PROCESS FOR THE PRODUCTION OF TRANSGENIC PLANTS

PROCEDE DE PRODUCTION DE PLANTS TRANSGENIQUES

English Abstract

GA/5-17599/=/ETH
Process for the production of transgenic plants
Abstract
The present invention relates to a novel, reproducible process for inserting genetic
material into cells of apical and axial meristems of plants using microinjection technology.
The present invention also relates to the use of the process according to the invention for
the production of transgenic plants, and to the transgenic plants themselves that are
obtainable by means of that process and to the progeny thereof.

Claims

- 46 -
What is claimed is:
1. A process for inserting genetic material into plants, which comprises microinjecting a
DNA-containing solution into cells of previously exposed apical and/or axial meristems of
seedlings and growing whole plants starting from said microinjected seedlings or from
parts thereof and, after interpolating one or more sexual or asexual propagation steps,
selecting the thus obtainable homogeneously transformed asexual or sexual progeny of
said heterogeneously transformed starting plants using known processes.
2. A process according to claim 1, in which said plants are seedlings that are at a stage of
development extending from the beginning of cotyledon development to secondary leaf
development.
3. A process according to claim 1, which comprises the following specific process steps:
a) germination of seeds of the particular target plant and selection of suitable stages of
germination;
b) exposure of the apical and/or axial meristem(s) for the subsequent microinjection;
e) microinjection of a DNA-containing solution containing one or more DNA fragments
into cells of the meristem(s) exposed under b);
d) growing the microinjected seedlings to complete, heterogeneously transformed plants;
e) production of sexual or asexual progeny of the heterogeneously transformed plants
obtainable under point d) and selection of the homogeneously transformed progeny.
4. A process according to claim 1, which comprises the following specific process steps:
a) germination of seeds of the particular target plant and selection of suitable stages of
germination;

- 47 -
b) exposure of the apical and/or axial meristem(s) for the subsequent microinjection;
c) microinjection of a DNA-containing solution containing one or more DNA fragments
into cells of the meristem(s) exposed under b);
d1) isolation and in vitro regeneration of microinjected apical and/or axial meristems
without selection pressure lo complete, heterogeneously transformed plants; or
d2) isolation and in vitro regeneration of microinjected apical and/or axial meristems
under selection pressure to complete, homogeneously transformed plants;
e) production of sexual or asexual progeny of the heterogeneously transformed plants
obtainable under point d1) and selection of the homogeneously transformed progeny.
5. A process according to claim 1, in which the microinjection operation proceeds in the
following steps:
a) fixing the seedlings in position using a special holding device
b) injecting the DNA-containing solution into cells of the apical and/or axial meristem(s)
using the microinjection capillary
c) turning the seedlings and injecting them again.
6. A process according to claim 5, in which the microinjection capillaries are
microelectrodes having sealed glass filaments of borosilicate.
7. A process according to claim 5, in which the microinjection operation is carried out
from 1 to 1000 times per meristem.
8. A process according to claim 5, in which the injection volume is from 1 pl to 100 pl per
injected cell.
9. A process according to claim 5, in which the DNA concentration of the injected
DNA-containing solution is from 0.1 µg/µl to 10 µg/µl.

- 48 -
10. A process according to claim 5, in which said DNA-containing injection solution
contains the transforming DNA in linearised form.
11. A process according to claim 5, in which said DNA-containing injection solution
contains the transforming DNA in super-coiled form,
12. A process according to claim 5, in which said DNA-containing injection solution
contains the transforming DNA in the form of a mixture of linearised and super-coiled
DNA.
13. A process according to claim 5, in which said DNA-containing injection solution
contains a recombinant DNA molecule that consists essentially of a structural gene that
codes for a useful and desirable property and that is linked in operable manner to
expression signals active in the plant cell.
14. A process according to claim 13, in which said structural gene imparts to the
transformed plant an increased tolerance or resistance towards pathogens, herbicides,
insecticides or other biocides.
15. A process for the production of pure-bred homozygotic transformants according to
claim 3, in which
a) heterogeneously transformed plants that are amenable to self-pollination, that is to say,
that have no self-incompatibilities, are repeatedly crossed with themselves in order to
produce in-bred lines; or
b) heterogeneously transformed plants that are not directly amenable to self-pollination
are, for the production of in-bred lines, repeatedly crossed with plants with which they
have a high degree of relationship.
16. A process according to claim 3, in which the seedlings are transferred directly into soil
after microinjection and are grown to whole plants in the same manner as normal
seedlings.
17. A process according to claim 3, in which, after microinjection, the seedlings are

