Genetic Transformation in Conifers

17. Genetic Transformation in Conifers

S.C. MINOCHA1 and R. MINOCHA2 'Deparfrtletrl of I'latrl Biology, Uttiversily of New I~amnpshire, Durlror~r, NfI 03824, USA. Enrail: [email protected]. 2LlSDA firesf Service, NERS, 1?O. Box 640, Dtrrlrom, NFI 03824, USA. E-Moil: rn~inocha@lro~~~~er.unl~.e~lrr

Inlmduction

Several attempts at the genetic improvement of tree species have been made, but in comparison with crop plants the efforts as well. as the results have been rather limited. The most comn~only used approaches have involved selection of superior genotypes from natural outbred populations, mutations, and intra- and inter-specific hybridization under controlled conditions. While the conventional methods have proven remarkably successful in yielding irnproved genotypes that could be stabilized by back crossing, the techniques of cloning, marker-aided selection, and genetic engineering when integrated with the conventional breeding programs, will dramatically improve genetic gains. Conventional methods of genetic improve- rnent involve a recornbi~latior~ of pre-existing gene pools within a limited range of sexually compatible taxa. The process of backcrossing and selection takes several generations before a desired set of genes can be transferred to a selected species. Genetic manipulation through recombinant DNA permits us to cross the barriers of inconlpatibility, not only among species and genera but also among kingdoms. Genetic engineering provides new tools for mixing genetic infonnatio~i in plants fro111 a vast pool of existing genes as well as genes designed by human intervention, i.e. synthetic gene sequences. Furthermore, undesirable genes in the plant genorile can be selectively siler~ced in the target tissues by the antisense approach (Uonrque, 1995; Lee and Douglas, 1997). Site-specific mutagenesis, homologous recombiiiation, and the use of specific pronloters provide a precise means of controlling specific gene expression and its manipulation to achieve optirnal genetic improvements.

Genetic 'Ikansfonn:~tioi~ in Plants

Several techniques are used to introduce biologically active genes into plant cells. 'I'hese include: direct uptake, microinjection, electroporation, liposorlle fusion, viral- and Agrobacteriunr-based vectors, and biolistic bombardment. Each one seems to have certain advantages and some major disadvantages. For a detailed tlescriptiorl of different techniques, see Jones (1995), Gartland and Ilavey (1995) and Christou (1996).

Direct uptake of DNA from the st~rrorlnding medium and the expression of foreign genes by plant cells has been demonstrated in several cases (see reviews by

S M . Jain, P K Cuplo, 1l.J. Newron (crls.), S,?ntar:c Ewbr),og.encsis a Il.%ru,/y J'larrr.~, Vohrmr 5, 221-3912 0 1999 Kl,rw,er Academic P~rblirlzrrs. Prinred bl Grpnr llrirnirr.

292 S.C. Mirroclm nnd R. Mirrochn

Shillito ant1 Potrykus, 1987; Lazzeli and Sliew~y, 1994). Furtlicr cnl~ancements of direct DNA uptake call be achieved by atldirlg polyethylene glycol (PEG) or co- precipitatior~ wit11 calciual pllospllate. Delivery of DNA into plant p~oloplasts by ~nicroi~~jection irnproves the efficiency of direct transfonnation, however, the technique is very cu~nbersome and inefficier~t in 1iandli11g large ~iurnbers of cells.

Electropo~atiorl, or the facilitation of DNA uptake under the influence of an electric field call dramatically improve tlie eflicier~cy of transformation (for ~eview see Nickoloff 1995; Lu~quin, 1997). The availability of an affordable electlo- poration apparatus also makes this method a viable option for no st laborato~ies. In the Iiposolne fusion method, nucleic acids are encaps~~lated in synthetic pliospholipid vesicles and under approp~iate incubation conditions are allowed to fuse with plant protoplasts. Advantages of the liposo~ne-mediated gene transfer include protection of ~lucleic acids from digestion by nucleases in the culture medium and tlie high efficiency of ~iucleic acid delivery into p~otoplasts Potentially several kb long nucleic acids can be delivered by this method. Rece~lt availability of a variety of co~n~nercial p~odiicts (e.g. Transfectam - Promega, Madison, WI, Lipofectin arid LipofectAMINE, Life Science Technol., Grand Island, NY, etc.) that aid in DNA transfer has made this a widely used method for transfectiorl in animal cells. For all of the above techniques, thc preferred target cells are protoplasts, which makes these techniques suitable o111y in cases where plant regeneration from protoplasts is possible. While protoplast isolation has been den~o~lstrated fron~ a variety of coriife~ous tissues, regeneration of whole plants fro111 protoplasts is not conimonly observed (Bekkaoui et al., 1995).

A 11unlber of viruses, particlllarly the Caulimoviruses and Geminiviruses, have been suggested as useful vector syste~ns for the trarlsfer of selected cloned genes into plant cells (Mushegian and Sl~epherd, 1995; Palmer and Rybicki, 1997). Whereas the DNAca~ried by a virus is stably expressed in the transfo~~ned (infected) plant, in no case the integ~at io~~ of the transferred gene into tlie host gellorile has been demonstrated. Unambiguous transtonnation of higher plants wit11 foreign genes lias been achieved with Agrobacleriurt~ tumejiacierts and A. rhczoger~es (for review see Weising and Rahl, 1'996). With the vast amount of infor~natior~ al~eady available regarding gene maps, the role of tlie vir and the T-regions, and the border fragments, it lias been poss~ble to greatly modify the plasmid to develop efficient gene transfer vectors for a variety of plant cells, including tree species (Zambryski, 1992; Han et a[., 1996; Gartla~id and Davey, 1995; Tinland, 1996). Genes transferred via Agrobacrer lurn show stable integration and Me~idelia~i i~ihe~itance.

