORAMIENTO GENETICO FORESTAL …colbun.utalca.cl/intercambio/.../mgforestal/Manual_Adobe/apunte002.pdf · Fco. Zamudio (PhD) Genética & Mej. Forestal Universidad de Talca

Fco. Zamudio (PhD) Genética & Mej. Forestal

Universidad de Talca

1

CURSO DE MEJORAMIENTO GENETICO FORESTAL MANUAL: INTRODUCCION A LA GENETICA FORESTAL CAPITULO 2

“BIOTECNOLOGIA: TRASFORMACION GENETICA Y SU USO POTENCIAL EN ESPECIES FORESTALES” (Versión en Ingles)

1. INTRODUCTION

Plant genetic engineering has been carried out for thousands of years and has generated a rich

variety of food crops such as corn, wheat, rice, soybean, potato, etc. But, breeding, as a

biotechnology practice, started being systematically applied to crop improvement since the

firm of Vilmorin in France began selecting for sugar content in beets in 1727

(Riemenschneider et al, 1987). Increases in agriculture productivity during the twentieth

century have occurred through use of original breeding strategies to produce crops that show

resistance to disease and pests, greater yield, and better performance in many environments.

Additionally, practices such as the use of pesticides, herbicides, and fertilizers, have

contributed to increase the crop productivity.

The most traditional approaches to plant genetic engineering are limited by the gene

pool of the species of interest and the generation time it takes to transfer a given gene by

breeding into a crop plant. But the past decade has produced a revolution in plant molecular

and cell biology. Now, it is possible to transfer foreign genes with ease to almost all major

crop plants and regenerate fertile, genetically engineered plants from tissue culture (Gasser

and Fraley, 1989). The ability to produce a new genetically engineered plant variety is

limited only by the ability to identify and isolate genes that control important qualitative and

quantitative plant characters.



2

Already, crop plants have been genetically engineered for insect and viral resistance

(Hain et al, 1993; James et al, 1992), delayed fruit ripening (Redenbaugh and Hiatt, 1993),

improved nutritional quality (Hildebrand et al, 1993), flower color (Johnstone, 1992),

chilling tolerance (Baertlein et al, 1992), and male fertility control (Strange, 1993;

Hernould et al, 1993). Now, researchers have shown that it is possible to use gene transfer

technology to redirect the innate biosynthetic pathways of plants. With genetic manipulation,

plants can be coaxed into producing more of the natural products that they normally make,

such as comestible oils, waxes, lipids, and starches, as well as products which are not in

their biochemical pathways, such as biopolymers, including polyesters (Poirier et al, 1992).

On the other hand, Tree improvement involves the identification, recovery, and

multiplication of unique, useful gene combinations that provide the desired growth, quality,

and stress resistance traits to forest trees. The long generation time of trees, incomplete

knowledge about juvenile-mature trait correlations, and difficulty in obtaining the transfer

and expression of desirable genes at high frequencies seriously limit the success of the

classical selection, breeding, and testing cycle.

Biotechnology has several advantages over classical breeding in forest tree

improvement (Ostry and Michler, 1993). This technology may reduce the time necessary to

introduce new traits into desirable species and can overcome long generation times typical

with forest trees for production of improved genotypes. But, the sum of critical differences

between forestry and agronomy suggests that forestry needs its own unique strategies for

applying biotechnology in tree genetics and breeding. For Haissig and Riemenschneider

(1993), such strategies do not exist in forestry or are deficient.

This paper reviews the methods currently used in genetic transformation and gives a

background on the present state of the transgenic studies in plants. It also addresses the

possible applications in forestry and discusses the strategic planning of transgenic

experiments in forestry, that would lead to a possibly more rapid genetic improvement in

economic traits than is now possible with conventional methods of tree breeding. The

differences with agronomy and the pitfall and difficulties are emphasized. A final conclusion

is also included.



3

2. REVIEW OF METHODS CURRENTLY USED IN TRANSGENIC STUDIES IN PLANTS

2.1 Background

Modification of crop plants to improve their suitability for cultivation has persisted for at

least 10,000 years. Early farmers produced better crops simply by saving the seeds of

desirable plants. During the last century, plant breeding become more rigorous in its

approach. But now, there exists a promising method of developing superior plants: genetic

engineering. By using recombinant DNA techniques, biologists can direct the movement of

specific and useful segments of genetic material between unrelated organisms. This approach

can add a significant degree of diversity to the total repertoire of traits from which the plant

breeder can select (Gasser and Fraley, 1992). In the laboratory, plants has been modified to

withstand insects, viruses, and herbicides, to produce fruits that resist spoilage, and to

provide grain more nutritious and economical.

Biologists created the first transgenic plant less than 10 years ago. Since then,

researchers have applied genetic engineering to more than 50 plant species (Gasser and

Fraley, 1992). In this section, I will describe the methods used at present to engineer plants

genetically. As it can be deduced, genetic engineering is nothing but plant breeding done

with high precision.

2.2 Methods

The first practical, and still the most widely used, system for genetic engineering of plants

relies on an innate ability of the plant pathogens Agrobacterium tumefaciens and A.

rhizogenes to induce crown gall and hairy root disease in many dicotyledonous plants. Each

bacterium contains a specific plasmid (Circular pieces of DNA found outside the

chromosome), the tumor-inducing (Ti) or the root-inducing (Ri) plasmid, respectively,

which are involved in the process (Manders et al, 1992). The Ti and Ri plasmids both

contain a region known as the transfer-DNA (T-DNA), which is excised from the plasmids

and which integrates into the host nuclear DNA. Virulence (vir) genes located on the Ti/Ri

plasmid synthesize enzymes which respond to chemical induction and excise the T-DNA and

mediate T-strand formation, although the precise mechanism of T-DNA transfer and

integration into the genome of plant cells remain unclear. After integration into



4

chromosomes of infected plant cells, genes included in T-DNA induce the cells to produce

elevated level of plant hormones, which causes the plant to form tumors or prolific root

masses that supply an adequate environment and nutritious source for the Agrobacterium

strain.

