CA2064017A1

CA2064017A1 - Particle-mediated transformation of woody plant species

Info

Publication number: CA2064017A1
Application number: CA002064017A
Authority: CA
Inventors: Dennis E. Mccabe; Brent H. Mccown
Original assignee: Individual
Current assignee: Agracetus Inc; Wisconsin Alumni Research Foundation
Priority date: 1990-05-24
Filing date: 1991-05-23
Publication date: 1991-11-25
Also published as: WO1991018094A1

Abstract

The creation of transgenic trees is disclosed through the use of a particle-mediated transformation procedure. The method is demonstrated to be effective with various tissue types of poplar, including protoplasts, internode and petiole segments, and, most efficiently, with nodule cultures. Transgenic trees were recovered which express sufficient levels of the insect specific toxin from Bacillus thuringiensis so as to provide significant toxicity to feeding insects.

Description

2 ~ 17 ~ 091/18094 PCT/US9~/03629 !

PARTICLE-MEDIATED TRANSFORMATION
OF WOODY PLANT SPECIES

Field of the Invention - - . .
The present invention relate~ to the genetic engineering of plant3 in general and relates, in particular, to a methodology u~ing particle-mediated transformation techniques to achieve the germ line genetic transformation of woody species of perennial plants such as trees.

Back~round of the Invention The technology of recombinant DNA manipulation and insertion has evolved to the point where it is now posslble to g~nstically engineer many crop plants. In the genetic engineering of crop plants, one or more foreign gQnetlc constructions are lnserted into the genomic DNA of the target plant species. The progeny plants produced through such a process carry in their genomic DNA the inserted foreign genetic construction which can thereafter be passed on to the progeny of the plant by normal plant breeding techniques. Using such tèchniquès, it has beco~e com~onplace to genetically engineer several model species, such as tobacco, j petunia, carrot, and potato. The technology techniques ~ of genetic engineering have recently been extended to ' : .
' .

W091/~809~ 206~ol 7 PCT/US91/03629 ~

enable the transformation of some important crop species such as cotton and soybean.
The genetic engineering of plants typically involve3 the insertion of the foreign genetic construction into a living plant cell or tissue, typically as fostered in so-called ti~sue culture.
Tissue culture refers to an in vitro culture of living plant cells. The most common technique utilized to transfer foreign genetic materialR into plant cells makes use of the common soil-dwelling plant pathogen bacteria, Aqrobacterium tumefacien~. A. tumefaciens natively harbors a plasmid, referred to as the Ti (tumor-inducing) plasmid, which has the inherent ability to transfer a portion of it~ DNA (T-DNA) into a target plant cell. By suitable ~anipulation of the Ti plasmids of Aqrobacterium tumefaciens, it ts po sible to in~ert a foreign genetic construction into tho T-DNA of the Ti plasmid, which is then transformed into succeptible plant cells in tissue culture by the bacteri~. Other techniques for transforming individual cells or cells in tissue culture include direct DNA in;ection and electroporation o~ plant protoplast cells.
The techniques of Aqrobacterium-medlated transformation of plant cells have baen applied to at lea~t one woody species. In U.S. patent number 4,795,855, a technique is described utilizing an Aqrobacterium tran~formation system, followed by regeneration of the tr~nsformed cells, which is de~cribed with regard to the dicotyledonous tree species poplar. ~One difficulty in the utilization of Aqrobacterium-mediated techniques for plant transformation is that they can be very dependent on the species specific interaction between Aqrobacterium and the cells of the target species plant. Anoth.er difficulty is that not all plant species can yet be regenerated from tissue cultures transformed by Aqrobacterium.

- - - , - . . . .
. .

- . - .~ : ~. . .
, . , . :
:: .

~`06~0~7 One alternative methodology for genetically engineering plants which has been suggested involves the coating of DNA onto small particles which are accelerated into cells. It has been previously reported with herbaceous species of plants that indi~idual cells can be transformed, much in the fashion of Aqrobacterium transformation, when D~A is coated onto small particles which are then physically accelerated into the cells of the target plant tissues. Klein et al., Proc. Natl.
Acad. Sci. U.S.A., 88, pg3. 8502-8505 (1988). A
technique of germline transformation of soybean by particle-mediated transformation has also been published. McCabe et al., ~io/TechnologY, 6, pgs.
923-926 (1988).
once it becomes pos6ible to create genetically engineered trees, it then become~ logical to inquire as to what for~ign genes may be inserted into trees to create more valuable trQes. One gene which has been inserted into herbaceous plants i8 a chimeric genetic construction coding for the expression of an insect-toxic portion of the delta-endotoxin protein produced by the 80il dwelling microorganism Bacillus thurinaiensis, or 3.t. This toxin has been previously found to be specifically toxic only to Lepidopteran insects, i.e., the larvae of moths and butterflies.
Since caterpillars are a primary consumer of leaves of tr~e species grown for fiber, lumber, or energy, the creation o~ trees having resistance to attack by Lepidopteran larvae by ~irtue of the insertion of a B.t.
geno would be of significant value.

Summary of the Invention The present invention is summarized in that the genetic engineering of woody species of plan~s involves the use of a particle-mediated transformation technique in which tissues of the woody plants are transformed with DNA carried on small carrier particles.

. .
, . . , . .. . . - . . .
-, - ,: , ., . , : . :
- : , . . . . . . .
.
.. . : ~ , , . ~

. . . . . . . . . .

: :`

4 2 0 ~ ~ 017 PCT/US91/03629 The present invention is also summarized in that poplar trees are created which express in their leaves an insect-toxic amount of the B.t. Lepidopteran specific toxin.
It is an object of the invention to enable the genetic engineering of tree species with useful foreign genetic traits, such as the B.t. toxin gene.
It is another object of the present invention to provide a methodology having broad applicability to woody species to enable the genetic engineering of many such species.
Other objects, advantages, and features of the preqent invention will become apparent from the following specification when taken in con~unction with the accompanying drawing~.

~rief De~cri~tion of tha Drawin~s Fig. l is an illu~tration o~ a particle accelaration apparatus useful within the procedure of the present invention.
Fig. 2 is an illustration and a restriction map of the plasmid pTVBTGUS.
Fig. 3 is a graphical repreqentation of the results of whole plant assays using the transgenic plants of the present invention.
Fig. 4 i9 a graphical representation of the survival of larvae feeding on parts of the transgenic plants created in accordance with the process of the pre~ent invent~on.
Fi~. 5 i9 a graphical representation of a whole leaf a~say utilizing the transgenic plants of the present invention.

