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Genetic transformation: a short review of methods andtheir applications, results and perspectives for forest

treesL Jouanin, Acm Brasileiro, Jean-Charles Leplé, G Pilate, D Cornu

To cite this version:L Jouanin, Acm Brasileiro, Jean-Charles Leplé, G Pilate, D Cornu. Genetic transformation: a shortreview of methods and their applications, results and perspectives for forest trees. Annales des sciencesforestières, INRA/EDP Sciences, 1993, 50 (4), pp.325-336. �hal-00882849�

Review article

Genetic transformation:a short review of methods and their applications,

results and perspectives for forest trees

L Jouanin ACM Brasileiro JC Leplé G Pilate D Cornu

1 INRA, laboratoire de biologie cellulaire, route de Saint-Cyr, 78026 Versailles Cedex;2 INRA, station d’amélioration des arbres forestiers, Ardon, 45160 Olivet, France

(Received 10 September 1992; accepted 11 February 1993)

Summary — This report reviews the state-of-the-art in plant genetic engineering, covering both di-rect and indirect gene transfer methods. The application of these techniques to forest trees has beendiscussed and a summary of the published results given. An overview of the possibilities of introduc-ing genes of agronomic interest to improve some characteristics such as resistance to pests andmodifications of phenotypic traits has been examined.

Agrobacterium I biotechnology I forest tree I genetic transformation

Résumé — La transformation génétique : résultats et perspectives pour les arbres forestiers.Cet article fait le point sur les techniques directes et indirectes de transformation génétique desplantes. Leur application pour la transformation des arbres forestiers est discutée et une liste des ré-sultats déjà publiés est établie. Les différents gènes d’intérêt agronomique qui peuvent être intro-duits afin d’améliorer des caractères comme la résistance aux pathogènes et des modifications duphénotype sont détaillés.

Agrobacterium / arbres forestiers / biotechnologie / transformation génétique

INTRODUCTION

Biotechnology includes tissue culture, mo-lecular biology and genetic transformation.This field of research can accelerate tree

improvement programs in a number of

ways. Tissue culture not only offers the

potential to multiply selected genotypes ef-ficiently and rapidly, but is also essentialfor the multiplication of transformed geno-types. Molecular biology and genetics pro-vide insight into the nature, organization,and control of genetic variation (Cheliakand Rogers, 1990).

* Present address: Embrapa/Cenargen, Sain Parque Rural 70770, Brazilia-DF, Brazil.

Transgenic plant recovery is a relativelynew domain and was first attained withmodel plants such as tobacco. The intro-duction and expression of foreign DNA ina plant genome requires several steps: in-troduction of DNA into a cell, selection andgrowth of this cell, and regeneration of anentire plant. Continuing progress is madein obtaining transgenic plants from annualcrops. However, it has been slower in tree

species which can be transformed but aremore difficult to regenerate, in part due toinefficiencies of in vitro culture systems.Thus, many public and private laboratoriesare working on improving tree culture sys-tems. In this paper, we provide some in-sight into the main transformation proce-dures developed for crop plants andreview the results obtained with foresttrees.

GENETIC TRANSFORMATIONMETHODS

Different systems can be used to introduceforeign DNA into a plant genome. Thesemethods include biological systems basedon the pathogenic bacteria Agrobacteriumfumefaciens and A rhizogenes, or physicaland chemical systems such as microinjec-tion, electroporation, chemical porationand microprojectile bombardment. Manyother ways of introducing DNA into the

plant cell have been tested, and havebeen recently reviewed by Potrykus(1991 ).

Agrobacterium-mediatedtransformation

A tumefaciens and A rhizogenes are con-sidered as natural genetic engineers dueto their ability to transfer and integrateDNA into plant genomes through a uniqueintergeneric gene transfer mechanism.

Both are phytopathogenic bacteria of theRhizobiaceae family. A tumefaciens is thecausative agent of crown gall disease andA rhizogenes is responsible for hairy rootdisease. These bacteria are pathogenic ina wide range of dicotyledons and in somegymnosperms (De Cleen and De Ley,1976, 1981). In particular, they have beenthe cause of problems in vineyards andfruit orchards in Eastern Europe. Monoco-tyledons are naturally resistant to Agrobac-terium infection (De Cleene, 1985).