The removing of the disease-causing genes from the Ti plasmid, alteration known as

disarming, makes the bacterium an effective and efficient vector for carrying new genes into

plants, this means DNA transfer (Moffat, 1992). Researchers at the Monsanto Corp.,

Washington University, the Max Planck Institute for Plant Breeding in Cologne, and State

University of Ghent in Belgium first accomplished the task in 1983. Disarming thus

eliminates the bacterium's ability to cause disease but leaves the mechanism of DNA transfer

unaltered.

The first engineered gene, constructed with Agrobacterium in the early 1980s by

groups at the Max Planck Institute in Cologne and at Monsanto, made plant cells resistant to

the antibiotic kanamycin, a growth inhibitor. This achievement showed that foreign genes

and proteins could be expressed in plants, and demonstrated that kanamycin resistance is

useful as a "marker". Because a small number of cells take up, integrate, and express

introduced DNA, marker genes helps to identify those cells into which genes have

successfully been introduced.

Because plant cells are totipotent, which means that cells can generate a whole

organism, a complete, reproductively competent plant can emerge from the transformed cell.

As a result, most methods today rely on the cells of explants, or small pieces of plant for

genetic engineering.

Although the method is simple and precise, many plant species, including rice,

corn, and wheat, are not natural hosts for Agrobacterium and so are not readily transformed

by the method. As a result, alternative systems have been developed. One of the first was

the introduction of DNA into plant protoplasts (plant cells that have had their cell walls

removed by enzymes). Protoplasts are used because the pores of cell walls are too small to

allow the easy passage of DNA, and the only barrier in protoplasts is the plasma membrane.

The most commonly used chemical delivery agent is polyethylene glycol. It penetrates the

plasma membrane and transport the DNA. Electroporation can also carry DNA across the



5

plasma membrane. In this process, short, high-voltage pulses briefly produce pores in the

protoplast membrane, and DNA uses these spaces to enter the cell.

The former are, in principle, general methods of transforming cells because these

procedures do not rely on any special biological interaction. But the regeneration of plants

from isolated protoplasts has proven problematic in different species, especially cereal

grains. Corn and wheat respond very poorly, usually yielding infertile plants. Consequently,

investigators have developed systems that introduce DNA into intact plant cells that still have

their walls. In 1987, researchers at Cornell University constructed a practical device that

used tungsten particles to bombard many plant cells with genetic material. They surmised

that small metal particles, with a diameter of one or two microns, previously coated with

DNA, and sufficiently accelerated, could penetrate the walls of intact cells and thus deliver

the DNA. The small holes produced by the "particle gun" in cell walls and membranes

rapidly close by themselves and do not compromise the integrity of the cells. The particles

remain in the cytoplasm but are too small to interfere with any cellular function. Researchers

at Agracetus in Midleton, Wis., developed a similar gun using gold particles propelled by

the vaporization of a water droplet. Both of these particle guns have produced transgenic

plants. On 1991, other efficient, consistently functioning particle gun systems for the

transformation of corn was independently developed by Monsanto and a group at DeKalb

Plant Genetics in Groton, Conn. Finally, the factors that affect the biolistic transformation

rate of embryogenic cell suspensions and its optimization have been recently discussed by

Hébert et al (1993).

Introducing DNA into cells is not the only step in the process of transforming plants.

The genetic fragments are to be manipulated to produce a useful phenotype, a plant variety

possessing the desired characteristics. The modular nature of genes facilitates this task.

Genes that encode, or produce, proteins are in a broad sense made up of only three regions:

a promoter sequence (which helps specify timing and location of gene expression); a coding

region (it contains the information that determines the nature of the protein encoded by the

gene); and the polyadenylation region (which ensures that the messenger RNA transcript

terminates correctly).

The assembling of components from different genes results in what are commonly

referred to as chimeric genes. In principle, the coding region of the chimeric gene can come



6

from any organism. This remarkable flexibility is the main advantage of genetic engineering

over more traditional methods, which can transfer genes only between closely related

species. Furthermore, by choosing various promoters, researchers can target gene expression

to specific organs such as leaves, roots, seeds and tubers, and in many cases, to specific

cell types within these complex tissues (Gasser and Fraley, 1992).

3. CURRENT STATUS OF GENETIC TRANSFORMATION RESEARCH IN PLANTS

As described by Rob Fraley of Monsanto Corp. (Moffat, 1992), the first area of application

for genetically engineered plants was the delivery to established crops of new, improved

agronomic traits, such as control of insects, weeds, and disease resistance. Then plants were

modified with improved traits that affect food processing, such as slowing down ripening in

tomatoes. Now, plants are being used to produce specialty chemicals and novel

biopolymers.

Additional advances in the simplicity and breadth of genetic engineering techniques

and increasing knowledge of plant biology promise to extend greatly the beneficial changes

that gene transfer can offer. Below, I give some examples.

3.1 Disease Resistance

Resistance to diseases is one of the most promising traits offered by gene transfer techniques

and stimulating results have been achieved in creating plants resistant to viruses. Researchers

at Washington University and Monsanto constructed a vector to introduce and express in

tomato and tobacco plants the coat protein of the tobacco mosaic virus (TMV). Plants so

modified were then inoculated with a heavy concentration of the virus. The plants were

found to be strongly resistant to infection.

Subsequent experiments have shown that the expression of the TMV coat protein

confers resistance only to strains of TMV and a few other closely related viruses. Still, the

mechanism appears to be generally applicable; expression of the coat protein gene of almost

any plant virus, at a sufficiently high level, protects against infection by that virus. Workers

have now engineered effective tolerance to more than a dozen different plant viruses in a

broad range of crop species (Gasser and Fraley, 1992).