The technique of biolistic bombardment with DNA-coated gold or tungsten particles has yielded high frequency trdnsfor~natioris in numerous species (for review see Christou, 1994; Chibbar and Kartlla, 1994). This technique does not rely on the availability of single cells, nor is the cell wall an i~npedinlent f o ~ transfer of DNA. T l ~ e technique has beer1 succeifully applied to t~ansfor~u cells of intact organs and tissues of plants as well as ani~nals (Christou, 1994).

Several important factors make it irnpossible to critically cornpare and evaluate the efficierlcy and the effectiveness of different methods of transformation for a given tissue. In no case have different methods beer1 tested with the same tissue,

Gerretic ~rurrsforr~tn~iort irr cortfirs 293

the same genelpromote~ construct, ant1 i n the sarnc laboratory. 'l'he single most i~nportant factor governing the applicability of a particular technique for a given species is the mode of regeneration of wholc plants from srngle cells in that species. Nevertl~eless, it is obvious that by using a variety of gene transfer systems, it is now possible to introducc foreign genes into alrnost any plant cell. The [tireign DNA is often integrated randomly in the plant genome, and major probleins still exist in achieving the integration of specific transgenes at specific sites in the host genome. (Flavell, 1994; Jorgensen et al., 1996). Further co~nplexities in the site of integration involve variable number of copies of the Iransgene, re-arrangements of DNA sequences, differential amplification of segments of the transgene, and selective silencing of the transgenes (Deroles ant1 Gardner, 1988). These variations lead to differential and unpredictable expression of the transgene, thus affecting the behavior of transgenic tissues/plants. Another, as yet unexplained con~plexity comes f ~ o ~ n 'co-suppression' where exp~ession of the native as well as the trans- gcne are often suppressed when the two share some common sequences (Matzke ancl Matzke, 1995). Koziel el al. (1996) have discussed several strategies tliat can be used to optimize the expression of transgenes in plants.

A variety of promoters from A. tunrefacie~~,%, cauliflower mosaic virus (CaMV), and higher plants have been fused with a number of reporter genes to study their expression in traosgenic plants. Among the promoter sequences used to achieve constitutive exp~ession of the foreign genes, the CaMV-derived promoters are a~nong the strongest ones. Numerous ri~odificatior~s of the CaMV 35s promoter have made it highly suitable for foreign gene expression in almost any plant. Although many of the crop plants have reached the stage of field trials and commercial plantations (Dale et al., 1993; Moffat, 1998), the need to extend the basic gcne transfe~ tecl~nology to woody plants still remains unfulfilled. This is particularly true for the conifers, which would benefit uniquely from these techniques for genetic improvement but pose certain unique problems for regeneration in tissue culture.

'Ihrisforn~ation in Conifers

Establisl~ment of routine cell and tissue culture tecl~niques wit11 herbaceous plants was a major step allowing for the rapid advancement of genetic engineering appli- cations wit11 these species. 111 contrast, while considerable effort for cell culture and regeneration of many tree species has been underway (see earlier volun~es in this series, Jain et al., 199Sa,b,c), the technical level of conifer tissue culture is far less developed than that of the herbaceous plants. The variety of tissues from which regeneration of whole plants can be routinely obtained is rather limited. While multiple shoots can be regenerated from juvenile tissues of some species, these tissues are not quite suitable for transformation by most techniqi~es due to the limited material available for use. The most reliable tissue in conifers tliat is capable of regeneration is the embryogenic cell masses obtained from the culture of zygotic embryos. Embryogenic cell masses can be initiated from juvenile

material or zygotic embryos on many conifers (Jain et al., 199Sa,b,c). In the case of pines, the starting material is often limited fi~rtl~er to a narrow window during the early stages of developmer~t of the zygotic embryos. Therefore, most of the studies on transformation of conifers have ernployed the embryogenic cell masses.

As described above, the techniques of direct DNA uptake, microir~jection, electroporation, and liposome fusion all depend upon the availability of protopiast regeneration, and thus are not well suited for transfomlation of conifers. Similarly, viral vectors, although quite useful for expression of foreign genes in many plants, are not reliable for integration of the foreign DNA and its expression in tissues not infected with viruses. Although A. funtefacietzs can infect most conifers, and thus should be a suitable vector for transformation (see review by Ellis, 1995), the lack of regeneration from callus creates a major problem for its use. Orily a few reports on the transformation of conifer tissues with A. tumefaciens have appeared in the literature, and in almost all cases the resulting transformed tissue was only a callus. Therefore, biolistic bombardment, which is capable of transferring foreign DNA to virtually any cell type, becomes the only viable approach for transformation of conifers.

Althor~gh genetic transformation in several species of conifers has been demonstrated (Table I ; see also Ellis, 1995), and the potential of such techniques in the genetic improvement of commercially important species has been discussed thoroughly, only a few reports on the production of transformed plants expressing commercially t~seful genes have been published to date. I b the best of our knowledge, no large scale field trial of genetically engineered coniferous species expressing a con~mercially useful gene has been undertaken. This is in contrast to the millions of hectares of cultivation of genetically engineered corn, canola, cotton, tomato, soybean and a few other agricnltural crops (Moffat, 1998). A review of literature on conifer transformation further reveals that most reports of conifer transformation only demonstrate the technique of transformation using a reporter gene and/or a selection marker gene. Only in a few instances, have the genes of potential commercial value been used. Among the reporter genes, the CiIJS (b-glucuronidase) expression under the control of a modified CaMV 35s promoter has been the preferred choice. In a few studies, the effectiveness of different promoter sequences has been compared either in transieni assays or in stable trar~sfor~nations.

C~lrrent Status of 'Ikansforn~ation of Conifers

A recent review by Ellis (1995) su~n~narized the published literature 011 reports dealing with transformation of gymnospernls, n~ostly conifers. Without duplicating the efforts of Ellis, the following discussion provides a technique-based summaly of the work with conifers, identifying achievements in each case and evaluating [lie current situation. Unfortunately, only a few detailed reports of the prodr~ctiori of transgenic conifer species have been published since 1995, although personal corntnu~~ications wit11 Dr. Cl~ristiari Walter (New Zealand Forest Research Institute, Rotorua, New Zealand) indicilte a great cfcal of progress in this area.