- 49 -
transferred onto a specific cultivation medium that has a composition suitable for the
further development of the seedling to the whole plant.
18. A process according to claim 4, in which, either immediately after microinjection or at
any desired later time, the microinjected apical and/or axial meristems are isolated, with or
without selection pressure, from plants that are amenable to regeneration starting from
isolated meristems, and the microinjected apical and/or axial meristems are grown to
morphogenic cultures from which, in turn, whole plants can then be regenerated in vitro
using known processes.
19. A process according to claim 18, in which the cultivation of the isolated microinjected
apical and/or axial meristems is carried out in a cultivation medium that stimulates the
formation of adventitious shoot buds or adventitious embryos and to which an osmotically
neutral thickening or gelling agent has been added.
20. A process according to claim 19, in which said thickening agent is a copolymer of
saccharose and epichlorohydrin having a molecular weight of from 70,000 to 400,000.
21. A process according to claim 19, in which said gelling agent is one selected from the
group consisting of agar, agarose, alginate, pectinate, Gelrite?) and gelatine.
22. A process according to claim 19, in which the culture medium contains
phytohormones from the group of auxins and cytokinins at a concentration of from0.1 mg/l to 20.0 mg/l in each case, depending on the stage of development of themorphogenic culture.
23. A process according to claim 19, in which the pH value of the culture medium is from
pH 5.2 to pH 6.5.
24. A process according to claim 19, in which the cultivation density of the meristems is
from 1 meristem/µl of culture medium to 50 meristems/µl of culture medium.
25. A process according to claim 19, in which the cultivation of the meristems is carried

-50-
out at least for a time in a two-compartment system.
26. A process according to claim 4, in which the meristem cultures, when they have
reached a specific stagc of development, are transferred onto solid culture media
customarily used for the regeneration of plants, for differentiation of the shoot and root
apices.
27. A process according to claim 4, in which the culture media contain a selective
substance that permits positive selection of the transgenic cells.
28. A process according to claim 4, in which the regenerated plantlets are planted in soil
and further treated in the same manner as normal seedlings.
29. A process for the production of transgenic plants according to claim 1.
30. Transgenic plants and the sexual and asexual progeny thereof, produced in accordance
with a process according to claim 1, in which the majority of the somatic cells and/or germ
cells of said plants and their progeny are transformed.
31. Transgenic seed of plants according to claim 30.
32. Transformed viable parts of plants according to claim 30, selected from the group
consisting of protoplasts, cells, cell clones, cell agglomerates, callus, and/or tissue cultures,
seed, pollen, ovules, zygotes, embryos and the progeny derived therefrom.
33. All hybridisation and fusion products containing the transformed plant material
defined in claim 30, that still contain the inserted genetic material.
34. All hybridisation and fusion products containing the transformed plant material
defined in claim 32, that still contain the inserted genetic material.
35. Variants and mutants of transgenic plants as well as of the sexual and asexual progeny
thereof according to claim 30, that still contain the inserted genetic material.

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36. Variants and mutants of transgenic viable parts of plants as well as of the sexual and
asexual progeny thereof according to claim 32, that still contain the inserted genetic
material.
37. Parts of plants, such as, for example, blossom, stems, fruit, leaves and roots, that
originate from transgenic plants or the progeny thereof that have been transformed
previously using the process according to the invention and that are therefore made up at
least partially of transgenic cells.