7

3.2 Insect Resistance.

Resistance to insect predation is another important goal for genetic engineering, especially in

cotton, potato, and corn plants. Traditionally, farmers have relied on the bacterium Bacillus

thuringiensis (Bt), which produces an insecticidal protein. Commonly used preparations of

Bt are highly specific to the caterpillar larvae of lepidopteran insects (moths and butterflies)

which are major pests. The Bt protein bind to specific receptors located on the gut

membranes of the target insects. The binding interferes with ion transport in the epithelial

cells of the gut, thus disrupting the insect's ability to feed.

In the mid-1980s researchers at several companies, such as Plant Genetic Systems in

Ghent, Belgium, Agracetus, and Monsanto succeeded in isolating from the bacterium genes

for the insecticidal protein. They used the particle gun and A. tumefaciens to insert the genes

into tomato, potato, and cotton plants. Monsanto scientists redesigned the original bacterial

gene to mimic more closely the plant DNA sequences. The changes dramatically enhanced

insect control. Gasser and Fraley (1992), mention that two years in field testing confirm that

the presence of these Bt genes within cotton plants effectively control all major caterpillar

pests. These genetically engineered plants should reduce the use of insecticides on cotton by

about 40 to 60 percent.

Scientists have screened extensively for naturally occurring B. thuringiensis strains

that are effective on insects other than caterpillar. One such strain led to the redesign of a

gene that is effective against the Colorado potato beetle. In the summer of 1991, Russet

Burbank potato plants expressing a beetle-control gene were tested at several sites from

Maine to Oregon. Results indicated that the potato plants were essentially immune to beetle

damage. Bt genes acting against plant parasitic nematodes and Bt genes active against

mosquitoes have also been identified (Gasser and Fraley, 1992).

3.3 Herbicide Tolerance

Crops are also challenged by weeds. Weeds that compete for moisture, nutrients and

sunlight can reduce a field's potential yield by 70 percent. Moreover, weed material in the

harvest significantly reduces the value of the crop, and weeds serve as a habitat for pests.

The use of genetic engineering has been strategically oriented to create plants that can

tolerate exposure to a single broad-spectrum, environmentally safe herbicide.



8

There have been two general approaches to engineering herbicide tolerance.

Researchers at Monsanto and at Calgene in Davis, Calif., have worked to enable plants to

tolerate glyphosate, the active ingredient of a herbicide called Roundup, a broad-spectrum

herbicide that control broadleaf and grassy weeds. The compound kills plants by inhibiting

the action of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a necessary enzyme for

the production of the aromatic amino acids that a plant needs for growing. Roundup is

particularly attractive because does not affect animals and it degrades rapidly in the

environment into harmless, natural compounds.

In 1983, a research group at Monsanto isolated the gene for (EPSP) synthase from

bacteria and plants, and also identified variants of the genes that produce proteins with a

reduced sensitivity to Roundup. Later, researchers constructed genes that produced higher

quantity of these proteins in plants and introduced them into tomato, soybean, cotton,

oilseed rape, and other crops. Field tests performed in the US, Canada, and Europe, since

1989, demonstrated that the crops were able to tolerate treatments with Roundup at levels

that effectively controlled weeds. Researchers at Du Pont have used a technically similar

approach to engineer plants that can tolerate certain kinds of sulfonylurea herbicides. The

agronomic performance of sulfonylurea-resistant transgenic tobacco plants grown under field

conditions has been recently reported by Drandle and Miki (1993).

Using another approach to herbicide tolerance, scientists at the German company

Hoechst isolated, from the microbe Streptomyces hygroscopicus, a gene for an enzyme that

inactivates a herbicide called Basta, which affect the glutamine synthase pathway in weeds

and thus interferes with their growth. Plants carrying this protective gene inactivate Basta

before damage can occur. Field tests performed on the Basta-tolerant plants demonstrate the

effectiveness of the protection.

3.4 Fruit Ripening

Researchers have already identified and isolated several genes that play a role in the

biosynthesis of ethylene, the signal molecule that triggers the ripening of fruits (Deikman et

al, 1992). Delayed spoilage would allow harvesting at a later stage than is currently practical,

which may improve the flavor and even the nutritional value.



9

To increase the shelf life of fruit, two genetic methods have been developed. The

first has been the insertion of antisense version of the ripening genes. Antisense molecules

binds with specific messenger RNA to turn off the genes. Researchers at the USDA in

Albany, Calif., and the University of Notthingham have shown that fruits of tomato plants

with the antisense genes resist ripening. In a different approach, researchers at Monsanto

have introduced a gene into tomato plants that induces them to manufacture an enzyme that

degrades the precursor compounds that form ethylene, thus retarding spoilage (Redenbaugh

and Hiatt, 1993).

3.5 Animal Proteins, Enzymes, and Antibodies

Researchers have shown that plants are very good at making several human and mouse

proteins that have potential application in therapeutic and diagnostic medicine. In late 1988,

a group at Michigan State University and at the Miescher Institute in Basel, Switzerland,

introduced the gene for human interferon (a protein with natural antiviral activity) into turnip

plants for testing the hypothesis that interferon could make plants more resistant to viral

infections. Although the transgenic plants were just as susceptible to infection by turnip

yellow mosaic virus as the control plants, the Swiss group noted that their transgenic plants

made large amounts of the interferon and, which is as important, the protein was active in

animals.

At about the same time, a group at the Scripps Research Institute in la Jolla,

California, began experiments aimed at improving the resistance of tobacco plants to tobacco

mosaic virus (TMV) and also to certain nematodes that are serious plant pests. The strategy

was to genetically engineer the plants to produce antibodies against the pests. The results

have shown that tobacco plants can synthesize large quantities of mouse antibodies and that

the plant-made antibodies behave just like normal antibodies, at least in test-tube studies

(Moffat, 1992). Of the variety of bioactive compounds expressed in transgenic plants,

antibodies may offer the widest range of possible applications. Production of antibodies by

plant cells offers a variety of new possibilities for basic research in plant biology as well as

for large-scale production of antibodies for use in therapeutic, diagnostic, or affinity

reagents. The capacity and flexibility of agricultural production suggests that antibodies

derived from plants may be significantly less expensive than antibodies from any other

source. Moreover, antibodies in plants may become useful reagents for isolating and



10

processing environmental contaminants and industrial biproducts (Hiatt, 1990; Hiatt and

Mostov, 1993).