Table I. Surnrnaiy of pohlished ~es~r l t s on transformation io conifers. f/a to table T=Traosient expression; S=Stable expression.

Species 'Tissue Gene TIS I'roduct Test Reference -.

Agrubactetium P.se~rrlo~or~oga Seedlings Opines S Callt~s Opine Morris et a[. 1989

r~~rnziesii I'irrrr.~ potrderosn Seedlings Opines S Callos Opine Morris et al. 1989 Errga hereropllylla Seedlings Opines S Ca1111s Opine Morris et al. 1989 Ahie.sprocern Seedlings Opines S Callus Opine Ellis el 01. 1989 Picea glaucn Seedlings Opines S Callus Opines, horrnones Ellis el al. 1989 Piceo errgeln~anr~ii Seedlings Opines S Callus Opines, l~ormones Ellis er 01. 1989 Picen sitclret~sis Seedlings Opines S Callus Opines, hor~uones Ellis cr a/. 1989 I'.seudor.sugrr Seetllings Opines S Callus Opi~~es, hor~rtor~es Ellis el al. 1989

~t~et~zie,sii Lorix decidua Seedlings Opines S Buds/plants Opines, Southern lluang et a/. 199 1 Lorix rlecidun llypocotyl HI, aroA S Plants Bt/glyphosate Sl~in et nl. 1994

resis. Turfs Drevifolia Sl~oots Opines S 'Turnours Opines llan el 01. 1994 Tmus haccara Sl~oots Opines S 'li~rnours Soutlrer~~ Han et al. 1994 Picea ahies Seedlings Opines S Roots Opines Magnussen el a/.

1994 Pirlus conrorta Seedlings Opines S Roots Opines Magnussen er a/.

1994 P ~ ~ I N S trigra Seetllings Opines S Rooting Opines Miltalijevic el a/.

1996 Lark kaerttpferi x Ernh. NPrll S Emblplants PCRISoulhem Ikvt5e er al. 1997

L. decid~~a Cells Picea sitclrerr.sis E~nb. Cells GUS T Cells Western/stain Drake el a/. 1997

Biolistic bo~~rbn rdn~ea t IJir~us roerln Cot. cells GlJS 62d 'Tissue Stain Stotr~p er al. 1991 Picea gfn~rca Emh/callus GUS T None Stain Ellis el a/. 1991

seedlings Picea nbies SE n~ature NPT/ T/S Cells PCR/Southcrn, Robertson el a/.

GUS slain 1992 Picen rrtnria~~a E~nb. cells GUS T None Stairt Duchesne arrd

Charest, 1991, I992

Larix (feeidria Etnb. cells GUS T None Stain Duchesne end Cltaresl, 1992

Lark x errrolepis E~nb. cells GUS T None Stain Duchesne at~d Charest, 1992

Lark x E~nb. cells GUS 5-6d None Stain Ducl~esl~e el a/. lc~~loe~rro/~ne 1993

Lark Irprolel/is Emb. cells GUS 5-6d None Stain Duchesne el 01. 1993

I'icea gla~tca Cot. SE GUS/ 2-8 Tissue Stain/Soothenr Bommineni et al. NIT nlo 1993

Picea glartca SE GUSIL3t T/S Callus, PCR, Southern, Ellis er 01. 1993 embryos Bt

Pitt~ts radialn Etnh. cells GUS 35tl Ci~llus Stain Walter et nl. 1994 I'iceo obies E~nb. cells GUS T None Stain Yibrah el a/. 1994

Contintled on next page

296 S.C. M h o c l m rzrrrl R. Mirtochn

7irble 1. Cot~tinoed

Species 'l'issoe Gene T/S I'roduct l'est Refeicncc

Pirros syh~esfr-is Uutls/c:llli GUS ' 7' None Slain Arorlerl et ol. 1994 Chnmarcyl~or-is Pollen GUS/ 'I' Noi~e ELlSA [lay el a/ . 1994 rroofkaferrsis NIrI' 7:rrrga heteroplrylln Pollen GUSINI'T T None ELJSA Hay e f a / . 1994 Pirrrrs Dnnksin~ta Pollen GUSINP'T 'I' None EI.ISA Ilay el a / . 1994 Picen rrinrinrro Pollell GUSINPT 1' None ELlSA llay el a / . 1994 I'irrirs syli~eslris Duds GUS ' None Stain Aroneo e f nl. 1994 Pirrrrs rudinm Cotyledoi~ GUS I ' None Stain Rey el a / . 1996

201d Picen rrroriarra SE/SE GUSINP'I' S Cells, Stairi, ELISA, Cl~arest rr al. 1996

tissue embryos, PCR plarlls

Pirrrrs syh~eslr-is Pollerl GUS 'I' None SlaiflCR I-laggrnan et 01. 1997

I'icen abies Pollen GUS I ' None StainIPCR Hiiggman et a / . 1997

Electn~pnratioe Picea glauca Protol3last GUSICAT T Protoplasls SlainICAT Bekkaol~i el 01.

1988 Picen rr~ariann Proloplast, CAT 'I' Protoplasts "CICXT assay Tautorus et rrl. 1989

Enib. cells Pirrus borrksinna Proloplast CAI' 'I' Protoplasts "CICXI' assay 'I'auton~s el a / . 1989

Ernb. cells Picen glnrrcn I'rotoplast GUSICA'T 'I' Protoplasls Stai11/CKr Wilson er a / . 1989 Picen glancn SEIlissne GUSICA'I' 'T Protoplasts SrsildCAr Bekkaotii e f nl.