These diseases are caused by thetransfer and integration into the plant ge-nome of a portion of large plasmids (150-200 kb) called pTi (tumor-inducing plas-mids) from A tumefaciens and pRi (root-inducing plasmids) from A rhizogenes (re-viewed by Charest and Michel, 1991 ;Hooykaas and Schilperoort, 1992 ; Wi-

nans, 1992 ; Zambryski, 1992). The geneslocated in the transferred region, called T-DNA (transferred DNA) are integrated intothe plant genome and expressed in the

plant cells. Some of these genes (onco-genes) promote hormone synthesis or

modifications in hormone content that alterthe growth regulator balance of the planttissue, thus changing their growth charac-teristics. The tumors obtained after A tu-

mefaciens inoculation result from the

expression of the auxin and cytokininsynthesis genes present on pTi T-DNA. Inthe case of A rhizogenes, expression ofseveral genes called rolA, B and C (root-including loci) induces root formation at theinoculation point. Up to now this root induc-tion mechanism has not been completelyelucidated.

The T-DNA genes are not involved in T-DNA transfer mechanism and can be re-

placed by other genes without affectingtransfer efficiency. Two direct repeats of24 bp at the borders of all T-DNA areneeded for their efficient transfer. Another

sequence named overdrive near the rightborder enhances the transfer. The other

essential part of pTi and pRi is the viru-lence region (vir). The vir genes are re-

sponsible for the processing of the T-DNAand its transfer to the plant cell. Figure 1

presents a schematic map of the Ti plas-mid showing the most important regions,the vir-region as already mentioned, the T-region (called T-DNA when transferred intransformed plant cells) and the regionsimplicated in the replication of the plasmidin the bacteria and in the conjugative trans-fer between bacteria.

For plant genetic engineering the onco-genes need to be deleted from pTi as theyare not compatible with regeneration. En-tire plants containing pRi T-DNA can be re-generated from transformed roots. Howev-er, the plants expressing pRi oncogenespresent a specific phenotype (wrinkledleaves, root plagiotropism and reduction ofapical dominance ; Tepfer, 1984) which isoften incompatible with their use in plantbreeding programs.Two different strategies can be used for

gene integration with the Agrobacteriumsystem. In a cointegrate vector (fig 2A ;Zambryski et al, 1983), pTi T-DNA onco-genes are replaced via homologous recom-

bination by a DNA fragment containing thegene(s) of interest and if necessary a mark-er gene flanked with vector sequences.This strategy can also be used with pRiwithout removing the oncogenes which al-low the root formation. However, the strat-egy used in most cases involves a binarysystem (fig 2B ; Hoekema et al, 1983). Inthis case, the agrobacteria used for trans-formation contain Ti or Ri plasmids with in-tact virulence regions but with deletion oftheir entire T-region (including the bordersequences). These are termed disarmedstrains. The gene of interest and if neces-

sary a selectable marker gene are clonedbetween the border sequences into a sec-ond small plasmid. For plant transforma-tion, the binary plasmid is introduced into adisarmed Agrobacterium. The most cur-

rently used technique to obtain transgenicplants is the cocultivation of plant explants,eg leaf, stem, or root fragments, embryoswith the Agrobacterium containing the

gene of interest in its T-region. During thiscocultivation step, the wounded plant cellsare in contact with the Agrobacterium andthe transfer of T-DNA occurs. Then the

agrobacteria are eliminated and the plantexplants are transferred onto a regenera-tion medium. In complement to the ele-ments needed for regeneration of shoots,the medium contains 2 kinds of antibiotics,one to kill the residual agrobacteria (de-contamination) and the other to select thetransformed plant cells. Figure 3 summar-izes the different steps in the proceduredeveloped for poplar stem fragment cocul-tivation according to Leplé et al (1991).

Direct gene transformation

Direct transformation techniques over-

come Agrobacterium host range limita-

tions. These methods are generally basedon the use of protoplasts or tissues fromwhich efficient regeneration can be

achieved. With these methods, transientexpression (expression of the introduced

gene without integration in the plant ge-nome) of the transferred gene is often ob-served. However, stable transformation af-ter integration in the plant genome canalso be achieved.

Different means can be used to render

permeable the plant protoplast membraneto allow uptake of naked DNA. Some au-thors have used polyethylene glycol (PEG)or polyvinyl alcohol (PVA), but the transfor-mation frequency has sometimes been low(Kruger-Lebus and Potrykus, 1987). An-other method which can increase the

transformation rate is electroporation. In

this method, after or without pretreatmentwith PEG or PVA, the protoplasts are sub-mitted to a high-voltage electric pulsewhich enhances DNA penetration into theplant cell (Crossway et al, 1986 ; Fromm etal, 1986).