Still another group, this one at the agriculture biotechnology company Mogen

International, NV, in Leiden in the Netherlands, found that genetically transformed potato

and tobacco plants could produce human serum albumin (HSA) that is indistinguishable from

the genuine human protein. HSA was chosen as a first target by the group because of its

relative high value, broad clinical use, and a worldwide demand estimated to be in ton

quantities (Pen et al, 1993).

But while the former results point up the potential importance of plants as factories

for human biologicals, the first marketable chemicals from transgenic plants will probably

not be drugs, which will have to undergo rigorous testing for safety and efficiency. Instead,

they are likely to be enzymes used for food processing and other industrial applications, such

as the starch-digesting enzymes alpha-amylases. The enzymes from bacterial and fungal

origins are widely used. For example, they are used for starch processing and alcohol

industries for liquefaction, in the brewing industry for producing low-calorie beer, in the

baking industry for increasing bread volume, in the juice and wine industry for clarification,

and in the detergent industry (Pen et al, 1993). Both Biogen and the US biotech firm

Biosource Genetics have shown that tobacco plants can make the protein. Transgenic plants

grew just as vigorously as control plants, and the plant enzyme was just as effective in

breaking down starch as the bacterial enzyme. Indeed, researchers did not have to purify the

enzyme, just to mill the seeds from the transgenic plants (Pen et al, 1993).

3.6 Redirected Pathways

Moffat (1992) refers to some research teams to show that now it is possible to use gene

transfer technology to redirect the natural biosynthetic pathways of plants. A group from

Michigan State University genetically engineered Arabidopsis to produce granules of

polyhydroxybutyrate (PHB), a polyester used for plastic containers normally obtained from

the bacterium Alcaligenes eutrophus. The team transferred two of the genes the bacterium

uses to make PHB into the plants. As a result, the flow of carbon through the plants'

metabolic cycles was directed away from the pathway that leads to production of plant

hormones into PHB synthesis (Poirier et al, 1992). The plant PHB was stored in granules,

much as it is in bacteria. Moffat also mention the results of a work group at Calgene Inc. of



11

Davis, Calif., that redirected canola plants to produce either of two different oils with unique

fatty acids compositions that are of value for the food and detergent industries. Moreover, in

other project, Calgene researchers introduced two genes into canola that should allow the

plant to express an enzyme that catalyzes the production of jojoba oil.

4. INTEGRATING GENETIC TRANSFORMATION IN FORESTRY. REVIEW OF APPLICATIONS

4.1 Background.

Forest trees are mostly selected for large size and high timber production. Other

improvements are based on the ecological adaptability of the trees to grow in marginal soils

and to exhibit increased resistance/tolerance to abiotic and biotic stresses. For example, it

would be desirable to create drought-tolerant root systems which can withstand extreme

levels of nutrient and toxic substances. In addition, the ability to utilize atmospheric nitrogen

would remove the necessity to use large amounts of costly nitrogen-based fertilizers. This

could possibly be achieved by inducing the association of nitrogen-fixing microorganisms

with tree root systems, as occurs naturally in some woody genera such as Alnus (Manders et

al, 1992). The same reasoning also applies to extensive application of herbicides and

pesticides. These compounds are not always effective due to such chemical agents being

equally toxic to both the trees and the weed or specific disease/pest targeted. Consequently,

it would be desirable to incorporate into trees heritable disease and pest resistance and

tolerance to commonly-used herbicides, leading to more efficient and restricted application of

chemical agents.

The major interest in biotechnology has arisen in forestry since the early 1980s,

although biotechnology-like or biotechnology-related research has been conducted for much

longer (Haissig and Reimenschneider, 1993). The potentials of biotechnology in tree

genetics and breeding is an important discussion issue for two main reasons. First, tree

genetics has been studied for at most 75 years, under conditions of minimal scientific

infrastructure, thus more knowledge is needed (Bey et al, 1986). Second, human needs have

progressed faster than product development through tree breeding, so more products are

needed (Farnum et al, 1983). The application of biotechnology may alleviate the two

mentioned deficiencies.



12

The belief that biotechnology will be a means to effectively increase genetic

understanding and to speed genetic improvement of trees is sustained in the optimistic vision

that better genetic knowledge and more robust genetic technologies will initiate a chain

reaction of genetic-based improvement in productivity, stimulated by ever increasing

biological knowledge (Haissig and Reimenschneider, 1993). Nonetheless, it is unclear to

some forest researchers how biotechnology should or could be cohesively applied to trees to

efficiently achieve that strategic vision or any other strategic vision.

The current situation is confusing because biotechnology has been rapidly

incorporated across fields within forest biology such as genetics, breeding, physiology,

biochemistry, and ecology, but it is not generally understood how knowledge and pre-

existing technologies from these separate fields should be combined to enhance our

understanding of forest tree biology. Uncertainty has increased with the advent of

biotechnology, especially because biotechnology has increased research interest in definitive

genetic approaches to problem solving, and such genetic approaches were not previously

available to tree molecular biologists, physiologists, and biochemists due to the slowness of

tree breeding (Riemenschneider et al, 1987).

Haissig and Reimenschneider (1993) mention that the higher plant biotechnology

community agrees on several points. Both authors reviewed a series of papers about future

applications of biotechnology to genetics and breeding of crops for agronomy and forestry.