1990 Pirrus hnrrksintra SEIlissue EMV T Plotoplasls SfaidCAT Bekkaoui ei 01.

1990 Picerr nrnrinna SWtissue EMV T P roloplasls StaidCAI' Dekkaolii et nl.

1990

The ability of A. tum~fac~erzs to infect conifeis was rel~orted as early as 1935 (reviewed in DeCleene and DeLey, 1976), but it was not until the late 1980s, Iiowever, that Agrobncter~uni was used expelinlentally to transform conifer tissues to p~oduce crown gall tuinors (Clapham and Ekberg, 1986; Vandekar et al., 1987). Morris el a / . (1989) ;~nd Ellis et a / . (1989) conlpared several strains of A. tun~efacietis for their ability to induce crow11 gall tunlor formation on a number of conifer taxa. 'l'heir results s11owed that sonle strains were sig~~ificantly more poterlt than otl~ers for the induction of tu~nours. It was furtlier established that the integration of T-DNA was not a problem in conifers, and that the A. tuniefacietis proinoters from nopaline syntllase (NOS) and octopine synthase (OCS) genes were furictional in the conifer cells. Picea species were generally found to be more susceptible than the pines.

Using binary vectors containing either luciferase or NPTII genes, Ellis et al. (1989) showed the effectiveness of the conlrnor~ly used CaMV 35s promoter for

C;mretic !rr~rrs/orr~r~~!io~r irr corri/crs 297

stable gene expressiorl in a Sew conifers. I-Iuang el (11. (19'91) latcr confirmetl the ability of A. rhizogenes for gene transfer in wor~ndcd hypocotyl tissues of Europea~i larclr (Larix decidua). Some other examples of translbrtnation oE conifers by Agrobacteriurrl-based vectors wl~ich used eitlier opine syntliesis, hormone autonomy, or a marker gene are listed in l'ablc 1. A detailed list of the conifer species that can be infected by Agmbactet-ium also appears ir~ Ellis (1995) and HIggnlan and Arol~erl (1996). The first cornmercially usefrrl genes (Bt for insect resistance arid aroA for glyphosate resistance) were transferred to European larch using A. rhizoge?zes (Shin et al., 1994). Iiegeneratiot~ of plants involved direct shoot-bud induction from the wountled hypocotyl region of zygotic embryos. Transformed plants were tested for the presence and exprcssiori of the foreign genes and were found to be tolerant to glyphosate. Wliile the Bt-transgenic needles were not toxic, the insect larvae consumed significantly smaller anrounts of the transgenic needles than the coritrol needles. Magnussen et (11. (1994) and Mihalijevic el al. (1996) demonstrated tlie use of wild-type A. rhizogenes to improve rooting ability of Pinus nigra explants. Two of the recent reports on trans- foniiatiori of coriifers by A. trcnrefacietrs include the expression of the GUS gene in Sitka spruce (Drake et a/., 1997) and the production of transgenic hybrid larch (Larix kaealpferi x L. decidua) plants from embryogenic cell masses (LevCe et al., 1997). Plant regeneration via somatic embryogenesis was reported in the latter.

Wilso~i et al. (1989) demonstrated the use of PEG-mediated I>NA transfer into wliite spruce (Picea glauca) protoplasts. Bekkaoui el al. (1988,1990) and Tautorus et al. (1989) studied the effects of voltage, temperature, plastnid concentration and conforn~ation, and different prornoter sequences on transformation of black spruce (Picea marina) and Jack pine (IJirru.s banksiarza) proloplasts by electroporation. They found that while the 35s and nopaline syrltllase promoters were equally effective in driving the expression of tlie cllloramphenicol acetyltra~isferase (CAT) gene in transient expression assays, a tandem repeat of the 35s prornoter was substantially better than a single copy. Significant differences were observed among different cell lines of t11e same species. No stable expressior~ was obtained in any of these studies. Earlier, Gupta el al. (1988) had shown that the 35s 1)ronloter was also active in protoplasts of Douglas fir (Pseudotsuga Menziesii) and loblolly pine (Pinus taeda).

'I'lie biolistic bo~nbardnlent method has been used successfully for both transient as well as stable expression of a number of genes in conifers. Ellis et al. (1991) conducted a detailed comparative study of the expression o f several promoters in three different white spruce tissues using an electrically discharged bombardment device. While several constitutive and inducible promoters from Arabidolxis, soybean, and maize were active in this species, 35s promoter was the most active. 't'he ernbryogenic callus cells showed the least arnount of foreign gene expression i11 this study as compared with seedlings and zygotic embryos. Stomp et al. (1991) reported the expression of the GUS gene under the control of 35s promoter in loblolly pine using a particle gun (model BPG-4). Haggman et al. (1997) have demonstrated the feasibility of pollen transformation in Pirlus sylvestris and Picea abies by biolistic bo~nbardment. I-Iowever, no trar~sformed plants were reported

298 S.C. Mirrocha arrd R. Minoclra

from the fertilized ovr~les using bombarded pollen. The first case of stable transformation of a conifer tissue (Nonvay spruce) by this method was reported by Roberts011 ef al. (1992), who used a 35s promoter attached to the GUS gene and the NPT 11 gene. Tissue capable of growth on kanamycin was shown to have stable integration but had lost its embryogenic potential.

Duchesne and Charest (1992) compared several different promoter sequences for expression of the GUS gene in ernbryogenic cell lines of Larix decidua x leprolepis and Picea mariarza, again using biolistic boml3ardment. While the wheat Em gene pron~oter (abscisic acid-inducible) was found to be better than most others, the two cell lines did not show significant differences. The study involved only transient expression. Duchesne et al. (1993) later reported a detailed analysis of the response of 22 haploid and tliploid cell lines of four different species of Larix to bombardment with the GUS gene-coated tungsten particles of four different sizes. There was little difference among the different species or cell lines.

Similar studies have been reported on comparisons of different promoter sequences and different embryogenic cell lines for Nonvay spruce (Robertson et al., 1992; Yibrah et al., 1994), white spruce, red spruce, black spruce and L,arin x elrrolepis (Charest ef al., 1993), cultured somatic embryos of radiata pine (Walter et al., 1994), cultured cotyledons of radiata pilie (Rey et al., 1996), pollen grains of lodgepole pine (Pir~rcs co~ztoria), yellow cypress (Charnaecyl~aris noofkatensis), wester11 hemlock (Tsuga heterophylla), Jack pine (Pinus Dar~ksiana), black spruce (Ilay et UI., 1994), Norway spruce (Martinussen et aL, 1994), and buds from mature trees of Scots pine (I'inus sylvestris) (Aronen el al., 1994, 1995). Tandem duplica- tions of the 35s promoter were Ibur~d to increase the effectiveness of the original promoter sequence in several cases. Genotypic influence has also been reported for some species. 111 none of these cases were any transgenic plants regenerated.