Description of the Inv _tion In accordance with the present invention, it has been discovered that the general approach of . . ~
,, i :, . .. .

2a6~0l7 . i ` ! ~

particle-mediated transformation of plant tissues can be successfully applied to the germline transformation of woody plants. Through the use of this approach, disclosed herewith in particular with the model tree species poplar, it now becomss possible to genetically engineer trees generally and to imbue them with characteristics favorable for efficient growth and production qualities. For example, also described here is the creation of transgenic tree~ which have been imbued with the property of toxicity upon ingestion to larvae of Lepidopteran insects, so that the trees become resistant to grazing by caterpillars.
It has been discovered herein that a wide variety of culture tissue types from woody species can be genetically engineered through a particle-mediated transformation proc~ss. It has been found that cells derived from leaf protoplasts can be transformed, that su~pension culture~ of cells can b~ transformed, that cultured int~rnodes can be transformed, and further that ; nodule cultures of tres specie~ can be tran~formed. All of these tis~ue types have yislded transformed callus, and the tran~formation of protoplasts and nodules has resulted in the regeneration of transgenic trees. It has been particularly discovered that the transformation of nodules is a particularly efficient system for the genetic transformation of trees using particle-mediated trans~ormation techniques.
Nodule~ are phenomenon of the tissue culture of plant cells. -Nodules are independent, spherical, dense cell~ c~ster~, ~or~ed in an ln vitro culture, which display a degreG of tissue differentiation which distinguish the cultures from callus and other forms of less differentiated cultures which have been created by dedifferentiated cells. Nodule forma~ion is particularly characteristic of woody species such as poplar, but have also been noted as occurring with other herbaceous plant species such as carrots, day lily, and :

. .. . ... ~ .

WO91/18094 2 a6 ~ 0l 7 PCT/US~ 3629 other woody plants such as citrus, spruce, and pine. A
complete description of the formation of nodules in poplar, and the charac~erization of such nodules, is contained in the text of "Nodule Culture: A
Developmental Pathway With High ~otential For Regeneration, Automated Micropropagation, and Plant Metabolite Production From Woody Plants," McCown et al.
in Genetics ~aniPulation of WoodY Plants, Ed. by Hanover et al., pg. 149-166 (Plenum Publishing 1988), the disclosure of which is hereby incorporated by reference.
The ease and efficiency of the transformation of nodules, and the availability of analogous nodule cultures for other woody species such as citrus and pine, suggests that the genetic transformation technique applied successfully here to poplar may also be readily applied to other woody and herbaceous species for which a nodule culture regi~en has been, or is, de~eloped.
Nodules are particularly attractive for use in this technique since thoy may be created, manipulated, and selected in relatively large nu~bers.
The process described herein is directed toward the introduct~on of exogenouY, typically chimeric, genetic constructions into the germ line of woody plant species.
For use within the present invention, ~uch an exogenous genetic construction i8 preferahly DNA from one or more other organisms, whsther of the same or different species, which i8 introduced into the transformed woody plant through hu~an manipulation by the artificial introduction of the exogenous genetic construction into the cells of the transformed woody plant. The method of transformation described herein is the use of particle-~ediated transformation in which small carrier particles carry the genetic construction into the cells of the plants. The exogenous genetic construction would normally include a coding sequence which codes for the production in the cells of the plant of a transcription product or a protein of interest. The exogenous genetic .

~ ' '' . ' '' :. .

, , j ', `
~g~`W09t/lR094 ' PCT/US91/03629 construction therefor typically includes flanking regulatory sequences effective to cause the expression of thQ protein, or the transcription product coded by the coding sequence, in the transformed cells of the woody plant species. Examples of flanking regulatory sequences are a promoter sequence sufficient to initiate transcription, and a terminator or polyadenylation sequence sufficient to terminate the gene product, whether by termination of transcription or translation.
It i~ also possible to include translational enhancers located between the promoter and the coding sequence to assist in the efficiency of the expression of the genetic product, especially the expression of p~otein products, in the tran~formed woody plant cells. It is specifically envisioned that gena product~ other than proteins may also be expressed by th~ inserted exogenous genetic con~truction. For exa2ple, the in3erted construction can expre~ a negative RNA strand, also referred to a~ an anti-sen~e ~trand, effective either to suppres the expression of an endogenous gene in the woody plant 6pecies or to inhibit a disease process by a pathogenic organism. It ha~ been spscifically found that the creation of chimeric exogenous genetic constructions, and their insertion into transgenic plants, results in traits which are inheritable by the normal sexual reproduction of plants thereafter in a conventional Mendellian fashion.
The procesq of the present invention is i~tended to maks particular use of a procedured apparatus utilizing an ad~u~able electric discharge device to physically accelerate DNA coated onto small par~icles into plant cells. An apparatus suitable for use within the present invention is illustrated in Figs. 1 and 2. The apparatus conslst3 o a spark discharge chan~)er 12 into which are inserted two electrodes 14 which are spaced apart by a distance of approximately 1 to 2 millimeters.
The sparX discharge chamber 12 is a horizontally , , : . : . .

.. ' : ,. .

W091/18094 2 ~ ~ 4 ~ 1 7 PCT/~S91/0362g ~ ~:
!

extending rectangle having two openings 16 and 18 extending out its upward end. The opening 16 is covered by an access plate 20. The opening 18, located on the side of the rectangle of the spark discharge chamber 12 opposite from the electrodes 14, i5 intended to be covered by a carrier sheet 22. The electrodes 14 are connected to a suitable adjustable source of electric discharge voltage. Such a source of electric discharge voltage would preferably include suitable electric switching connected to a capacitor of the one to two microfarad size range, with the amount of the voltage of the charge introduced onto the capacitor being adjustable, such as through the use of an autotransformer, through a range of perhaps 1 to 50,000 volts. Suitable high voltage electric switching (not shown) i8 provided 80 that the capacitor can safely be discharged through the electrodes 14 so that the apparatus can be used conveniently by a user.
The carrier sheet 22 intended to be placed upon the opening 18 in the spark discharge chamber 12 is a planar sheet of relatively stiff material such as a sheet of aluminized saran coated mylar. Above the opening 18 in the discharge chamber 12, positioned approximately 5 to 10 millimeters above it, i~ a retaining screen 24.
Placed above the retaining screen 24 at a distance of approximately S to 25 millimeters above the retaining screen, i5 a target surface 26. The target surface 26 can be any sultable culture surface onto which the material to be transformed may readily be placed such as, most~conveniently, an overturned petri dish into which the plant tissues have been positioned for culture. Copies of the exogenous foreign genetic construction intended to be transformed into the plant tissues i~ prepared by suitable DNA preparation technique~ well known to those of ordinary sXill in the art and multiple copies of the genetic construction are made. The copies of the foreign genetic construction, ' .
'~ ' .