They concluded that forestry and agronomy papers have differed little because most have

dealt with the array of technologies that are or soon will be available for vegetative

propagation or genetic modification of all higher plants. Ergo, many scientists agree that

most of the technologies can be used (perhaps with differing efficiency) with most higher

plants. Secondly, most scientists agree that biotechnology and breeding are necessary to

fulfill certain objectives, ranging from purely scientific to the purely practical. Thirdly,

most scientists (and financiers) agree that few of the remarkable promises of biotechnology

have been realized with either agronomic or forestry crops. Short time-frames of research,

and research and development are often cited or alluded to as the reasons that the potentials

have not been realized.

Most forestry biotechnology papers suggest that biotechnology is a more effective

substitute for conventional genetics and breeding; problems are not categorized by species,



13

except perhaps between angiosperms and gymnosperms (Dunstan, 1988). In comparison,

agronomic papers are more species-oriented and tend to better explain how biotechnology fits

or should fit into existing strategies for gaining knowledge to help genetically improve crops

(Cohen, 1988).

Haissig and Reimenschneider (1993) also state that a certain euphoric blindness

pervades the scientific literature and meetings about biotechnology. Perhaps forest

biotechnologists have been necessarily distracted in an initial exploration of the newly

available technologies, but the exploratory phase is rapidly approaching its end.

4.2 Transformation of forest trees.

It is now possible to transform genetically a variety of commercially important forest trees

species which opens the way for directed improvement of woody plants. Transformation has

already permitted agronomically useful genes to be inserted into forest trees, such as the

insertion of glyphosate resistance and the Bt endotoxin gene for insect resistance into hybrid

poplar explants. These advances were achieved following inoculation of explants with

Agrobacterium and bombardment of cells with DNA-coated particles, respectively,

illustrating how both well established transformation technologies are being applied to forest

tree species. Other methods of achieving transformation have involved chemical and/or

electrical uptake of DNA into isolated protoplasts.

Manders et al (1992) mention that, in spite of the recent advances in applying

transformation strategies to tree species, there are still few reports of the regeneration of

transgenic trees. The authors state that, in part, this is related to the difficulties associated

with obtaining reproducible plant regeneration from explants, via organogeneis or somatic

embryogenesis. The problems in achieving regeneration from isolated protoplasts of trees are

considerably more difficult to overcome. The continuation of studies in tissue and protoplast

culture is a basis for the application of genetic manipulation techniques.

A brief description of some of the transgenic studies in forest tree species is given

below.

Fillatti et al (1987) were the first researchers to insert an economically important

gene into a forest tree. A binary oncogenic strain of A. tumefaciens with a plasmid

containing a gene coding for bacterial EPSP synthase was inserted into Populus alba x P.



14

grandidentata leaf disks. The regenerated transgenic plants conferred kanamycin resistance

and the later field testing confirmed an increased tolerance to the commercial formulation of

glyphosate, Roundup (Riemenschneider and Haissig, 1991).

Successive work has been done to genetically transform hybrid poplars using stem

pieces from Populus trichocarpa x P. deltoides and Allocasuarina verticillata and mediated by

A. rhizogenes (Manders et al, 1992), and recently Howe et al (1994) recovered

transformants when suspension cultures of hybrid poplar (P. alba x P. grandidentata) were

inoculated with A. tumefaciens.

To date, most reports of direct gene transfer into protoplasts of woody plants have

explored the conditions for optimizing transient expression of reporter genes (Mander et al,

1992). For example, transient expression of the firefly luciferase (luc) gene in protoplasts of

Loblolly pine and Douglas fir was obtained following electroporation (Gupta et al, 1988).

This provides an alternative method of transformation to that involving the use of

Agrobacterium targeted to intact plants or explants (Sederoff et al, 1986). Transient

expression of the chloramphenicol acetyltransferase (cat) and gus genes has also been

reported using protoplasts from several forest tree species (Manders et al, 1992).

McCown et al (1991) presented the first report of the recovery of transgenic plants of

an economically important tree species following the bombardment of tissues with high-

velocity microprojectiles. Leaf mesophyll protoplast-derived cells, nodular callus and stem

segments of the poplar hybrids Populus alata x P. grandidentata and P. nigra x P. trichocarpa

were bombarded with gold particles coated with DNA. The plasmid used contained the gus,

ntpII, and Bt genes, the later being the endotoxin gene that confers on plants resistance to

lepidopteran larvae which normally feed on leaf and other tissues. Interestingly, one of the

plants exhibited a high resistance to feeding by two lepidoptera.

High-velocity bombardment of microprojectiles coated with nucleic acids has been

also used to obtain transient expression of the gus gene when introduced into cotyledonary

cells of Loblolly pine (Stomp et al, 1990) and into embryos of white spruce (Picea glauca)

(Ellis et al, 1990). Direct gene transfer methods have also been used to introduce the gus

gene into protoplasts (polyethyene-glycol mediated uptake, electroporation) and intact cells

(microprojectile bombardment) of yellow-poplar (Liriodendron tulipifera) (Wilde et al,



15

1991). In addition to other parameters, the physiological age of the protoplasts was found to

be an important factor in the transient expression of gus.

5. STRATEGIC PLANNING OF TRANSGENIC EXPERIMENTS IN FORESTRY. PITFALLS AND DIFFICULTIES

A possible pitfall in forest biotechnology experiments would be to copy exactly the major

leads of agronomy in strategic planning (Haissig and Riemenscneider, 1993), for two

reasons. First, agronomy worldwide deals with few, mostly annual (herbaceous) species;

existing genotypes have already been highly tailored to specific purposes; substantial genetic

knowledge is already available; entire crops are replanted frequently; and huge genetic

improvement programs are in place, along with strong supporting programs in soils,

entomology, and pathology. For each of the foregoing, almost the exact opposite is true for

forest tree species. Second, a major strength of agronomy has been breeding, which has not

been the case in forestry, mainly because of insufficient scientific infrastructure (Haissig and

Riemenscneider, 1993) and because flowering of trees can be a late-life trait of many forest

tree species.

Biotechnology is supposed to speed the development of products and knowledge that

tree breeding has been unable to accomplish because of various constraints, but genetic

manipulation will almost certainly require substantial breeding to verify new knowledge or to

develop products.