Microjection permits direct and precisedelivery of DNA into the plant protoplastsusing a microsyringe containing the DNAin solution. However, this technique is ex-tremely delicate and requires the use ofexpensive equipment (Reich et al, 1986).

Microprojectile bombardment is a noveltechnique in which small tungsten or gold

particles coated with DNA are acceleratedwith a gun to velocities that permit penetra-tion of intact cells (Klein et al, 1987 ; Chris-tou et al, 1988 ; Sautter et al, 1991). Theuse of intact cells or tissues is a majoradvantage because it bypasses the needfor regeneration procedures from proto-plasts. Moreover, this technique allows thestudy of gene expression in organized tis-sues without the need to regenerate entiretransformed plants.

Many other techniques have also beentested with the aim of introducing DNA intoplant cells (laser microbeam, pollen tube-mediated delivery, ultrasonication, etc) but,in most of them, only transient expressionor non-reproducible results have been ob-served (Potrykus, 1991). All of these tech-niques have their limitations. The transfor-mation method selected will depend on thespecies and characteristics of the plant tobe transformed.

MARKER GENES

Two strategies can be used to recover

transgenic plants after transformation:

screening of all regenerated plants for ex-pression of a reporter gene, and/or selec-tion of transformed plants for resistance toa selectable agent. The marker genes arechimeric constructions containing plantexpression signals fused to the codingsequence of a gene of bacterial or other

origin. These regulatory sequences (pro-moter and polyadenylation signal), allow-ing expression in plant cells, are generallyderived from genes of the pTi T-DNA (nop-aline synthase, octopine synthase, manno-pine synthase, etc) or from the 19S and35S transcripts of the cauliflower mosaicvirus. Among the more frequently used re-porter genes, the β-glucuronidase (GUS)gene is very useful since its enzyme activi-

ty can be easily visualized by formation of

a blue precipitate in the presence of XGluc(5-bromo-4-chloro-3-indolyl glucuronide) inhistochemical assays or measured by fluo-rimetry in the presence of MUG (4-methylumbelliferyl glucuronide) as substrate (Jef-ferson et al, 1987). The introduction of aplant intron into the coding sequence ofthe GUS gene prevents its expression in

Agrobacterium. This characteristic permitsthe first steps of the transformation to befollowed, since it allows easy visualizationof the transformed plant cells without theproblems caused by the presence of agro-bacteria at the inoculation point (Vancan-neyt et al, 1990).

Among the selectable markers used toselect transformed cells on the culture me-

dia, the neomycin phosphotransferase(NPTII) gene (Fraley et al, 1983 ; Herrella-Estrella et al, 1983) is widely used. Theexpression of this gene confers resistanceto different antibiotics (kanamycin, neomy-cin, paronomycin, geneticin). The activityof this selectable gene product is easilydetectable. Hygromycin phosphotransfe-rase (HPT, Waldron et al, 1985) is also

very efficient but less frequently used andcan constitute an alternative when 2 mark-ers are necessary or when the selectionwith kanamycin does not work well.

Genes conferring herbicide resistancecan also be used for selection of trans-formed cells. In this case, the selective

agent confers a new agronomically impor-tant trait to the transgenic plants. Herbi-cides that have been used for selection oftransformed woody cells are phosphinotri-cin (De Block, 1990) and chlorsulfuron (Mi-randa Brasileiro et al, 1992). The resis-tance to the former herbicide is conferred

by the expression of the detoxification

gene bar for Streptomyces hygroscopinuswhich encodes a phosphinotricin acetyl-transferase enzyme (PAT) preventing theaction of the herbicide (Thompson et al,1987). The resistance to the latter herbi-cide is conferred by a gene isolated from a

mutant Arabidopsis thaliana line encodinga chlorsulfuron-resistant acetolactate syn-thase (Haughn et al, 1988).

PRELIMINARY RESULTSWITH FOREST TREES

After excision from the plant, tumors or

roots obtained following wild-type Agrobac-terium inoculation are generally able to

grow on a hormone-free medium. Such re-sults have been reported for many foresttrees including conifers (reviewed in

Charest and Michel, 1991) and have notbeen reviewed in this publication. Theseexperiments show the ability of Agrobacte-rium to transform forest tree cells. Similar-

ly, most of the results obtained by directtransformation procedures concern the

transient expression of genes = 24 h afterDNA introduction (reviewed in Charest andMichel, 1991). These results demonstratethat DNA has been introduced into the

plant cell but probably without stable inte-gration in the plant genome. Moreover,there is a distinct difference between theobservation of tumor formation after inocu-

lation, transient expression after electropo-ration or microprojection, and the regener-ation of an entire transformed plant.