Description

;~0175~
GA/5- 1 7599/=/ETH
P o_ss for the production of trans~enic plants
The present invention reliltes to a novel, reproducible proeess for the productioll of
lrarlsgenic plants which is based on the insertion of gcnetic m tteri;ll into cclls of nl)ic.ll
and axial meristems (adventitious meristems) of pl,mts ~lsing IlliCrOilljCCtiC)Il teChllOIOgy
and which is distinguished by its universal iapplicability.
The present invention also relates to the use of the process according to the invenlion for
the production of transgenic plants and to the transgenic plants themselves that are
obtainable by means of this process, and to the progeny thereof.
A large number of processes and techniques that are already being used as a matter of
routine in many laboratories have in the meantime become available for genctic
manipulation of the plant genome using recombinant DNA technology.
One of the best researched and most frequently used processes is, undoubtedly, the
A~robacterium transformation system. A~robaeterium cells have on their Ti-plasmid a
large DNA fragment, the so-ealled T-DNA region, which, in natural transformation of
plant eells, is integrated into the plant genome.
After earrying out various modifications, this natur;ll gene-tr~nsfer Systclll can be uscd its
a gene veetor system for speeifie tlly eontrolled transfonnation of plants [Chil~on, MD,
1~83~. The Agrobacterium transformation system has, however, the crucial disadv;lntage
that the rnnge of hosts for the agrobacteria is restrieted to speeifie dicotyledonous pliallts
and to a few representatives of the monocotyledons (Hernalsteens et ah, 1984;
Hookas-Vall-Slogteren et al., 1984) which, however, are unimportant from the point of
view of agriculturill economics. This mc tns tll.lt the most important cultivated plants are
not amenable to an effective gene transfer.

201754
-2-
In addition, the agrobacteria used are pathogens that induce characteristic disease
symptoms in the form of tumorous tissuc grow~hs in thcir host plants and thetcforc m;ly bc
hiandled in the laboratory only if strict safcty precialltiolls arc taken.
errl;ltive lransformlltion systems that hilVC bccll devcloped ~o offsct thcsc dis.l(lv.lllt;lgcs
of ~he ~cterium transforlllation sys~cm and ~ha~ aim a~ inser~ing C~OgCIliC l)NA inlo
pl;lnt prOtOpl.lStS, SllCIl as the direct gelle trallsfcr of vec~or-frcc DNA in~o pro~oplas~s
(Pas~kowski et al" 1~84, Potrykus et ,ll" 1986) iand the microin)ecti(!n of vector-free DNA
into protoplasts (Spangenberg et al, 1986; Steinbiss and Stabel, 1983; I~/lorikaw,l al-d
Yatnada, 1985) or cells (Nomura and Komamine, 1986), must be regarde(l as problel~ tic
in so far as for a large number of plant species, especially of the Gramineae group, the
regeneration of whole plants from plant protoplasts at present still entails numerous
problems.
Investigations carried out by Yamada et ah, 1986, and Toriyma _ al., 1986, inter alia for
the regeneration of rice protoplasts, and by Rhodes et al., 1988 (maize) and Harris et al.,
1988 (wheat) have shown the first signs of progress in this field.
A further disadvantage of these alternative transformation systems is the rates of
transformation, which are still relatively low and are at present from 1 % to 5 %. These
low rates of transfonmation also make it necessary to provide the DNA tll;lt is to be
inserted with markers (for example antibiotics resistance gcncs) which pcm1it rapi(l
selection of the transformants from the lar~c numbcr of untransfonnc(l cclls~
This means, however, that there is at prcscnt no satisf;lclory trallsforll1;ltion process th.lt
pem1its a production of trallsOellic pl.allts llaving novel alld useflll propertics th.lt is
efficient nlld inexpcnsive even from the commcrcial point of view, this applyill~ cspcci;llly
to plmlts from tlle Monocotvledoneae group.
It must therefore be regarded as a priority task to develop processes th;lt permit rapid,
efficient and reproducible transformation of all plants, regardless of their taxonomic
category and the characteristics associated therewith, and therefore ensure a production of
transgenic plants that is effective and economical even from the commercial point of view.