Haissig and Riemenschneider (1993) summarize that agronomy will supply with

useful examples but not with exact (or even nearly exact) templates for designing solving-

problem experiments. They suggest there is a persistent danger of transferring

biothechnologies developed for agronomy to forestry along with the agronomic research

goals that spawned the techniques.

The following section discusses the elements which should be considered in the

strategic planning of biotechnology experiments applied in forestry.



16

5.1 Elements of the strategic planning.

The strategic planning of experiments should involves the consideration, among others, of

the following elements:

(1) Strategic Vision - A clear strategic vision for transgenic experiments is needed to

consolidate support within the forestry community and to gather external support from

related areas of science and the public. The research will not develop efficiently unless its

vision includes obtaining knowledge for problems such as diseases, insects, wood

characteristics, growth characteristics, etc. The research vision needs to link

the development of knowledge and products so they are more logical and understandable by

scientists and the public alike.

Time-frame is one of the main points to be considered and, probably one of the main

pitfalls. In research, the time-scale for accomplishment is to be a prime consideration and

the project should be terminated if not completed in a "reasonable" period of time.

Everybody assume that there is a finite amount of time in which to demonstrate the efficacy

of an experiment in forest biology, whether for the generation of knowledge or products, or

both. Haissig and Riemenschneider (1993) consider that biotechnology can be flexibly

applied to tree genetics and breeding over a 10-year period; a longer period would be

unrealistic. Ten years is adequate for obtaining important new technology, and is about as

long as public and organizational support can be expected for any unproven research

program. But, the strategic plan should allow enough room to make modifications during

the planned period, and to compensate for unforeseeable change and to allow for

innovations.

(2) Goals - The best developing goals are those with high expectation but without a

concomitant great risk and probability of failure. A major pitfall in goal setting for

experiment planning is that the junctures of technology and specific problems are many times

chosen or made the focus of discussion before there is adequate supporting knowledge.

Overall, it is suggested to develop knowledge through applying biotechnology in important

problem arenas, communicating this to interested parties, but without having preconceived,

highly specific problem targeted.



17

(3) Species Selection - It is suggested that whether or not economics is initially the primary

basis for species selection, at the end the species selected will be the most economically

valuable because they are the ones that will be planted most or need the most protection from

damaging agents (as in agronomy). But it is questionable whether genetic engineering can

produce plants cheap enough, regardless of improved character, even for the commercially

most important species. Forestry is a consistent, low financial return, long-term investment.

Successful experimental research will only enhance the investment opportunity in forestry if

close attention is given to selection of species. The potential value of transgenic studies can

only be achieved if the genetically better clone or population is much planted or better

protected, or both. Transgenic research is going to be economically viable only if the cost

per plant is acceptably small to amortize research and development costs.

(4) Trait Selection - Riemenschneider and Haissig (1991) theorize that an appropriate target

trait for trees would achieve a characteristic that cannot be equalled or exceeded by

manipulation of other organisms in the ecosystem and/or that would result in such high added

value that economic opportunity would drive deployment of the genetically different trees. In

addition, the most suitable target trait would have four characteristics: 1) Definition based

on biochemistry rather than morphology; 2) Commonness to various plant species, thus

having a substantial knowledge base; 3) Importance in either maturation or secondary

vascularization (wood formation); and 4) Association with tree response/resistance to biotic

or abiotic damaging agents (stresses).

Bonga and von Aderkas (1992) also suggest that four criteria be met when selecting

traits to be manipulated by genetic engineering of conifers: 1) The trait should preferably be

controlled by a single gene and be dominantly expressed; 2) The molecular basis of the trait

should be known in detail to facilitate identification and isolation of the gene(s) that control

the trait(s); 3) The transformed trait should be sensitive to the conditions expected in the

plantation; 4) It would be convenient to select traits that are not of high survival value in

natural stands because the less a trait has been optimized by generations of natural selection,

the more likely it is that it can be improved.

(5) Means of Gene Transfer - McCown (1991) remarks that any gene transfer system,

whether utilized for trees or any other crop, has four requirements that must be fulfilled

before the transgenic experiment be generally successful: 1) A microculture (tissue culture)



18

system for the plant; 2) A methodology to transfer the DNA to the cultured tissues; 3)

Genes, preferably isolated and characterized; and 4) Control of the gene function in vivo.

Microculture has been and continues to be a necessity of all the most utilized genetic

transformation experiments, but developing of an effective and efficient selection system is

probably one of the most difficult parts of the microculture system assay. The selection of

the transformed cells among the masses of living, non-transformed tissue, is a critical step

most efficiently conducted in vitro. Since all gene transfer occurs at the individual cell level,

regeneration into normal plants is also essential in the experiment.

The most common vector used for transfer of specific foreign genes into the nucleus

of the recipient cell is Agrobacterium, but for most tree species the scope of such

introduction has been experimental rather than practical (Bonga and von Aderkas, 1992). As

pointed out earlier, the use of Agrobacterium as a vector still presents problems for the

recovery of transgenic plants in many forest species (Manders et al, 1992). Often these

difficulties result from the limited susceptibility of the plant tissue to specific Agrobacterium

strains and/or the inability to regenerate shoots, even where transformation of tissues has

been successful. Therefore, direct gene transfer techniques should be considered to be

explored in the time-frame of the research.

(6) Plant Regeneration - Regardless of the gene transfer system, regeneration of plants is

required after genetic modification, yet regeneration has been difficult to achieve with forest

trees. A serious limitation to the development of transgenic studies in forestry is the lack of

high-frequency regeneration in vitro, which is common to many forest tree species,

especially conifers and some angiosperms (Riemenschneider et al, 1987). For example,

some poplar can be regenerated from organ explants, callus, cells, and protoplasts, but

conifers have only been regenerated at high frequency from juvenile tissues such as

hypocotyls, cotyledons, or embryos (Riemenschneider et al, 1987). In addition, direct gene

insertion methods may require regeneration of plants from protoplasts or cells. Cellular-level

regeneration of conifers and angiosperms is improving but still deficient.