Indeed, all of the regeneration proce-dures so far described involve a tissue cul-ture regeneration system. This regenera-tion can be based on organogenesis froman explant (leaf, root, stem) or from an em-bryogenic culture (directly or through proto-plast isolation).

The most rapid advances in genetic en-gineering to data have been obtained withwoody angiosperms such as poplars. Hy-brid poplars are good models for foresttree transformation since they are easilymicropropagated in vitro, are generallyvery sensitive to Agrobacterium, and ableto regenerate entire plants from differentexplants. Several publications report the

obtention of transgenic hybrid poplarsmainly using Agrobacterium (Fillatti et al,1987 ; De Block, 1990 ; Klopfenstein et al,1991 ; Miranda Brasiliero et al, 1991,1992 ; Devillard, 1992 ; Leplé et al, 1992 ;Nilsson, 1992). Transgenic trees have alsobeen reported for walnut via Agrobacteri-um transformation of somatic embryos(McGranahan et al, 1988, 1990 ; Jay-Allemand et al, 1991). Recently micropro-jection has been used with poplar leaves(McCown et al, 1991) or embryogenic cellsof yellow poplar (Liriodendron tulipifera ;Wilde et al, 1992) followed by the produc-tion of transgenic trees. Table I summariz-es the published results for different foresttrees and the characteristics of the trans-

genic plants.

Regarding the recovery of transgenicconifers, up to now only transgenic larches(Larix decidua ; Huang et al, 1991) via Arhizogenes transformation and transgenicembryos and plants of white spruce (Piceaglauca) via microprojection (Ellis et al,1993) have been reported. In conifer spe-cies, many publications report tumor for-mation after Agrobacterium inoculation,and transient expression via protoplastelectroporation or via microprojection of

embryogenic tissues (reviewed in Charestand Michel, 1991). Recently, Robertson etal (1992) have reported the obtention ofstable transformed calli of Norway spruce(Picea abies) by microprojectile bombard-ment of somatic embryo explants. Conifertransformation and regeneration is a rela-

tively new field and different approachesare being tested.

POTENTIAL TRAITS TO INTRODUCE

An important question is that of which

genes to transfer in woody species. Fun-damentally, introducing genes into a foresttree genome would help in elucidating as-pects of gene control or expression andmetabolism. For angiosperms, gene regu-lation is probably similar for woody andnon-woody plants. However, very little in-formation is available on gymnosperms(conifers). The ability to introduce a geneor its regulatory sequences into coniferswill advance our understanding of the roleof genes, promoters or control regions. Upto now, there has been a lack of under-

standing of the structure and function ofconifer genes, since only few of them havebeen characterized. Some of these ques-tions could be solved by using transientexpression assays via protoplast electro-poration or by microprojection of organizedtissues.

Practically speaking, transgenic treescould constitute part of tree improvementprograms. Many potential applications ofnew traits conferred by a single genecould be envisaged such as resistance toherbicides and to diseases, as well asmodifications in phenotypic characterssuch as sterility or wood quality. Differentgenes able to confer new properties al-

ready used in annual plants could be intro-duced into forest trees.

Herbicide-resistant trees could be bred

by different strategies: introduction of amutant gene coding for a modified enzyme(resistance to glyphosate and chlorsulfu-ron), overproduction of the target enzyme(glyphosate) or detoxification of the herbi-cide (phosphinotricin, bromoxynil). Asweed problems are mostly found in tree

nurseries, this application should provide aroute for more efficient establishment of

young trees in nurseries, and an improve-ment in nursery management techniques.Two strategies for obtaining insect-

resistant trees could be tested: expressionof δ-endotoxin genes of Bacillus thuringien-sis (Bt) or of proteinase inhibitor (PI) genesinterfering with insect digestion. Bt geneswith activity against lepidopteran, coleopte-ran and dipteran insect species (Höfte andWhiteley, 1989) have been isolated. Up tonow some bio-insecticides containing Bt

preparations have been used against for-est phytophage insects. Expression of thecorresponding gene in a transgenic treecould enhance its resistance against thispest. Genes coding for different types ofprotease inhibitions are available and theeffect of their expression on insect pestscould be tested. Moreover, they could betested in combination with Bt genes(Brunke and Mensen, 1991).