;~0175~
This matter concerns especially planls from the Monocotylcdoneae group, cspcci.~lly ~hose
of the Gramineae f:amily, which includes the cultiva~ecl plan~ tha~ arc ~he ItlOS~ impOrt;lnt
-
from the point of view of a~ricultural economics, such as wheat, barlcy, ryc, oats, m.li~.c,
rice, millet, ctc., and which are thercfore of very special economic intcrcst, cspcci.llly
sincc therc have hitllcrto becn no satisf.lctory processes available for the prodllcli()ll of
~ralls~enic monocotyle(lon()lls plantx.
Vslrious transfortn.ltion processes whicll h;lve only recently been devclopcd cons~itllte the
first steps in this direc~ion. One yrocess involves the injec~ion of exogcnic DNA into the
young inflorescence of rye plan~s (de la Pena et al., 1987) and another involves an
mediated virus infection of maize plan~s with lMaize Streak Virus
(Grimsley et al., 1987). However, even these newly developed processes have
disadvantages; for example, it has been found that the first-mentioned process is not
reproducible.
Neuhaus et aL, 1987 describe an in vitro process for the transformation of rape plants by
the microinjection of DNA solutions into embryoids which are kep~ in a microculture (~
vitro) and, when transformation is complete, are regenerated to whole plants.
The present invention may be regarded as a further developmen~ of ~he microinjection
process described by Neuhaus et ah, 1987. The principal advan~agc of ~he processaccording to ~he invention over ~hc proccss describcd by Ncllll,ms ct al. rcsides in the usc
of whole plants as staning material for ~he microinjcc~ion of DNA-con~aininL~ solutiolls.
I-lere and hercill.lf~er, "DNA-conl.lillillg sollltioll" is no~ ~o bc in~crprctcd in the chclllic.ll
sense as a true solution but as a li(lui(l, cont.lining thc DNA, ~hal may bc eithcr a true
solution or a suspension, emulsioll, etc.. As a rcsult of using whole plants, it is possible to
dispense witll the sometimes timc-collslllllillg, and thercfore cxpcnsive, prcparation alld
cul~ivation of suitable embryonal stages. Working with whole plants also renderssuperfluous the regeneratioll step, which, a~ presen~, in somc cases is still complex and
involves diverse problems, depending on ~he plant material used.

Z0175~
Within the scope of the present invcntion, it has surprisingly now proved possible to
develop a highly efficicnt transformation system for plants, regardless of thcir taxonomic
catcgory and the characteristics associated thercwith, which SystclIl call scarccly bc
sllrpasscd in ;ts simplicity and gener.ll applicability and which does nOt have thc
previollsly mentioned limitations of lhc processcs known hitllcrto and is tllcrcforc
outstandingly suitable for routinc use.
In particular, the system is a process for inserting genetic material into plallts, whicll
comprises microinjecting a DNA-containing solution into cells of previously exposed
apical and/or axial meristems of seedlings and growing the microinjected scedlings to
whole, complete and preferably fertile plants. Tt-e exposure of the meristems, whicll can
be effected, for example, by removing the cotyledons or pulling them apart, does not
impair the further development of the plants so that, after microinjection, they can be
further cultivated in the same manner as normal seedlings.
Especially preferred within the scope of the present invention is the use of so-called
"vector-free" DNA, that is to say, DNA that does not have sequences that are known to be
involved functionally with the insenion or the integration of foreign DNA into plants, such
as, for example, the corresponding sequences from the Ti- or Ri-plasmids of agrobacteria.
Since the primary transforrnants obtainable by means of this process a~ chimaeras, which
contain the microinjected genetic material in only some of ~heir cells, thc .ISCXIIIII or
sexual progeny of said primary transformants are usc(l for thc productiol) of
llomogeneously transformed plants.
Especially preferred within the scopc of the prcscnt invcntioll is thercfore a process for
inserting genetic material into plants, which compriscs microinjecting a DNA-containing
solutioll into cells of previously exposed apical and/or axial meristcms of scedlings an(l
growing whole, and especially whole fertile, plants starting from said microinjectcd
seedlings or from parts thereof and, after interpolatillg one or more sexual or asexual
propagation steps, selecting the thus obtainable homogeneously transformed asexual or
sexual progeny of said heterogeneously transformed startillg plants using known
processes.

;~0175~
Alternatively, it is possible, espcci.llly in the casc of plants that are amenable to
regeneration starting from isolated merislems, to isolate the microilljecte(l apical lln(l/or
axial meristems cither immcdiately after microinjection or at any desircd latcr timc with or
without selection prcssure and to grow them to morphogenic cul~urcs from which, ill tUrtl,
wholc plants can then bc rcgenerated USillg known processcs.
The present invention also relates to the production of transgcnic wholc plallts th;lt havc
been transformcd using thc process according to the invenlioll with a rccombill,lllt DNA
molecule which, in the transformed plant, leads to novcl and desirable propcr~ies, alld ~o
~he transgenic plants themselves which are produced in ~hat manner and ~o ~he progeny
thereof, and also to mutants and variants of said transgenic plants.
The present invention also relates to plants that have been regenerated from transformed
cells or other plant par~s that contain said recombinant DNA molecule, and to the seeds
thereof, and to the progeny of plants that have been regenerated from said transgenic plant
material, and to mutants and variants thereof.
Within the scope of this invention, progeny of transgenic plants shall be understood as
being progeny of the transformed parent plant that is produced either sexually or
asexually, and also progeny of the microinjected and isolated apical and/or axial
meristems that is produced using cell culture and/or tissue culture techniques.
The present invention also relates to ~hc usc of thc transformation process accor(ling to ~hc
invention for the rapid and reprodllciblc pro(luction of transgenic plants having novcl an(l
desirable properties.
In the following description, a numbcr of terms are used that are custom,uy in
recombinant DNA technology and in plallt genetics. In order to ensure a clear and
uniform understanding of the description and the claims and also of the scope to be
accorded to the said terrns, the following def1nitions are listed:

~17S~
Apieal meristem: zone in the region of the tip of the shoot a~is where cells are formed and
divide.
~v~: mt,ristt,ms th;lt havc no conncclion with nn apieal
meristem but are newly formed from tissues of the shoot axis If or exalllple leaf axill.lry
buds (;r.~eristems)l.
Chim~!era: cell, group of cells, tissue or phmt, composed of cells having different genetic
infomlation ("hetero~eneously transforrned plants").
Plant material: parts of plants that are viable in culture or that are viable as such, such as
protoplasts, cells, callus, tissue, embryos, plant organs, buds, seeds, etc., and also whole
plants.
Plant eell: struetural and physiological unit of the plant, eomprising a protoplast and a cell
wall.
Protoplast: "naked" plant cell that has no cell wall and has been isolated from plant cells
or plant tissue and has the potential to regenerate to a cell clone or a whole plant.
Cell elone: population of genetieally identieal cells produeed from one cell by eontinuous
mitoses.
Proembrvo: preeursor stage of the embryo before polar differenti.ltioll into the above
struetures eharaeteristie of the embryo.
Plant tissue: group of plant cells organised in the form of a slmclllral antl funclion;ll unil.
Plant or,~an: structural nnd filnctional unit comprising several tissues, such as, for
example, root, stem, leaf or embryo.
Ileterolo~ous gene(s) or DNA: a DNA sequence that codes for a specific product or
produets or fulfils a biologieal funetion and that originates from a speeies other than that

201~75~/~
into which the said gene is to be inserted; said DNA sequence is also referred to as a
foreign gene or foreign DNA.
I lomologoll~erle~r DNA: a DNA sequcllcc that codes for a specific prodllct or
. _
products or fulrlls a biologic;ll function and th;lt origin;ltes from thc salllc spccics as th.lt
into which tlle said getle is to be inscrted.
syn(hetic ~ene(s~ or DNA: ,l DNA se(luencc that codes for a specific product or pro(lllcts
or fulfils a biological function and that is produced by synthetic mc.lns.
Growin~ cone: zone in the tip of a shoot axis in which the fonnative tissue is centr;llly
__
located.
Plant promoter: a control sequence for DNA expression that ensures the transcription of
any desired homologous or heterologous DNA gene sequence in a plant, in so far as said
gene sequence is linked in operable manner to such a promoter.
Termination sequence: DNA sequence at the end of a transcription unit that signals the
end of the transcription process.
Over-producin~ plant promoter (OPP): plant promoter that is capable, in a tr;3nsgenic plant
cell, of bringing about the expression of any opcrably linked functional gcne sequence(s)
to a degree (measured in the form of the amount of RNA or the amount of polypcptidc)
tllat is markedly higher than th tt observcd in the n;ltllr.ll state in host cells th;lt havc not
been transformed with saicl OPP.
3'/5' ulltr;lllsl;lted re~ion: sections of DNA locale(l dowllstrt alll/uystre;llll of the codillg
region whicll, although tr;mscribed into mRNA, .~re not translated into a polypeptide. 'I l-is
region contains regulatory sequel-ces, such as, for exa-nple, the ribosome bin(lil-g site (S')
or the polyadenylating sigll;ll (3').
DNA expression vector: cloning vehicle, such as, for example, a plasmid or a
bacteriophage, containing all the signal sequences necessary for the expression of an