(7) Field Testing - The gene transfer techniques may increase the efficiency of tree

improvement, but it does not replace the classical selection, breeding, and field testing

cycle. Field testing will remain a critical link between the in-vitro genetic manipulation and



19

selection phase and the release of the improved tree for future research and breeding. But

field testing has other reasons to be performed.

Concerns have been expressed by the scientific community as well as by the general

public, regarding possible problems that may follow the release of genetically engineered

material in the environment. Crawley et al (1993) state that the concern center on three

conjectural risks, which constitute main pitfalls for transgenic studies: that transgenic crop

plants will become weeds of agriculture or invasive of natural habitats; that their engineered

genes will be transferred by pollen to wild relatives whose hybrid offspring will then become

more weedy or more invasive; or that the engineered plants will be a direct hazard to

humans, domestic animals or beneficial wild organisms. Therefore, guidelines have been

established for transformation research and for the release of genetically engineered material.

These should be consulted before field tests of new genetic variants are started (Bonga and

von Aderkas, 1992).

Because of the long rotation cycle of trees, the risks involved in planting genetically

engineered trees is even greater than when transgenic, short-lived agricultural crops are

planted. For example, it has been argued that large scale planting of trees that have been

made insect resistant by gene transfer could lead to the development of new insect biotypes

that are more damaging than the ones previously encountered. When planting genetically

engineered trees, measures should be taken to prevent them from shedding their pollen

because this could transfer the introduced genes to other populations. One way to tackle this

problem would be by making the transformed trees male sterile by additional genetic

engineering.

According to Bonga and von Aderkas (1992), field testing is important because: 1)

It has to be established that the mutant or vectored new genes are expressed under field

conditions and are switched on and off at the appropriate moments; 2) foreign genes may be

eliminated in the sexual process or may cause unexpected changes in the original genotype of

the tree; 3) Some physiological interaction in the tree may be affected in unpredictable ways

by foreign genes. A tree genetically engineered to grow fast may be highly susceptible to

frost damage or insect and disease attack; and 4) Many genetically engineered organisms

may be less fit in a competitive environment than the non-engineered parent.



20

6. CONCLUSIONS

Concerns about food safety, the environmental impact of management in agricultural crops

and a rapidly changing farm infrastructure have combined with a general lack of

understanding about new technologies to practically eclipse the long-term need for

economical, high-quality food products. But, biotechnology can be one of the few new

solutions to the problem of feeding an ever-growing population in the next decades.

Genetic engineering can offer the product of the very latest technology to farmers or

foresters in a very traditional package: the seed. As a direct result, even the most

impoverished regions of the world could have access to the benefits of biotechnology without

the need for high-technology supplies or costly materials.

Plant scientists emphasize that they are not creating radically different species. The

plants coming out of the laboratory are changed only in subtle and well-understood ways,

but an exhaustive field testing should be accomplished, mainly on trees because of their long

rotation cycle, before the release of new genetically engineered crops. In the case of forest

tree species, the highest chances of succeeding may be limited to traits that are probably

controlled by single or few genes. However, multiple-gene interactions are probably

involved in the response of trees to stress in a competitive environment, and these

interactions should be studied before the release of transgenic trees.

Genetic improvement of plants, especially trees, requires much time. Breeding will

remain important for improvement of trees and other plants but genetic transformation is

expected to accelerate genetic improvement directly and indirectly.



21

7. LITERATURE CITED

Baertlein, D. A.; Lindow, S. E.; Panopoulus, N. J.; Lee, S. P.; Mindrinos, M. N.; Chen, T.

H. H. 1992. Expression of a bacterial ice nucleation gene in plants. Plant Physiology 100:

1730 - 1736.

Bey, C. F.; Houston, D. B.; Dinus, R. J. 1986. II. The business of tree improvements. Tree

genetics and improvement. Journal of Forestry 84: 45 - 55.

Bonga, J. M.; von Aderkas, P. 1992. In vitro culture of trees. Dordrecht. The Netherlands.

Kluwer Ac. Press. 236 p.

Cohen, J. I. 1988. Models for integrating biotech into crop improvement programs.

Bio/Technology 6: 387 - 392.

Crawley, M. J.; Hails, R. S.; Rees, M.; Kohn, D.; Buxton, J. 1993. Ecology of transgenic

oilseed rape in natural habitats. Nature 363: 620 - 623.

Deikman, J.; Kline, R.; Fischer, R. L. 1992. Organization of ripening and ethylene regulatory

regions in a fruit specific promoter from tomato (Lycopersicon asculantum). Plant Physiology

100: 2013 - 2017.

Drandle, J. C.; Miki, D. L. 1993. Agronomic performance of sulfonylurea-resistant

transgenic flue-cured tobacco grown under field conditions. Crop Science 33: 847 - 852.

Dunstan, D. I. 1988. Prospects and progress in conifer biotechnology. Canadian Journal of

Forest Research 18: 1497 - 1506.

Ellis, D. D.; McCown, B. H.; Russell, D. R.; McCabe, D. E. 1990. A transient assay to test

heterologous promoter activity in Picea glauca (white spruce) using electrical discharge

acceleration. Journal of Cellular Biochemistry Supplement 14E, 279.

Farnum, P.; Timmis, R.; Kulp, J. L. 1983. Biotechnology of forest yield. Science 219: 694

- 702.

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.

Gasser, C. S.; Fraley, R. T. 1989. Genetically engineered plants for crop improvement.

Science 244: 1293 - 1299.

Gasser, C. S.; Fraley, R. T. 1992. Transgenic crops. Scientific American 266: 62 - 69.

Gupta, P. K.; Dandekar, A. M.; Durzan, D. J. 1988. Somatic proembryo formation and

transient expression of a luciferase gene in Douglas fir and loblolly pine protoplasts. Plant

Science 58: 85 - 92.