Several strategies tested in annual

plants, such as the expression of the viralcoat protein, antisense RNA and interfer-ence with subviral RNA molecules (re-viewed by Gadani et al, 1990 ; Szybalski,1991) have been shown to be efficient inthe control of virus diseases. Such strate-

gies could be tested for virus protection intrees.

In poplar, enzymes encoded by wound-responsive genes that could be involved inpathogen resistance (chitinases and trypsininhibitors) have been isolated and charac-terized (Bradshaw et al, 1989 ; Davis et al,1991). Since introduction of a chitinase

gene in tobacco and rapeseed was found toenhance resistance to a fungal pathogen(Broglie et al. 1991), this strategy could betested in trees. Likewise, different strategiescould be tested to obtain trees resistant tobacterial diseases (Lamb et al, 1992).

Another possibility is to modify pheno-typic characteristics. One approach is to in-

terfere with the physiology of the plant byreducing the expression of a gene via anti-sense RNA strategy (Van der Krol et al,1990). This strategy could help to modifyexpression of a gene, thus changing thephenotype. However, the prerequisite forsuch an approach is the identification andisolation of genes that affect the characterin question. Up to now, very few forest treegenes have been isolated and character-ized. Several research projects are under-way to obtain this information. In particular,poplar genes involved in the lignin biosyn-thesis pathway are available, such as

those encoding O-methyltransferase (OMT ;Bugos et al, 1991 ; Dumas et al, 1992) andcinnamyl alcohol deshydrogenase (CAD ;van Doorsselaere et al, unpublished re-

sults). Reduction of the activity of OMTand CAD enzymes could be studied usingthe antisense strategy and lead to modifi-cations in the lignin content or in its com-

position. As part of the same approach, an-other project is to express an antisensechalcone synthase gene (CHS) in walnutin order to modify its content in phenoliccompounds and thus indirectly modify rhiz-ogenesis (Jay-Allemand et al, 1991). More-over, since most of these enzymes are im-

plicated in pathogen interaction, the effectof their over expression could provide in-formation on their possible role in plant de-fense against pathogens.

Several publications report on the pro-duction of transgenic poplars expressinggenes of interest. Most of them refer to

plants which express genes conferring re-sistance to herbicides: glyphosate (Fillattiet al, 1987), phosphinotricine (De Block,1990 ; Devillard, 1992) or chlorsulfuron

(Miranda Brasileiro et al, 1992). However,insect-resistant poplars expressing a Bacil-lus thuringiensis toxin gene have also

been obtained (McCown et al, 1991).The potential impact of the release of

transgenic trees in the fields is differentfrom that associated with annual crop

plants, due to the long life cycle of treespecies. In particular, we may question themost appropriate way of propagating thenewly introduced trait. Problems will varydepending on the species. In the case ofclonal or multiclonal strategy for produc-tion, forest trees such as hybrid poplars,which are mostly propagated by cutting,are easily multiplied to obtain stable trans-genic clonal propagations. The problem isnot so easy to solve for forest specieswhich are propagated by seed. Indeed,how will it be possible to stably incorporatethe trait? At present, not all the elements toanswer this question have been obtained.

Perhaps most importantly, if geneticallyengineered trees that can reproduce sexu-ally are used in reforestation programs,should one be concerned about the trans-mission of foreign DNA into the wild popu-lation (Cheliak and Rogers, 1990)? For

example, it is conceivable that the intro-duction of a herbicide-resistant gene couldbe transferred by sexual reproduction towild trees (Keeler, 1989). To avoid this

spread, technology to obtain sterile trans-genic trees may be envisaged using, for

example, destruction of pollen by expres-sion of a gene coding for an RNAase intapetal cells, as already attained in tobac-co and rapeseed (Mariani et al, 1990).

Finally, the introduction of pest resis-

tance in trees could involve the develop-ment of tolerance by the attacking organ-ism. This is critical for long-life forest treeswhich have to maintain defensive capacityagainst pathogens, despite enormous dif-ferences in generation times (Raffa, 1989).The problem is to determine at what pointthe attacking pest will develop tolerance(Bishop and Cook, 1981).

Moreover, at the present time it is diffi-cult to determine which government regu-lations will be put in place regarding therelease of transgenic trees in the field. De-spite the potential power that transforma-

tion technology can provide, many aspectsstill need to be considered. However, it isclear that transformation technology will

participate in the advancement of tree im-provement programs in the future.

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