2~6~t 7 091/18094 PCT/US9~/03629 _g_ !
in aqueous solution, are then coated onto small particles of a durable dense carrier material, such as gold, the carrier particles typically being in a size range of l to 3 microns. The carrier particles with the exogenous genetic construction coated thereon are then placed upon the carrier ~heet 22 which is inserted at the proper opening on the top of the spark discharge chamber 12. The target surface 26 including the living plant material thereon is then placed in position above the retaining screen 24. A small droplet of water, preferably 2 to 4 microliters in size, is then placed bridging between the ends of the electrodes 14. The access cover 20 is placed in position on top of the spark discharge chamber 12. At this point the entire apparatus is enclosed in a vacuum chamber and a vacuum i9 drawn until it i~ in the range of approximately 500 ~-millimeters of mercury. A supply of helium is continuously bled into the vacuum chamber to replace the atmosphere in the spacQ between the carrier sheet and the target with helium to take advantage of the lower relative density of helium.
At this point, the initiation o~ a spark discharge may be init~ated by the u~er between the electrodes 14.
This is dona by means of the appropriate electric switching which applie~ the voltage stored in the capacitor acros~ the terminals of the electrodes 14.
The ~OrCQ 0~ this ~lectric discharge bridges the spark discharg~ gap between the electrodes 14 instantly vaporizing the s~all droplet of water previously placed -thersbe~ween. The force of the vaporization of that water creates a shockwave within the sparX discharge chamber 12 which radiates outward in all directions.
The impact of the radiating shockwave upon the carrier sheet 22 propels the carrier sheet 22 upward with great velocity. The upwardly traveling carrier sheet 22 accelerate~ until it contacts the retaining screen 24.
The use of the helium within the vacuum containment for , , . : . . ., . ., . .: , .. ,~ , .

WO 91 /lR094 ~ Q 6 ~ ~ 17 P~/U591 /036~:9 ~

the apparatus provides less drag on the flight of the carrier sheet 22 as well as less force necessary for the shockwave to propagate the carrier particles to the target tissues. At the retaining screan 24, the carrier sheet 22 is retained, and the carrier particles coated with the exogenous genetic construction previously coated thereon fly off of the carrier sheet and travel freely onward toward the target tissues. The small carrier particles then proceed into the cells of the target tissues placed on the target surface 26 and pass freely into the cytosol of the cells placed thereon.
The actual momentum of the carrier particles as they impact the surface of the target tissues is adjustable, based upon the voltage of the initial electric discharge applied to the electrodes 14. Thus by varying the amount of the electric discharge applied across the electrodes 14, the velocity by which the particles impact the target can be adjusted, and thus the depth of penetration of the carrier particles into the tissue of the target tissues can be adjusted continuously throughout the range of adjustment provided for the electric voltage applied acro~s the electrodes 14.
The apparatus of Figc. 1 and 2 has previously been demonstrated to be useful for the transformation of tissues of various herbaceou~ plants. It has been found through the work disclosed herein that this apparatus and the proc~dure for using it may also be appropriately applied to the transformation of woody plant species and can result in-the germline transformation of complete whole, intact and sexually mature trea species.
Therefore, the target tissues to be placed upon the target surface 18 in the apparatus of Fig. 1 for use within the present invention would preferably include a regenerable tissue sample of the woody plant species to be transformed. It has been found that a particularly advantageous tissue type for each transformation is a nodule culture of the woody plant species. Other usable - , . . . : ~ -:
:, . ,: . . . .. . : :
-: .. . . . .. .. .
- ' . ,. , , . . : . . .
., - - : . . :
.; , ~.

4 ~ 1 7 091/1~094 PCT/US91/03629 tissue types include stem segments, protoplasts, and suspension cell cultures. Since the germline transformation of woody species is an object of this invention, it i9 therefore desirable that the particular tissue types placed upon the target surface 28 be one which can be regenerated either readily, or at least with a sufficient frequency, so that whole, intact plants can be recovered through the use of this technique. Such cultures exist for a variety of woody species, such as the model species poplar disclosed and discussed herein.
As will be apparent from the following examples, the technique of particle-mediated genetic transformation can be adapted to a wide variety of tissue types in woody species. The use of a tunable electric spark discharge for the motive force in the particle acceleration has proven also to be of great advantage, sinc~ the acceleration imparted to the particles can be readily and ea~ily tuned using this technique, thereby facilitating adaptation of the technique to different tissue type~. Note ~hat unlike Aqrobacteriu~ trans~ormation methods, the present technique can be u~ed either with or without a selectabl~ marker. In the examples described below, the selectable markor of kana~ycin resistanc~ has been used since the re~i3tance gene provides good selection in poplar, and thus add~ to the efficlency of the procedur~. Note that the selection step could have been omitted, sinca a readily detectable marker gene, beta-glucuronldase was included in the trans~ormation vector. ~he omission of selection would require the regeneration of more tissues, since the tissues would have to be screened for the presence of the marXer, but the transformation of the germ line of the woody plant species could still be achieved. Other selection agents and marker genes are also known to those of skill in the art.

. .
: - , . . , , . , , . : ~
;: .