--- z~ 5~
inserted DNA in a suitable host cell.
DNA transfer vector: transfcr vchicle, s~lch as, for cxample, a Ti-plaslnicl or a virlls, tl~at
permits the inscrtion of gcnctic m"terial into a suitable host ccll.
Mut.lnts, va nts of tr,~c ~: dcrivative of a transgenic plant th.l~ is prodllccdspont~meously, or artificially, using known process measures, sllch as, for exalllple, UV
treattnent, treatment with mutagenic agents, etc.,
and that still has the features and properties of the starting plant that are essential to the
invention.
The transformation process according to the invention for the direct insertion of genetic
material into plants is based on the microinjection of a DNA-containing solution into cells
of apical and/or axial meristems of plants. Microinjection into the apical meristems
(growing points) is especially preferred.
The shoot and root growing cones accommodate a group of cells that are referred to as
primordial meristem cells and that are the starting material for the development of the
whole plant body. These primordial meristem cells differ in characteristic manner from
the cells surrounding them. Like the embryonal cells from which they are directly
derived, they are relatively small and approximately isodiametric. Their cell walls, which
are generally perpendicular to one another, are very delicate and still deficient in cellulose.
The cells of the primordial meristem are densely packed. The cell caviîy is filled with
dense protoplasm containing a relatively large cell nuclcus. This area of thc shoot an(l
root tips, which is referred to as the ;lpiC;II mcristem or growin~ point, compriscs only a
few hundred cells and is concentr.lted ill ;lll arca of approximately 100 ~,lm x 10() ~Im.
Within the scope of the present invelltioll, it has surprisingly now been found that these
cells which are concentrated in the area of the shoot and root growing point are amenable
to rnicroinjection when suitable process measures, which are described in detail below, are
used.

The microinjection of a DNA-containin~ solution one or more times into cells of the
apical meristem (grvwing point) of plants leads to the formation of transformed primordial
meristem cells which s~lbscquently further differcntiate to transfonrlcd cells and tissues
all(l thereforc Ica(l to thc formation of hetero~cneously transfonnc(l plallts (chhtlacras) lf
thc DNA-containing solution is microinjectcd into an arca within thc gfOWill~ point ~h.lt iS
directly or in(lirectly involv~d in the formation of the blossom meristcms tllcll the
incorporated genetic material can pass into the gerrn-linc of thc plant and thercforc be
transmit~ed to the sexual progeny of the hetcrogeneously transformed parcn~ plan~s.
Since the sexual progeny origina~es from one cell (~he zygote) the said progeny i5
homogeneously transformed plan~s ~ha~ con~ain the genetic material inserted by means of
microinjection s~ably incorpora~ed in their genome.
In the manner described above ~he forma~ion of transgenic chimaeras which is in some
cases undesirable can therefore be avoided.
Preferred within the scope of this invention is the microinjection of a DNA-containing
solution into the growing cones of monocotyledonous plants but especially into the
growing cones of cereals and other grasses for which no satisfactory transforrnation
system has hitherto been available.
Surprisingly within the scope of the present invéntion it has now becn possiblc for ~he
first time by using specific process measures ~o microinjec~ gcnelic matcrial in vlvo in a
specifically controlled manner into cells of apical mcristcms (growing concs) of scedlirlgs
and ~o grow homogeneously transfonned plan~s from ~he plan~s so trca~cd.
The process according ~o ~he inventioll is not restricled to apical meristems but c.tn also be
used in exactly the same manner on axial meris~ems of ~he plant.
The microinjection of the DNA-con~aining solution into apical and/or axial meris~ems is
carried out according to the invention using a microinjection apparatus equipped with
manually controlled or motor-controlled micromanipulators.

- lo -
The basic construction and the equipment of said microinjection devices are widely
described in the literature. Microinjection apparatus have in the mcantime also been
offered for sale as complete cquipment and already form part of the standard equipmellt of
many laboratories.
Apicsll and/or alxinl meris~ems of see(Jlings nre the prcfcrrcd plant starting materinl for
microinjection. Af~er c,uTying out the microinjection described above and depending on
the plant speeies used, either these meristems are placed directly in soil and grown very
rea(lily to whole plants using the customary cultivation mcasures, or the microinjected
apical and/or axial meristems or parts thereof are isolated from the seedlings, in so far as
the plants in question are amenable to regeneration from isolated meristems, and can then
be grown very readily in vitro to morphogenic cultures and regenerated to whole plants
using known process measures.
The plants obtainable initially using the process according to the invention are chimaeras,
that is to say, heterogeneously transformed plants only some of whose tissues contain the
microinjected genetic material stably incorporated in their genorne. In order to produce
homogeneously transformed plants, therefore, chimaeras are preferentially selected that
possess transformed germ-line cells and whose sexual progeny therefore has the desired
homogeneous transformation. This selection according to obvious features can readily be
carried out by the person skilled in the art.
Alternatively, the chimaeras can be cultivatcd in an in vitro cultivation s~ep under
selection pressure, in so far as plants are uscd thnt can be regencrated to whole plnnts
starting from isolated apic~l and/or nxial meristcms. The exposed and microinjected
apical meristems are hrst isolated from the sccdling, cullivated in the form of in vltro
cultures and then regenernted to wl~ole plants. The cultures arc prefcrably subjected ~o a
specifically controlled selection pressure so th;lt only plants that express the injected
genetic material are regenerated.
This alternative process for the production of homogeneously transformed plants, which
can be used especially with plants that can be regenerated to whole plants starting from