Ellis el al. (1993) were the first to obtain regeneration of transfor~ned plantlets from en~bryogenic tissue of white spnlce bombarded with DNA-coated gold particles. 'l'he embryogenic cells and the plantlets showed the expression of GUS, NP'I'II, and the Bt genes. All three genes were integrated into the plant genome. The callus and the seedlings both showed sublethal levels of Bt protein when tested with spruce budworm feeding experiments. Soon after, Bon~mineni et rrl. (1993) observed stable expressiorl of the GlJS gene in white spruce ernbryogenic tissue. Christian Walter's group at the New Zealand Forestry has s~~ccessfi~lly used biolistic bombardment of the embryogenic tissue to produce transformed plants of radiata pine (Walter et al., 1994 and personal communications). 111 all of tiiese cases, duplicated CaMV35S-tlerived promoters were used to drive the expression of GUS gene.

Charest el 01. (1996) obtained transgenic plants of black spruce using a similar approach. They found 110 phenotypic effects of the iotegration and expression of the GUS and the NPTIl genes on rcgerlerated plants. I'ersonal co~nmunications with Dr. Clrristiarr Walter at NZ Forestry indicate that embryogenic cell lines of several pine sl~ecies have been transformed and the trausge~iic plants have beet1 brought to the greenhouse stage.

Genetic frurtsforrrruriort in corrfirs 299

Targets for Genetic Matlipillation

Numerous areas have been the target of genetic manipulation in crop plants; the most obvious areas of success being insect resistance, virus resistance, herbicide

.

tolerance, fruit ripening, and ornamental features. Most of these areas are equally important targets for transforn~atior~ of the con~n~ercially valuable coniferous species. A brief discussior~ of some of the target areas relevant to conifers is given below.

Resistance to Irzsect Pests

Insects contribute a rnajor threat to trees, causing severe damage to natural as well as cultivated forests. Insect control has generally been achieved by the use of insecticides, many of which have severe negative inipact on the enviror~mellt and often become ineffective due to the development of resistance in insect popnla- tions. Thus the development of insect resistant genotypes of commercially valuable conifer species will be of tremendous value both eco~lolnically and environ- mentally. Considerable success has been achieved in imparting insect resistance in many crop plants through genetic engineering. Three commonly used strategies are: (1) the overexpression of &endotoxin proteins of the bacterium Bucillus thluringieizsis (Bt), (2) over-expression of protease inhibitor genes found in some plants, and (3) the production of proteins in the plant cells that interfere with insect development andlor metabolism. The Dt endotoxins are naturally occurring toxins, which are Itighly eKective against a variety of insect pests. These toxins ;Ire qrrite specific for different insect species and, therefore, have been used s~~ccessfully to kill specific pests of a crop with no major environmental harm. Overexpression of the natural endotoxin genes or their mutated forms have been used to produce transgenic plant varieties that have shown resistance in a number of field trials. Some of these crops have been approved for cornrnercial production (Brunke and Meeusen, 1991; Koziel et al., 1993). There is no reasou to believe that the sanle strategies cannot be employed to generate insect-resistant conifers of commercial importance. Furthermore, it has been suggested that this strategy should be quite effective in the control of nematodes, miles, and all types of insects. The expression of genes coding for neuropeptides and otller enzymes that may interfere with insect development or matindfeeding bel~avior has also been suggested as a means to achieve insect tolerance in plants. A major concern for the use of any of these approaches is the potential for clevelopmer~t of resistance in the insect populations against these compountls, t11us rendering the whole crop vul~rerable to increased losses (RaEa, 1989; McGaughey and Wltalon, 1992). However, the concurrent use of Inore than one approach in the same genotype sl~ould mini~nize the risk of developing resistance by the insects.

Resistuizce to Viral, Bacteriul and Fungal Pafllogeirs

Among the most devastating pathogens of plants, viruses are the hardest ones to control by chenlical methods. However, viral resistance has also been one of the

300 S.C. Mirrochn n~rri I<. Mirrocho

easiest targets for genetic i ~ n p r o v c r ~ ~ e ~ ~ t of pla~its tll~ough genetic engineering. For quite some time it had beell known that virus i~lfection in pla~lts results in cross protection against related viruses. This led to the demonstration of virus resistarice in trarlsger~ic tobacco pl;~rits expressing the coat protein of tobacco mosaic virus (Powell-Abel er al., 1986). These observations were soon followed by reports o11 the use of either co~nplete or partial coding secluences of coat proteins of a number of RNA and DNA viruses to impart viral resistance in several plaot species. Later studies showed that in addition lo transgenic exprcssiori of the viral coat protein, a llu~nber of other strategies could be equally effective in iniparti~lg resistance to viruses using transgenic techniques. 'l'hese irlclude the use of virus replicase (RNA-depe~~dent RNA polymerase) genes, viral protease genes, ~nutated viral- rnoverner~t protei~t genes, antisense strarlcl gene expression, and satellite RNAs (for review see Kavanagh and Spillarle, 1995; Pappu e f a/ . , 1995). Considerable progress has also been made in u~lderstanding the host response to viral infection, leading to the use of this information to overexpress host genes involved in this response. 'The examples ir~clude pathogenesis-related (I'IC) proteins, systemic acquired resistance (SAR) proteins, ant1 anti-viral or ribosome-inactivati~ig (RIP) proleills (Kai~l and Winter, 1995). It has also been suggested that ~nammalia~~, rnor~oclonal alltibody (1nAb) producing genes and ~nammalian 2'-5' oiigoadenylate synthetase gene-based mecha~lis~ns rnay be used in the future to achieve either strain-specific or broad-based viral resistance in plants. The fact that several transgenic rnajor crop species have been field-tested for virus resistance, and that some of these have been approved for co~li~nercial production, provides a strong basis for tlie use of this approacll for virus resistance in conifers.