W09t/t8094 2 0 6 ~ ~ 1 7 PCT/US91/03629 ~ ~

ExamP-les 1. Description of Expression Cassette The exogenous genetic construction utilized in the examples described herein was the plasmid pTVBTGUS. An illustration of the coding sequences and a restriction map of the plasmid pTVBTGUS is illustrated on Fig. 3.
The plasmid pTVBTGUS includes expression cassettes for three separate coding sequences found to be effectively expressible in the cells of plant tissues. One expressible genetic construction is for the gene aminoglycoside phosphotransferase II ~neomycin phosphotransferase) gene (NPT II or APH II) commonly used as a selectible marker for kanamycin resistance in plant transformation procedures particularly those utilizing A~robacter~um-mediated transformation technigues. The plasmid pTVBTGUS also includes a gene coding for the expression of the beta-glucuronidase gene (GUS), which codes for the expression of an enzyme which can be readily detectible by a colorimetric assay as described by Jefferson, Embo J., 6:3901-3907 (1987).
Also in the plasmid p~V~TGUS is a plant expression vector cassette coding for the expreesion of B.t., i.e.
the delta endotoxin from Bacillus thurin~iensis. The toxins produced by the soil-dwelling microorganism Bacillus thurin~isnsi~ have long been recognized to have .
potential value a~ sQlective biological insecticides due to their uniqu~ and speclfic toxicity only to Lepidopteran insects. The entire DNA sequence of this gene, lncluding flanking regions and inferred amino acid sequence, have been previously published, as have various physical char~cteristics and features of the expression in its natural host. It has also been reported that deletion analysis of the full length protoxin coded for by the native gene sequence reveal that the amino ter~inal portion of the gene is sufficient for toxicity. As de~cribed in published PCT
patent application WO 89/04868, an amino-terminal -: .; . . . ~ ;
., . . .......... - , . . . .
~, .... - . ;. . . , : ~ :

.. :. . , 206~ol 7 0 91/18094 ' i ` ' PC~r/US91/03629 portion of the B.t. delta endotoxin gene can be constructed which codes for the expression of a truncated toxin protein which retains the toxicity of the protoxin. The truncated toxin has been found to be effectively expressed in plant cells to confer those cells with toxicity upon ingestion to Lepidopteran insects.
The B.t. toxin coding region in pTVBTGUS codes for the same amino acid as the corresponding B.t. coding region found in plasmid pAMVBTS, described in the above-identified PCT patent application W0 89/04868, but the actual nucleotide sequence i9 different. The first 141 codons have been altered to codons which conform to plant codon usage frequencies. This was accomplished by creating overlapping synthetic oligonucleotides for the desired sequence, and then linking the oligonucleotides together to form th~ ~irst 141 codons of the coding sequence, which was then inserted into the plasmid pAMVBTS.
It ~ay bQ appreciated by one of ordinary skill in the art that the plasmid pTV4 incorporated into the expression plasmid pTVBTGUS dsscribed herein contains the laft and right border sequenc~s from A~robacterlum tumefaciens which arQ necessary to effect plants for transformat~on us~ng an Aqrobacter~um-mediated transformation techniqu~. It is bel~eved that those le~t and r~ght border se~uence~ are unnecessary in an expres~ion cassette utilized within the method of the present lnventlon. However, since this plasmid was construc~ed for other A~robacterium-based transformation experiment3 with other plants, this expression plasmid was conveniently available for use within the practice of the present invention a described herein.
Tho actual construction of the plasmid pTVBTGUS was an evolutionary one as plasmids constructed for certain experiments were modified conveniently for other experiments. Hence the derivation of this plasmid - ~

.
. . . . ~. . ,: : . .
; . : . : , .
,, . : , .

W09t/18094 2~4~17 PC~/U~9~362~ ~

involved several modifications not relevant to the examples illustrated here and several procedures which would not be repeated if synthesis of this plasmid directly was the objective. How this plasmid may be readily directly ascembled from depo~ited plasmids and sequence information is described below.
The plasmid pTVBTGUS thus has, in addition to antibiotic resistance markers ~Ampr and Sur) three plant expression cassettes. These expression cassettes are for kanamycin resistance (NPT II~, beta-glucuronidase gene (GUS) and for the B.t. toxin. The inclusion of both the NPT II and GUS genes would be redundant for most applications, since either selection or screening of transformant tissues would normally be performed to identify transgenic tlssues, but not both. The plasmid pTVBTGUS l~ a large one, of approximately 18,687 base pairs, and the a~ility of the proceture described here to insert such a large DNA insert into transgenic plants is indicative of the ability to achieve the same result with other large constructions.

2. Transformation o~ Isolated Cells The isolation and growth of protoplasts was conducted in accordance with the method described in "Recovery of Plant~ from ~eaf Protoplasts of Hybrid-Poplar and Aspen Clone~," Rus~ell and McCown, Plant Cell RePorts, 7:59-62 (1988), the text of which is heraby incorporated by reference. The particular germ plasm used within the practice of this invention were two genotype~ of commercially significant hybrid poplar trees, NC5339 which is a hybrid of Populu alba by P.
qrandidentata "Cranden" and NC5331, which is a P. niqra L "betulifolia" by P. trichocar~a. Tissues of NC5339 were used in the protoplast, nodule, and stem transformations discussed below, while NC5331 was also used in the nodule transformations. Cells were cultivated in suspension cultures taken from uniform and : : .
~ . , .

: . .,~ . . ....................... : . , :
.- . : . , ' ' '. : - - :

~ 10~ `7 091/18094 ` ~-. PCT/US91/03629 rapidly growing cultures at the pre-log and early-log phase of growth which was typically three to seven days after subculture.
Protoplasts and suspension cultures were plated at a density of 25,000 to 60,000 cells per milliliter which was deemed to be a population high enough to give adequate viable population after blasting and losses during later development and a low enough density to permit rapid cell division and cell colony singulation.
Floating discs in the manner described in Russell et al., suPra were created with attached cells derived from protoplast~ or overlaid suspension cells derived from early log-phase cultures (3 days old) growing in liquid MS medium (Murashige and Skoog, Phvsiol. Plant, 15:473-497) supplemented with lmg/l 2-4,D and 0.05 mg/l BA. The discs wore placed on flve centimeter petri dishes with one dl~c platad per platQ. The plating medium consisted of lO milliliters o~ the ba~ic WPM
medium (Lloyd and McCown, Proc. Intl. Plant ProP. Soc., 30:421-427 (1980)) as modified with hormone~ or sugars suitable for the growth of protoplast-derived cells (Russell et al., s~pra) or 3uspension cultures (l.0 micromolar NAA and 0.1 micromolar ~A). The discs were blotted with filter paper prior to the transformation technique. Tho petri dishes were then used as the target surface 26 within the apparatus of Fig. l.
Each set of plated protoplasts or suspension culture cells were subject to a single transformation event at a voltage of six kilovolts for the spark dischargo. The carrier particles utilized were l to 3 micron gold particles. The loading of the particles onto the carrier sheet was in a range of 0.025 to 0.05 milligrams per square centimeter. The density of the copies of the foreign genetic construction DNA applied to the carrier particles themselves was approximately 0.1 micrograms of DNA of pTVBTGUS per milligram of gold beads.