;~0175~
isolated apical and/or axial meristems, therefore consists especially in the identification
and selection of transformed cells or tissue from heterogeneously transformed pl~mts iand
in their in vitro regeneration, preferably in cultivation media that stimulate the fonmatioll
of adventitious shoot buds or adventitiolls cmbryos, to wholc, homogeneously transformed
plants, usinL~ known process measures.
Processes for the In vitro re~ener.ltion of transformed plant material are describe(l, for
example, in the following publications: Pas~kowsky and Saul, "Dircct Gene 'I'rallsfer to
Plants" in: Methods for Plant Molecular Biology, eds. A ~ ~l Wcisbach, Acadcmic Press,
1988, pages 447-463; Rogers SG _ ah, "Gene Transfer in Plants: Production of
Transformed Plants Using Ti-Plasmid Vectors", in: Methods for Plant Molecular Biology,
eds. A & H Weisbach, Aca~emic Press, 1988, pages 423-436.
An important advantage of the process according to the invention is that it can be used
universally, regardless of the taxonomic category of the particular target plant and the
associated characteristics and technical difficulties that occur when using the
transforrnation processes available hitherto, for example in the form of a restriction to
specific dicotyledons when using the A~robacterium transformation system or a hitherto
insufficient ability of monocotyledonous plants to regenerate from protoplasts when using
the direct gene transfer processes.
This universality of the process according to the invention is due to the fact that genctic
material is transferred by a physical method (microinjection) into a structure (growing
cone) obtainable from any plant regardless of its taxonomic category.
In addition, by growing plants directly from scedlings, the negative effects of "somaclonal
vari,ltion" that normally occur in other transfonnation processes, especially in those whose
regeneration step is via a callus phase, are completely or at least partially avoided, which
is of crucial importance for the purposes of plant breeding.
Thus, within the sc.ope of the present invention, it is now for the first time possible,
regardless of the above-mentioned restrictions, to produce, with a high degree of
efficiency, transgenic plants, especially also those from the Monocotvledoneae group,

20175
- 12-
having novel and desirable properties by microinjecting a DNA-conlaining solution into
cells of apical meristems (growing points) ancVor axial meristems of seedlings and then,
using known cultivalion measures, growing the microinjccted seedlings or parts thereof to
whole, complclc pl~nts.
In (le~ail, thc process accor(ling lo lhc invell~ion compriscs thc followin~ specific proccss
steps:
a) germin.ltion of seeds of the particular target plant and selection of suitable stages of
germination;
b) exposure of the apical and/or axial meristem(s) for the subsequent microinjection;
c) microinjection of a DNA-containing solution containing one or more DNA fragments
into cells of the meristem(s) exposed under b);
dl) growing the microinjected seedlings to complete, heterogeneously transformed plants;
d2) isolation and in vitro regeneration of microinjected apical and/or aYial meristems
without selection pressure to complete, heterogeneously transformed plants;
d3) isolation and in vitro regeneration of microinjccted apical and/or axial mcris~ems
under selection pressure to complete, homogeneously transformed plants;
e) production of sexual or asexual progeny of the heterogcneously transfonncd plan~s
obtainable under point dl) or d2) and selection of the homogcneously transformcdprogeny.
For the preparation of suitable seedlings, sceds of the p~rticular target plants are
germinated preferably under sterile conditions. The germinatioll period depends on the
germination activity of the seed material used alld is generally from ~ to 10 days.