Whereas the use of transgenic approaches to impart insect and viral resistance in plants are example of success, engineering tolerance against major bacterial and fungal pathogens 11as not beer1 practical thus far. 111 the case of bacteria and fungi, the pathogen genes are not quite suitable for transfer to the host plant. Tlie strategies used in some cases have involved the overexpression of genes that interfere with growth and development of the pathogen, or degrade the pathogen cell wall material, or courlteract the effects of the toxin produced by the pathogen. Sorne of these approaches for bacterial pathogens include: (I) the expression of anti- ~nicrobial cysteine-rich thionines (Bohlmarln and Apel, 1991), (2) the expression and secretion of avian 01- T4 bactcriupllage lysozy~r~e (Trrudei et al., 1992; Diiring et al., 1993), and (3) expression of a tabtoxin-inhibiting acetylase (hr~zai et a/. , 1989) or a toxin-resistant target enzylne (De La Fuente-Martinez et al., 1992). In all cases only partial reversal of pathogenesis-related symptoms was achieved. For fungal pathogens, the transgenic approaches have involved (I) increased production of phytoalexins (Hail1 et al., 1993), (2 ) increased productiorl of antifungal proteins, such as chitinases, P-1-3-glucanases (Broglie et al., 1991; Neuhaus el al., 1991), and (3) increased i11ductio11 of I~ypersensitive reaction i~lvolvir~g programmed cell death (Martini et a!., 1993; Strittmatter and Wegener, 1993). A recent report on tlie c lo~~ing of a chitinase gene from several pine species (Wu et

al., 1997) should open the way for overexpressing a liomologous gene to achieve fungal resistance in pines and other conifers. A possible future approach for

antimicrobial resistance n ~ a y involve the p~oduction of p12kllt antibodies (also called pla~itibodies) directed against sl~ecific bacterial or fungal proteins. Tlie cu r re~~ t status of the success of genetic engineerir~g approaches to produce palhogen resistant plants has been revicwetl by Ilerre~a-Estrclla ant1 S i~npso i~ (1995). So far, no sitigle stlategy seems to have resulted in tile productior~ of pathogen-resistant plants that have been tested at the field Icvcl.

Ilerb~cide resistance was one of the earliest targets of genetic enginee~ing in plants and cot~siderable progress has been achieved with nrany cornrnercial crops. Genes for resistance agair~st 2111 major groups of hc~bicides have been c l o ~ ~ e d and later used in overco~nir~g the toxic effects of difre~ent herbicides. 171e ditiereut strategies have been based upon tlre ability to increase ~net:~bolism of the he~bicide (detoxification), overexpression of the alter~tale patl~way enzymes where the herbicide inte~feres with the blosynthesis of an essential metabolite, and introduction of a mutated gene for the target enzyme/protein. Field trials have been coinpleted for a nurilber of transgenic clop plants and comiirercial production has been approved (Dale et al., 1993). W h ~ l e some initial success with sucll genes has been demonstrated in some hardwoods (e.g. Populus; IIan et al., 1996), such research with conifers has barely been started. In fact the use of herbicides in cultivated forestry is relatively ~ e c e r ~ t and the need is only now being recognized.

Tolerance fo Abiotic Str-esses

In additior~ to facing attacks fioni pathogens, plants are constantly being exposed to a variety of natural and man-made abiotic stress factors which have a sigr~ificarit negative impact on giowth and development. Some of these stress factors include salinity, d~ought, cold, heat, chemical pollutants, and Ilerbicidal, fungicidal and insecticidal treatments. As the stress factors are varied, so ate the plant tesponses to them. Flar~ls also show sorne systemic ger~eral responses to stress. The complex stress response must begin with stress perception, transduction of some signal, and ever~tually clranges at the cellillar and ~nolecular level. A number of stress-induced gems and other corr~pour~ds have been identified, some of which are similar in plants, algae, fungi and bacteria (Ingram and Bartels, 1996). A better underslanding of this response sl~ould lead to potential genetic manipulation of the plants to nriniinize the impact of stress on their growth and development. For example, the halophytic bacteria and plants have ~nechanisnrs to exclude Na+ from the cells while accumulating high levels of K+. This is achieved by regulating antiporter activity of the plasma membrane and tonoplast. In recent years, a number of genes for ion pumps have become available thus opening the door for their genetic ~nallipulatio~~ in plants (Jia et al., 1992; Bohnert et al., 1995). An al te~~la te aplxoach to tolerallce of both high salt and osmotic stress is the accumulation of o~ganic oslnolytes and other nretabolites, such as glycerol, arabitol, mannitol, sugars, proline, glyci~ie-betaine, and probably polyamines (Rliodes and Hanson,

302 S.C. Mirloclra and R. Minocha

1993; Ober and Sharp, 1994; Chiang ant1 Dandekar, 1995; Ye et al., 1997). The biosynthetic pathways for these protlucts have been elucidated, providing a great potential for plant improve~l~ent through genetic engineering (Bartels and Nelson, 1994; lngram ant1 Bartels, 1996). Although the feasibility of genetic manipulation of the cellular levels of many of these c o ~ n p o ~ ~ n d s has been experimentally demonstrated, and the effectiveness of this approach in achieving stress tolerance in the transgenic plants implied from laboratory data, no large scale field studies have been reported thus far (mannitol - Tarczynski et al., 1993; Thomas et al., 1995; fructans - Pilon-Smits et ml., 1995; glycine-betaine - Ishitani el al., 1995, L i l i ~ ~ s et al., 1996; proline - Kavi Kishore el al., 1995; trehalose - Romero et al., 1997). In a similar way, genetic ~nanipulation of the aquaporins ( a group of water channel proteins that regulate watcr balance in plants - Chrispeels ant1 Maurel, 1994; Kaltlenlioff ei al., 1995) may provide a practical way to achieve drought tolerance in plants. Other commonly observed proteins that are intluced in response toosmotic stress and other stresses are the LEA (late embryogenic abundant) proteins, dehydrins, osmoti11, 111etallotl1ionein-like proteins, calrnodulin, proline- rich proteins, heat-shock proteins, etc. Coding sequences for many of these proteins have been clo~led and characterized and thus can be used for their genetic mat~ipulation. Abscisic acid appears to be a comrnon factor for the transduction of stress signal in many cases.