, - :
,. ,;
'`.?
, ~ ' :. '' '' ` , :. . ' ` ' '- . , .. ' .' ::
'.' ' ' WO91/18094 2 ~ 6 4 01 7 PCT/US91/0~629 ~ , The process for coating the DNA onto the carrier particles was to suspend l microgram DNA in 2 microliters 0.2M ~DTA and l0 microliters of Tris, pH8.
l0 milligrams of gold beads were also introduced into the suspension. The suspension was vortexed and dried under a nitrogen stream. Then the dried particles were resuspended in ethanol, coated onto the carrier sheet, and air dried.
Once the particle-mediated transformation event had occurred, the culture~ were moved to a liquid growth medium in a multi-well plate in the case of protoplasts or were kept on the target plates in the case of suspension cells. All tissues were kept in the dark for 48 hours after blasting. The protoplast cells were cultured on this medium with continual medium refreshment and o~moticum reduction i~ needed every 7 to 10 days. As the mediu~ was refreshed and antibiotic selection regimen was included at a rate of 20 milligrams per liter of kanamycin added to the standard WPM medium.
After recovery of the tissues was noted due to the resumption of cell d~vision activity, the dish wa~ moved to a solidified mediu~ con~isting of WPM medium supplemented with 0.l micromolar BA (benzyladenine), 0.l ~icromolar NAA (naphthal~n~ acetic acid) 0.l micromolar THI (thidiazuron) and 50 milligrams per liter kanamycin.
After the plant tissues were ob erved to grow, the plates were flooded with liquid kanamycin in aqueous solution at a-volume equal to 10% of the total volume of the pla~e and in a concentration of kanamycin of 50 milligramc per liter based on total volume of medium in the plate.
When the green colonies grew to a stage large enough to manipulate, they were removed from the culture wells and placed on similar plates for development and/or differentiation. All plates were continuously ~ . ~ , . ' : ., ,, . . :

.

2 ~ 1 7 VO9t/t~094 ,~,j,1 , PCT/US91/03629 flooded with kanamycin at the rate of 50 milligrams per liter based on the total media volume in the vessel.
Large nodular green colonies in excess of 3 millimeters in diameter were divided and part were sacrificed for analysis of genetic construction using a polymerase chain reaction analysis. The remaining portions of the green colonies were put on a regimen of l.0 micromolar zeatin or O.l BA, or no hormone for differentiation to shoots.
The colonies which were identified as positive by polymerase chain reaction (PCR) analysis were deemed to be transformed with the foreign genetic construction and the colonies were continually grown on and analyzed for expression of GUS and were further analyzed for presence o~ thc genetic construction by additional PCR analysis and by Southern blots taken on the t~ssue~ of the resulting cultures. Calli~ were recovered and utilizing te~hniques described by Russell et al., supra, the callus tis~ues were regenerated into plants. Two whole plants were recovered through this technique.
Overall transformation rates were calculated to be between one and three percent of the protoplast cells surviving the transformation treatment.

3. Internodes and Petiole Transformation Pieces were taken from actively-growing shoot culture~ a~t~r four to six weeks of growth in ln vitro culture.
The pieces to be subject tc a transformation event wera pr~-treated in multiwell plates with three to five pieces per plate by a liquid medium supplemented with 0.1 micromolar BA, O.l micro~olar NAA and O.l micromolar THI. The treated pieces were shaken on a rotary shaker so that the pieces were moving in the inoculating liquid pre-treatment cultures.
After ten days of such treatment, the pieces were grouped on target plates with the pieces arranged for ,, . : , .

`; : `
WO ~l/lXO9~ 2 0 6 ~ ~17 PCr/US9~l0~.9 ~

maximum accommodation within a one square centimeter in the middle of a solid plate dish. The pieces were oriented so that the heaviest concentration of "meristematic zones" of each individual culture piece were sxposed to the transformation particle path. The plating medium consisted of 10 milliliters of solidified medium as described in conjunction with the transformation of the protoplasts and suspension cell cultures above. Again the tissues were blotted immediately before the tran~for~atlon event.
These tissues were subjected to a particle-mediated transformation event utilizing the apparatus of Fig. 1 from one to five replicates per sample, spaced at six to twelve hour intervals. The voltage utilized in the electric discharge was approximately 12 kilovolts. The rate of loading of DNA onto the particles and of the carrier particles onto the carrier sheet was the same as with the protoplasts.
Again the tissues were kept and cultivated in the dark for forty-eight hours after the last transformation event. After that the cultures were plated in light and when growth wa~ visible, the plates were flooded with 50 milligrams per liter kanamycin. Some of the cultures were return~d to a liquid medium in multi-well plates under 20 to 50 m$11~gram~ per llter kanamycin selection.
Nodular growth in green callus and shoots were observed. The cultures which survived kanamycin expression were moved to new plates under selection for further development and testing. Growing callus cultures, of a type previously regenerable, were achieved which were resistant to kanamycin selection.
No further analy~iq or regeneration o~ these particular cultures wa~ taken.