Hain, R.; Reif H.; Krause, E.; Langebartels, R.; Kindl, H.; Vornam, B.; Weise, W.;

Schmelzer, E.; Schreier, P. H.; Stöcker, R. H.; Stenzel, K. 1993. Disease resistance results

from foreign phytoalexin expression in a novel plant. Nature 361: 153 - 156.



22

Haissig, B. E.; Riemenschneider, D. E. 1993. Strategic planning for applying biotechnology

to woody plant genetics and breeding. In: M. R. Ahuja (Ed.) Micropropagation of woody

plants. Kluwer Ac. Pub. The Netherlands. pp. 485 - 500.

Hébert, D.; Kikkert, J. R.; Smith, F. D.; Reisch, B. I. 1993. Optimization of biolistic

transformation of embryogenic grape cell suspensions. Plant Cell Reports 12: 585 - 589.

Hernould, M.; Suharsono, S.; Litvak, S; Araya, A.; Mouras, A. 1993. Male-sterility

induction in transgenic tobacco plants with an unedited atp9 mitochondrial gene from wheat.

Proc. of the Nat. Ac. of Sc. U.S. 90: 2370 - 2375.

Hiatt, A. C. 1990. Antibodies produced in plants. Nature 344: 469 - 470.

Hiatt, A. C.; Mostov, K. 1993. Assembly of multimeric proteins in plant cells: Characteristics

and uses of plant-derived antibodies. In: Hiatt, A.C. (Ed.) Transgenic Plants: Fundamentals

and applications. M. Dekker, Inc. N.Y. pp. 221 - 237.

Hildebrand, D. F.; Deng, W.; Grayburn, W. S. 1993. Use of genetic transformation to alter

fatty acid metabolism in plants. In: Hiatt, A.C. (Ed.) Transgenic Plants: Fundamentals and

applications. M. Dekker, Inc. N.Y. pp. 3 - 21

Howe, G. T.; Goldfarb, B.; Strauss, S. H. 1994. Agrobacterium-mediated transformation of

hybrid poplar suspension cultures and regeneration of transformed plants. Plant Cell, Tissue

and Organ Culture 36: 59 - 71.

James, D. J.; Passey, A. J.; Webster, A. D.; Barbara, D. J.; Viss, P.; Dandekar, A. M.;

Uratsu, S. L. 1993. Transgenic apples and strawberries: advances in transformation,

introduction of genes for insect resistance and field studies of tissue cultured plants. Acta

Horticulture 330: 178 - 184.

Johnstone, R. 1992. Blue genes for red roses. Far Eastern Economic Review 155: 70.

Manders, G.; Davey, M. R.; Power, J. B. 1992. New genes for old trees. Journal of

Experimental Botany 43: 1181 - 1190.

McCown, R. H. Genetic transfer of trees via direct gene transfer. In: Ahuja, M. R. (Ed.)

Woody plant biotechnology. New York, N. Y. Plenum Press. NATO ASI series. pp. 207 -

211.

McCown, R. H.; McCabe, D. E.; Russell, D. R.; Robinson, D. J.; Barton, K. A.; Raffa, K. F.

1991. Stable transformation of Populus and incorporation of pest resistance by electric

discharge particle acceleration. Plant Cell Reports 9: 590 - 594.

Moffat, A.S. 1992. High-tech plants promise a bumper crop of new products. Science 256:

770-771

Ostry, M. E.; Michler, C. H. 1993. Use of biotechnology for tree improvement in Populus

model systems. In: M. R. Ahuja (Ed.) Micropropagation of woody plants. Kluwer Ac. Pub.

Netherlands. pp. 471 - 483.



23

Pen, J.; Sijmon, P. C.; van Ooijen, A. J. J.; Hoekema, A. 1993. protein production in

transgenic crops: Analysis of plant molecular farming. In: Hiatt, A.C. (Ed.) Transgenic Plants:

Fundamentals and applications. Marcel Dekker, Inc. N.Y. pp. 239 - 251.

Poirier, Y; Dennis, D.; Klomparens, K.; Somersville, C. 1992. Polyhydroxybutyrate, a

biodegradable thermoplastic, produced in transgenic plants. Science 256: 520 - 524.

Redenbaugh, K; Hiatt, W. 1993. Field trials and risk evaluation of tomatoes genetically

engineered for enhanced firmness and shelf life. Acta Horticulture 330: 133 - 140.

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.) Woody plant biotechnology. New York, N. Y. Plenum

Riemenschneider, D. E.; Haissig, B. E.; Bingham, E. T. 1987. Integrating biotechnology

into woody plant breeding programs. In: J. M. Hanover and D. E. Keathley (Eds.). Genetic

manipulation of woody plants. New York, N Y. Plenum Press. pp. 433 - 449.

Sederoff, R.; Stomp, A.; Chilton, W. S.; Moore, L. W. 1986. Gene transfer into loblolly pine

by Agrobacterium tumefaciens. Bio/Technology 4: 647 - 649.

Stomp, A. M.; Weissinger, A.; Sederoff, R. 1990. Transient expression for microprojectile-

mediated DNA transfer in Pinus taeda. Journal of Cellular Biochemistry Supplement 14E,

291.

Strange, C. J. 1993. Gene advances replace mechanical emasculation. Bioscience 41: 9.

Wilde, H. D.; Meagher, R. B.; Merkle, S. A. 1991. Transfer of foreign genes into yellow

poplar (Liriodendron tulipifera) In: Ahuja, M. R. (Ed.) Woody plant biotechnology. New York,

N. Y. Plenum Press. NATO ASI series. pp. 227 - 246.

Documents

CURSO DE MEJORAMIENTO GENETICO FORESTAL …colbun.utalca.cl/intercambio/.../mgforestal/Manual_Adobe/apunte002.pdf · Fco. Zamudio (PhD) Genética & Mej. Forestal Universidad de Talca