For achieving heat ant1 coltl tolerance, the use of heat shock proteins and antifreeze proteins has been discussed (Ilightower et cil., 1991; Murata et al., 1992; Wallis et al., 1997; Grahain et al., 1997).

Wood c111d Pulp Qttality

?Lvo major users of conifers are the pulp-paper industry and the timber irldustry. Both require very different wood prodncts, ant1 in Inally ways, the requirement of the two are somewhat mutually exclusive. Whereas the pulp/pnper industry requires fibers that are rich in cellulose arid low in lignins, particularly those lignins that are hard to rernove during pulping, the timber industry uses different criteria for wood quality. As our untlerstanding of the biosynthesis of dil'ferent types of l ig~~ins increases (Boutlet et al., 1995; Whetten and Sederoff, 1995; Douglas, 1996; Ci~mpbell and Sederoff, 1996; MacKay ei nl., 1997), we should be able to manipulate the an~ou~lts as well as the chemistry of lignins in wood. 1-ignin production is a very complex process with a tren~e~ldous amount of variatior~ among different plant species. I t is an extremely heteroge~leous polymer, the hiosynthesis of which is affected by environmental, nutritional arltl other factors that affect growth and development. Prese~~tly, it is difficult to anticipate the consequences of genetic manipulation of the biosynthetic pathways of lignin on growth, develop~ner~tal and morphogenetic behavior of pli~nts. Several attempts have bee11 made to genetically ma~lipulate lignin biosynthesis via the transge~~ic approi~cli (Sederoff et ( I / . , 1994; Atanasova et al., 1995; Doorsselaere et al., 1995; Kajita et al., 1997). In addition to co~~tributing to the strength of wood, lignin also plays a crucial role in the ability of wood to resist pathogen infection and rotting.

Genetic fransJorma~iorr i r t co11iJers 303

This will require alteration of lignin chemistry in ways quite different from those that are needed for wood strength or pulping quality (Yao et a]. , 1995).

Like lignin, tlie deposition of cellulose in the secondary cell wall is also quite important in determining wood quality and its use. 111 contrast to lignin, however, cellulose chernistry is highly conserved in plants. While its chernistry is simple in that only a few steps are involved in its biosynthesis, it is only recently that we have identified the enzymes involved in cellulose biosynthesis (l<udlicka and Brown et al., 1996b; Arioli et al., 1998; Delmer, 1998). The most difficult and variable aspect of cellulose deposition is tlie underlying matrix that determines the cellulose fiber length, orientation, thickness of the secorldary cell wall, the composition of other cell wall components, and other structural features of tlie cell wall. While increased cellulose biosynthesis in plant cells has been achieved through genetic engineering, other parameters of cellulose deposition have not been an easy target. It is expected that during the next few years, we will have a better understanding of the regulation of cellulose biosyntl~esis and deposition in the cell wall in plants. The production of cellulose in genetically engineered bacteria, and stociies with it1 vitro cellr~lose biosyntliesis in cotton ovules, should provide extremely useful information to increase cellulose production in appropriate plant tissues and also its deposition in the cell wall. This is an area where conifers can be the prime target for genetic manipulation. Pulping and timber quality probably will dictate different approaches to ~nariipulation of cellulose deposition in a given conifer species.

A major concern for the field plantations of genetically engineered plants is the risk of spreading the transgene or the transge~lic plants and its impact on natural plant populations. This becomes even more critical for the conifers which have large populations of natural forests and spread their pollen grains through wind pollination at great distances. While the spread of tlie seeds of transgenic plants may be controllable to some extent, cross fertilization of the wild populations via wind-spread transgenic poller~ cannot be controlled. 'l'he only viable options to li~nit the spread of tlie transgenic pla~its by seeds or the transgene by pollen are to i~npact complete male sterility, if not a complete lack of fertility, in the transgenic trees. The question then is, will the advantage of limiting the spread of transgene in this way outweigh the advantages of breeding of conitnercially useful genotypes with the transgenic material? In other words, li~nitir~g the fertility of transgenic trees will force us to depend almost entirely on their propagation by cloning. Tliis will also lirnit our ability to amplify the gains of genetic engineering for a given trait by incorporating it into a broad genetic background of tlie species, which can be done only by co~lventio~lal breeding. The best hope to achieve a practical solution to this problem will be to use fertility co~itrolling genes under the regulation of a promoter that car1 be experimentally induced or suppressetl so that infertility can be reversed when desired for breeding purposes. While a number of potential mechanisms for controlling male sterility have been discussed (some

304 S.C. Mir~ocl~n nrtd R. Ati~zocha

have also bee11 sl~own to be quite effective) and solne piogress in cont~olling t l~e flowering pl~enomenorl has also bee11 made (see Stlauss et al., 1995 for rev~ew), stlitable promoters to achieve the reversal of infertility have yet to be identified ; I I I ~

tested for tlarlsgenic expression. Anotller comn~e~cially useful target will be the genetic nianipulation of ea~ ly

~naturation and c o ~ ~ c productior~ in conifers for use irr fast breeding cycles to incorporate the technology of genetic engirleering into traditional breeding and seed p~oductiorl tecl~nolvgies. Our understandirlg of the process of flowering 11;~s tren~endously increased over the past few years. A nurnbe~ of gcnes cont~olling the timing of Slower productio~l, flower morphology, and the developmei~t of f lowc~s have been identified in several plants and some have been tested under transgenic conditions (both sense or antise~lse expression) in heterologous hosts These genes should help us produce transgenic conifer tlces which show early ilowering, thus e~ihat~cing o u ~ capability of reducing the breeding cycle and, in turn, inc~easing the bleeding potential. Sirnilar genes need to be identified and characteriled in i~npol- tant conifer species. This will aid in i~lcleasir~g the genetic background of rriaterial used in agrolorestry and allow 11s to n~anipulate cornplex characteristics of growth, yield and morphology of the t~ees.