4. Transformation of Nodules Nodules were cultivated in accordance with the procedures described by McCown et al., su~ra. The most - - . . . . . , . -- . . ........................................ . :
- . . .. -. ' :.- .- ' ' :

2 1~ 7 ~;N091~t8094 , I PCT/US91/03629 successful nodules utilized were those created from stabilized shoot culturea and the nodule cultures created in this fashion could be serially and continuously salf-replicated to make continuing ln vitro cultures o~ nodules. Nodules were also created from nodular growth of other tissues including stems and callus cultures, and microcalli derived from protoplastq .
If the nodules were from established cultures, prior to the particle-mediated transformation event, the nodule~ were pre-treated with the cytokinin THI. This was done after preliminary results indicated a lower level of transformation for nodules harveqted directly from a high auxin/low cytokinin medium. The remaining nodule cultures were treated from two to six weeks with 0.1 mlcromolar THI and basic 0.1 micromolar ~A and 0.1 micromolar NAA medium. Nodules which were larger than 1.0 millimeters in diameter were harvested, cut in half, and grouped togather in one square centimeter target areas in the middle of petri plates in the same fashion as were the internodQs. The cut surfaces of the nodules were upwardly exposed when they were plated into the petri plate ~o as to be exposed to the transforming particle stream. The medium onto which the cut nodules were plated i~ solidified pre-treatment medium. Again the surfaco of the nodules was surface blotted with blotting paper prior to the transformation event for drying.
The nodule3 were subjected to a particle-mediated tran~formatlon event between one and ten replicates.
The timing between replicates was six to twelve hours.
The apparatu~ was used with an electric discharge voltage of fourteen kilovolt applied between the electrodes. The carrier particles used were amorphous crystilline gold powder (Englehard), and consisted of one to five micron particles. The DNA of pT~TGUS was loaded on the carrier particles at a rate of G.l :
. ,, ~ .
- ~ . .. . . . . . .
- , , -:. :
. . ', : . ' WO91/18094 2 0 ~ ~ O 1 ~ PCT/US91/036~9 ~
-20- t microgram DNA per milligram gold and the coated gold particles were applied to the carrier sheet at a loading of .025 mg per square centimeter.
After the nodules were sub;ect to the particle-mediated transformation event, the plated nodules were kept in the dark for forty-eight hours after the last transformation event. The nodules were kept on plates and were subseouently flooded with fifty milligrams per liter kanamycin when initial growth from the cut surface was observed, typically in a time period of seven to ten days. The kanamycin was refreshed as needed.
The new green nodules and calli surviving the kanamycin selection were removed from the surface~ of parent nodules when two to three millimeter~ in diameter and were cultured separately in tho same solidified m~d~um. As is charaGteristic of such nodules, the nodules were cultivated through to further differentiation including shoot and root development into plantlets. The regeneration of ~uch nodules is through organogenesis. Nodule shoot differentiation was stimulated by THI administered as a pulse treatment at a rate of O.l micromolar. Adventitious rooting followed resulting in plantlets. The resulting plantlets were cultivated into trees which appeared morphologically normal exhibiting the normal growth characteristics of poplar trees regenerated from nodule culture.
Some regenerate plants were positive for kanamycin resistance, but not for the B.t. toxin gene, as determined by PCR analysis. However, some kanamycin resistant plants assayed poqitive PCR for both the 5' and 3' ends of the B.t. gene.

5. Assay for Insect ToxicitY
Growing tree~ of poplar were recovered from the nodule transformation procedures described above for . - .: , . . . . ~ :
.: - . . ~ . . .
- . - . : . : , . . ~ . .. :.

. : :: . . , :
, .. .. . . ..
.. . . .. .. .
.

206~0~7 both varieties NC5339 and NC5331. The plants were grown to a height of one to two feet in greenhouse cultivation. The resulting plants were tested by insect bioassay for insect toxicity activity. The assays were conducted both on the whole plant by live larvae feeding, by whole detached leaf in petri dish larval feeding, and by leaf di~k feeding also in petri dish.
Shown in Figures 3 - 4 are graphical representations of the results of the insect bioassays.
As shown in Fig. 3, a control plant consisting of a regenerated poplar tree was compared to transgenic poplar plants deslgnated BT-l and BT-2. The whole plants were assayed. Live larvae of forest tent caterpillar (Malacosoma dis~tria) were placed onto the whole plants to feed upon the leaves. The larvae were placed at a density of ~ix larvaR per plant. The larvae were given no other choice for feeding activlty and the number of larvae surviving at the end of six days and the mean weight of thQ larva~ was measured for the three plants in the assay. As illustrated in Fig. 3, in the control plant, in excess of 60% of the larvae continued to survive after the exporimental period, while only less than 20% o~ the larvae o~ each of the transgenic plants survivQd the feeding assay. Also indicated in Fig. 3 is the r~an wQight of the larvae per plant after six days indicat~ng a dramatically reduced mean weight on the larva~ feeding on the transgenic plants.
Shown in Fig. 4 is a graphical representation of th`e survival of larvae from the whole plant assay, a whole l~af a~aay, and the leaf disk assay. The whole plant assa~ represented the same replicate as indicated in Fig. 3. The whole leaf assay was an assay conducted in a petri plate wlth four larvae per dish, no feeding choice, and a feeding time of four days. The leaf disk assay was conducted with two larvae per dish with a choice of disk for the larvae to feed upon, and with a time period of thirty hours. As ~ay be seen by ' W091/18094 2 ~ ~ 01 7 PCT/U591~03629 reference to Fig. 4, there seem to be little difference in the survival of larvae in a short term triai such as that conducted on the leaf disk while in both the whole plant and whole leaf assays significant differences in survival rates for the transgenic plants as compared to the control plants were evident.
Illustrated in Fig. 5 is a graphical representation of the whole leaf assay in more deta~l. The left-hand bar graph of Fig. S illustrates the mean weight of the four larvae per di~h after the four days of treating on the whole leaf from the control or the transgenic plants. Of perhaps more ~ignificance is the right-hand bar graph in Fig. 5 which illustrates the area eaten in square centimeters of the leaf in the assay by the feeding larvae. For example, even though the larvae feeding on the leaf of plant BT-2 experienced no mortality during the four days o~ the trial, the amount of tho leaf eaten wa~ a very small ~raction o~ the amount of the lea~ tissue eaten from the control plantO
In other words, the larvae were not killed since they had an avoidance behavior to eating the leaves in question. A8 far aQ field suitability of such a plant, it may be insignificant as to whether the larvae are killed or ~erely avoid the transgenic plants for which insect resi~tance is intended.
Whole leaf assays were also conducted with Gypsy moth caterpillars, Lymentria dispar. Six L2 Gypsy moth larvae (about six days old), selected for robustness, were allowed to feed on each leaf for 5 days.
Survivorship was recorded and the survivor group was weighed. The leaves were recovered and the consumed area was measured. The following table illustrates the results both for tent caterpillar and for Gypsy moth larvae.
' -. -. ., . - . . . -. . - .. .