Biontass and Other Characteristics

'Iota1 biomass of a tree is determined by overall growth rates and organic mattel accumulation in various tissues of the t~ce . These arc highly complex pl~cnornena w11ich are dependent upon the interplay of plant ho~mones, primary productivity, pa~titioning, t~ansport aud cellular ~nctabolisln. In rnost cases, a sirlgle gene may not contribute significantly to the ovcrali biomass yield of a plant due to the conl- plex interplay of l~o~noeostatic controls of plant metabolism. Iiowever, the results of liobsor~ et al. (1996) on the genetic e~~girleering of harvest index (total leaf biomass) in tobacco by overexpressior~ of a phytochrome gene indicate the possi- bility of increasing some aspects of productivity of plants by a single gene transfer

Genetic improve~nent of the embryogenic potential of cell cultures may be another area of great sig~~ificance lor the t~ansger~ic app~oach. The derno~lst~atior~ of improved and prolonged somatic embryogenic potential of carrot cell lines through gejlelic manipulation of poiya~nilre ~netabolism (Bastola and Minocha, 1995) may providc the basis Sol targeting certain metabolic patllways that play a11 important role in developmer~t. ?'his approach would require a better understanding of the molecular and biocl~ernical events associated with sonlatic embryogenesis.

Some of the other areas that call be targeted for genetic improvelnent of corlifers include: juvenility and matu~ity, fast growth of onlamental trees, shape and branching pattern of on~amental [lees, aging and senescence, better utilizatiori of nutrients, 1nyco1111izal interactions, heavy metal toleral-rce, frag~ance, etc. For many of these cl~aracteristics, genes have already been identified and cloned, and thus should become available for transgenic expression.

F111ure Perspectives

Recent advances in transfer of multigene constructs with polycistronic DNAs (Lough et al., l997), the transfer of large (sevcral Ilundretl kb) DNA molecules (I-latnilton el crl., 1996), targeting of the transgene to specific sites in the genome, manipulation of (he 5' and 3' untranslated regions of the gene to optimize gene expression (Icoziel et al., 1996), site directed mutagenesis, tlie use of scaffold attachment region (SARs) genes (Allen et a[. , 1993), gene disruption ant1 replace- ment tlirough Iiomologous recombination (Merloll and I-Iooykaas, 1995; Miao artd Lam, 1995; Kempin el al., 1997), and a multitude of other approaches to regulate the transfer and expression of foreign gelles in tile recipient cells/l)la~~ts, s11ould provide exciting new ways to produce liighly desirable plants, including conifers. An obvious need in this regard is a concerted effort at testing the tech~lology with econoniically important tree species using genes of cornrnercial value, some of wliich are already available and many more are being clotled and characterized. Ultimately, the selection of tile gene(s) and the plarlt will be determined by a balance of economic gains and potential risks of environmental damage or purturbance in tlie biodiversity of natural populations.

The long-tenn (25-50 years) rotation cycle for production of useful tree products is a problem for planning of specific targets for genetic engineering of trees. 'l'l~e changing tech~iology for the usage of timber, productiori of pulp and paper, and the usage of tree biomass for energy production (all requiring different types of plantations), raise the most fundamental question 'What trees should be planted today to meet the needs of mid 21st century'? Pathogen evolution and developrnerlt of resistance over the years could make the plantatiorls highly prone to damage after decades of growth in the field.

The testing of genetically improved stocks typically requires at least 5-10 years for fast growing conifers, e.g. Piruts radiafa, and even more tinie for most other species. Quantitative trait markers associated with desirable cliaracteristics are currently not available to aid in early selectioil and evaluation of the improved genotypes. Thus, a broad spectrunl of genetically improved stocks (cell lines) must be maintained in the storage banks for rapid cloning ancl plantation at a later date. Al t l~ougl~ cryopreservatiorl can help in this situation, this technology has not been optimized for most conifer species (Day ailti McLellan, 1YY5).

Plantation forests during their maturation cycle often serve other hunlan rieeds sucli as recreation, wildlife feeding and hunting grounds, and a multitude of urban and rural tourism-associated activities. We must develop policies and strategies to treat densely-planted agroforestry areas at par with agricultural land lo potentially limit its usage for other needs.

Entire populations of genetically engineered stocks must be clonally propagated because the normal reproductive cycles of most conifers are too long to take advantage of heterosis. Thus the transferred gene will often be in a heterozygous form in most of the tree plantations.

Ultimately, the success of genetic engineering of conifers will depend upon economic gaitls to tlie forester which, due to their long harvesting periods, requires

long-term investn~ent into controlled research, deve lop~r~ent and evaluation of this technology.

At present, the best that can be said is that w e have started to think about and plan for using the gene transfer technology for genctic improvement of trees. There have also been sporadic demons t ra t io~~s of success in the use of presently available techniques of transformation. Soon, we must demonstrate phenotypic improvement of the product at t l ~ e field level and demonstrate its ulti~nale gain to the forester. This is likely to come first with the hardwood species, such a s Populus and Eucnlyptirs, which are relatively faster growing than the conifers but stlare a number of commercially useful traits wit11 them (IJan et a/., 1994).

The authors would like to thank Dr R. 0. Blanchard, Dr Pierre Charest, M s S. Bains and Ms I3 Crlasl~eeri for helpfit1 suggestions to improve the manuscript. Scientific contribrttion Number 1985 from the New Hampshire Agricultural Experiment Station.

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