. - . ; . . : .-: . . .. .. .. . :
-, . . ... .
- : . .: . , : . . .
: .. , : ...

2 ~ 1 7 ~WO91/18094 - ` PCT/US91/03629 Larval Insect Leaf Area Survival Weight Con~umed Insect & Plant (5 days~ Gain ~) (cm ) _ Malacosoma disstria 92 46.2 31.3 & control plant Malacosoma disstria 40 9.8 0.14 & transgenic plant Lvmentria disPar 97 17.6 ll.l & control plant LYmentria dis~ar 76 4.2 0.08 h transgenic plant Accordingly, the graphical results in Figs. 3 - 5 make clear that the transgenic poplar trees created through the process of the present invention exhibit ignificant toxicity to insects upon inyestion when compared to control plants thus indicating the efficacy of the inserted genetic trait~ into the trees in question.

6. Convenient Assemblv of ~TVBTGUS
Although the plasmid vector pTVBTGUS wa~ not constructed using thi~ exact procedure, the following is a description o~ how it may be constructed from deposited plasmids and publicly available sequence information. Thi~ information is presented so that this procedure may be u~ed by those of ordinary skill in the - art to construct either a B.t. expression vector, or an expression vector effective to express another desired gene in woody plant species.
A pla~mid pTV4AMVBTSH has been previously deposited, at ATCC Accession No. 53636. This plasmid is a cointegrate of two progenitor plasmids, pTV4 and pAMVBTS, as more fully described in published application WO B9/04868. To recover the two component plasmids, the plasmid pTV4AMV3TSH may be digested with Xho I, a restriction enzyme that separates the two component plasmids in the cointegrate. Religation of : -. '.' "
, .
',:
. . ~

: ' W09t/t8094 2 ~ 6 ~ O 1 7 ` PCT/US9~/~3~2g ~

the digested DNA under dilute conditions (1-10 microgram/milliliter of DNA) will close the plasmids. If the products are transformed into E. coli and properly selected, the two cointQgrates oan be isolated.
Colonies that are sulfadiazene resistant but ampicillin sensitive will have pTV4 while colonies that are ampicillin resistant but sulfadiazene sensitive will yield the plasmld pAMVBTS. correct structures may be confirmed by plasmid mini preps. The plasmid pAMVBTS
has also been deposited with the ATCC, Accession No.
53637.
The plasmid pTVllO0 is a cointegrate of progenitor plasmids pTV4 and pCMCllO0. The plasmid pCMCllO0 has also been deposited with the ATCC, Accession No. 67641.
The plasmid pCMCllO0 was derived from pAMVBTS by substituting for the B.t. toxin coding region a coding region for the beta-glucuronidas~ gene (GUS). To cointegrate the two plasmids, DNA of each plasmid may be separately digested with Xho I, the linearized DNA
combined, and then the plasmids religated under concentrated D~A conditions (10-50 micrograms/milliliter of DNA). If th~ products are transformed into E. coli, the desired cointegrate plasmid will be harbored in colonies which are resistant to both sulfadiazene and ampicillin and confirmed by plas~id mini preps. The cointegrates will be of two possible orientations. The desired orientation being the plasmid that has the ligated Xho I site 5' to the nopaline synthase promoter of the kanamycin resistance gene (NPT-II or APA-II) from pTV4 to the Xho I site 5' to the CaMV 35s promoter on the GUS gene from pCMC1100. This provides a "head-to-head" orientation of the two genes on this plasmid, with the NPT-II and GUS genes oriented away from each other and the ampicillin resistance gene of pCMCllO0 adjacent the Ti plasmid right border sequence from pTV4.

' -' ' ' . . ' " " ' ' ' ' ' ', ' . '.. "' : ' ~
.

- . . .
,: , ~:

To create the synthetic B.t. coding region, one may begin with pAMVBTS, which includes a cassette of the CaMV 35S transcriptional promoter, a 5' noncoding mRNA
leader sequence homologous to the 5' noncoding region of alfalfa mosaic virus coat protein mRNA, a DNA fragment of the first 644 codons of the wild-type B.t. gene from B.t. strain HD-1-Dipel, two terminal proline codons, and the nopaline synthase polyadenylation region. A
synthetic coding region for this protein has been derived, based on substitution of codons fro those found in the native sequence to those which are found most commonly inplant genes. This synthetic sequence was substituted for the first 138 codons of the native sequence from the amino terminus. The entire DNA
sequence of plasmid pAMVBT4 is set forth in Fig. 6, where the altered synthetic sequence, which is the only different between pAMVBTS and pAMVBT4, is between nucleotide 480 (an Nco I recognition site) and nucleotide 897 (a BSP 1286 recognition site). The synthetic sequence was constructed as six overlapping single stranded oligonucleotides which were annealed into three overlapping duplex stands which were each inserted in turn into pAMVBTS. This procedure may be repeated using pAMVBTS and the sequence of Fig. 6 to make pAMVBT4. This codon substitution has previously been found to enhance the expression of this protein in plant cells.
To make the plasmid pTVBTGUS from pTV1100 and pAMVBT4, first the plasmid pTV1100 may be linearized by digestion to completion with the restriction endonuclease Sal I, which cuts pTV1100 at a plasmid unique site between the 3' end of the GUS expression cassette and the 3' end of the gene for ampicillin resistance. The linearized plasmid may then be partially digested with Xho I, which can cut the plasmid in two places, one place being between the NPT-II and GUS genes and the other being immediately adjacent the WO91/18094 2 ~ 1 7 PCT/US91/03629 Ti right border sequence and near the 5' end of the ampicillin gene. The latter site is the intended cut.
The partial digestion should be designed to result in a significant portion of DNA cleaned only once. The desired fragment, which is linear DNA of pTVllO0 with the ampicillin re~i~tance gene removed, can be purified from the other fragments by agarose gel electroph~resis.
The four primary DNA fragments to be expected from this digestion include a full-length pTVllOo with no cuts, fragments cut at the desired Xho I site to detach the ampicillin resistance gene from the GUS cassette, fragments cut at the Xho I site between the GUS cassette and the NPT II cassette, and fragments receiving two cuts at both Xho I sites detaching both the GUS cassette and the ampicillin resistance gene from the NPT II
vector. The purified desired fragment will contain the sulfadiazene-resistant vector with the NPT II and GUS
cas6ettes attached, with 2xposed Sal I and Xho I sites at the ends. Sinc~ the exposed end~ are compatible in ligation, treatment of this DNA with phosphatase is recommended to avoid self-ligation.
The plasmid pAMVBT4 is then digested to completion at a unique Sal I site, located immediately 3' to the polyadenylation region of the B.t. toxin expression cassette. The linearized pAMV~T4 may be combined with the phosphatased fragment recovered from pTVllO0, and the two plas~ids ligated. If the products are transformed into E. coli and celected for resistance to both sulfadiazene and ampicillin, two alternative plasmids~will be obtained, one being pTV~TGUS and one being a pl~smid of the same DNA with the pTVllO0 fragment insert ~eing in the opposite orientation. The correct orientation can be identified by plasmid mini prep analysis.
In order to enable others to repeat this procedure, the following plasmids hosted in E. coli have been deposited with The American Type Culture Collection, -: . '' ,' .. . . . . .
' 236~017 PCTtUS91~03629 `NO91/~8094 12301 Park Lawn Drive, Rockville, MD, U.S.A. under the terms of the Budapest Treaty, with the following accession numbers.

Plasmid ATCC Accession No. Date of Deposit pAMVBTS 53637 June 24, 1987 pATV4AMVBTSH 53636 June 24, 1987 pCMCllO0 67641 March 1, 1988 The present invention is not to be limited in scope by these plasmids deposited, since these plasmids are but a single embodiment of one aspect of the invention.
Indeed various modlfications of the present invention in addition to those shown and described herein will become apparent to tho~e akilled in the art ~rom the foregoing description. It i~ also to he understood that all nucleotide sizes given are approximate and that the sequences given, while believed correct, may have occasional errors due to limitations in present technology.

. ' ., '

Claims

1. A method of creating transgenic plants of a woody plant species comprising the steps of creating an exogenous genetic construction including a coding region and flanking regulatory sequences effective to express a genetic product in cells of the plant species;
coating copies of the exogenous genetic construction onto carrier particles of a sufficiently small size so as to be able to be inserted into cells of the plant species without killing the cells;
placing regenerable tissue of the woody plant species on a target surface;
initiating an explosive electric spark discharge in proximity to the carrier particles such that the carrier particles are physically accelerated at the target surface so that carrier particles are introduced into the cells of the target tissue, the acceleration of the carrier particles being determined by the voltage of the electric spark discharge;
regenerating whole plants from the treated tissue;
and assaying for the presence of the exogenous genetic construction in the whole plants.

2. A method as claimed in Claim 1 wherein the woody plant species is poplar.

3. A method as claimed in Claim 1 wherein the carrier particles are gold.

4. A method as claimed in Claim 1 wherein the carrier particles are coated onto a carrier sheet which is accelerated by the spark discharge.

5. A method as claimed in Claim 1 wherein the regenerable plant tissue includes cells derived from protoplast or suspension culture.

6. A method as claimed in Claim 1 wherein the regenerable plant tissue are plated sections of internode or petiole segments.

7. A method as claimed in Claim 1 wherein the regenerable plant tissue includes in vitro nodule cultures of the plant tissue.

8. A method of genetically engineering woody plant species comprising the steps of creating an exogenous genetic construction including a coding region and flanking regulatory sequences effective to express a genetic product in cells of the plant species;
coating copies of the exogenous genetic construction onto carrier particles of a sufficiently small size so as to be able to be inserted into cells of the plant species without killing the cells:
introducing tissues of the woody plant species into tissue culture and culturing the tissues into the formation of nodules in in vitro culture;
physically accelerating the carrier particles into the nodules of the woody plant species;
selecting the nodule tissues for transformed tissues;
regenerating the transformed tissues into whole plants carrying the exogenous genetic construction therein.

9. A method as claimed in Claim 8 wherein the step of physically accelerating the carrier particles is performed by use of an electric spark discharge.

10. A method as claimed in Claim 8 wherein the exogenous genetic construction includes a genetic trait of resistance to a selection agent normally toxic to cells of the woody plant species and the selecting step is performed by exposing the nodule tissues to the selection agent.

11. A method as claimed in Claim 10 wherein the selection agent is an antibiotic resistance marker.

12. A method as claimed in Claim 8 wherein the nodules are regenerated through organogenesis.

13. A method as claimed in Claim 12 wherein the nodules undergoing organogenesis were treated with pulses of thidiazuron.

14. A method of genetically engineering woody plant species comprising the steps of creating an exogenous genetic construction including a coding region and flanking regulatory sequences effective to express a genetic product in cells of the plant species;
coating copies of the exogenous genetic construction onto carrier particles of a sufficiently small size 80 a to be able to be inserted into cells of the plant species without killing the cells;
introducing tissues of the woody plant species into tissue culture and culturing the tissues into the formation of nodules in in vitro culture;
physically accelerating the carrier particles into the nodules of the woody plant species;
regenerating the transformed tissues into whole plants;
assaying the whole plants thus produced for plants carrying the exogenous genetic construction therein.

15. A method as claimed in Claim 14 wherein the exogenous genetic construction includes a marker gene expressing a gene product readily detectable by assay and the assaying step is performed by assaying for the presence of the marker gene product.

16. A method as claimed in Claim 15 wherein the marker gene is beta-glucuronidase.

17. A method as claimed in Claim 14 wherein the nodules are regenerated through organogenesis.

18. A method as claimed in Claim 17 wherein the nodules undergoing organogenesis are treated with pulses of thidiazuron.

19. A transgenic poplar tree comprising in its genome an exogenous genetic construction including 5' to 3' a promoter effective in poplar cells, a coding region and a polyadenylation region effective in poplar cells, the coding region coding for the expression of an insect toxic amino-terminal portion of the delta-endotoxin crystal protein from Bacillus thurinsiensis, the exogenous genetic construction effective in the cells of the poplar tree to provide toxicity to insect upon feeding on tissues of the tree.

20. Poplar seed which upon cultivation yield the tree of Claim 19.