~-~)175~
Seedlings that are at a stage of development extellding from the beginning of cotylcdon
development to secondary lcaf devclopmcnt arc prefcrred within the scope of the present
inven~ion. Seedlings havinL~ fully developed cotyledons .are espccially preferred.
Within the scope of the present invention, it is a(lv.lntageous to keep the shoot axis of the
sëëdling short. This can be achicved, for examplc, by suitable illumination of the
germinating secds or the young seedlings. The light intensi~y is advantageously
approximately from 5 to 145 Einstein-s-l-m-l, and especially from 45 to 60
Einstein-s-l m-l.
When the seedlings have reached a specific stage of germination suitable for
microinjection, the growing cones, or axial meristems, are first exposed. In the case of the
shoot growing cone, this can be achieved, for example, by removing one or both
cotyledons.
A further possibility is to pull the cotyledons apart to the extent that the apical meristem is
visible.
The preparation of the growing cones is preferably carried out using fine scalpels and fine
needles or finely drawn forceps and special clamping devices while monitoring with a
microscope.
The clamping device is used in parlicul,lr for fixing the cotylc(lons in posilion~ which
greatly facilitates the
subseqllent exposure of the growing cone.
Seedlings whose apical meristems are readily accessible, such as, for example, the
seedlings of soybeans, cotlon, deciduous trees, conifers, etc., are especially suitable for
use in the processes according to the invention.
The seedlings so treated can then be used for microinjection.

;~017S4
- 14-
The mîcroinjection of the DNA-containing solution into apical and/or a~;ial mcristems is
carried out using processes known per se which arc dcscribed, intcr ali~l, by Steinbiss alld
Stabel, 1983; Neuhnus et al, 1~84, 19~fi, 1987; and by Moril;.lwa and Yamad,l, 1')~5
In detail, the microinjection intl) individu.ll cells of thc apical mcristem is carried out
wllile monitoring with a microscope and using a specific microinjection app,lr,ltus whose
main equipment is micropipettes that are controlled by micromanipulators.
The diameter of the tip of the injection capillary is from <l ~tm to lO0 ~lm. Injcction
capillaries the diameter of whose tips is from 0.5 ~m to 1 ,um are very especially suitable
for use in the process according îo the invention. Within the scope of the present
invention, there are used as microinjection capillary preferably microelectrode capillaries
having sealed glass filaments of borosilicate glass. These capillaries have an outside
diameter of from 1.00 mm to 2.00 mm. A diameter of from 1.2 mm to l.S mm is preferred.
It is, of course, also possible to use any other types of glass or other materials suitable for
effecting the microinjection. The rnicroinjection pipette is generally connected v~a a
pressure^regulating unit, the so-called microinjector, to a gas source, especially a nitrogen
source or a compressed air source, which ensures a very accurately metered transfer of the
injection solution from the micropipette into the plant tissue.
When the tip of the micropipette has penetrated into cells of the apical and/or axial
meristem(s), the DNA-containing injcction solution is relcased.
The injection volume varies accor(linL~ to thc ccll size from I pl to 1()() pl pcr injectcd ccll.
Within the scope of this invcntion, an injcction volumc of from 10 pl to 40 pl pcr injccted
cell is preferred.
In order to ensure as unifom1 a transfon11.ltion as possible of all the cells making up the
multicellular structure to be injected, these cells are injected from l to 1000 times, but
preferably from 4 to 60 times, depending on the existing number of cells.

2017S4~
- 15-
This can be achieved, for example, by turning the seedlings to be injected under the
microinjection device between the various injection operations so that as large a number
of differcnt cells as possible is microinjected.
DNA solutions suitable for microinjection into plant cells are, for example, buffer
solutiolls tllut eontain the tr.msforrning DNA in a ~uitable concentration.
The DNA coneentration preferred within the seope of this in~ention is from 0.01 ~1 to
10 ~lg/~LI, but espeeially from ().1 ~g/l,ll to 5 ~lg/~,ll. A DNA concentration of from 0.5 ~g/,ul
to 1.5 ~lg/~l is very especially preferred.
The pH of the buffer solution can vary from 6 to 8; a pH range from 7.5 to 7.8 is prefcrred
aceording to the invention.
For the integration of the foreign gene into the genornic DNA of the plant cell, it is
advantageous if the transforming DNA is flanked by neutral DNA sequences (carrier
DNA). The neutral DNA sequences flanking the transforrning DNA either may be of
synthetic origin or may be obtained from naturally occurring DNA sequences by treatment
with suitable restriction enzymes. For example, naturally occurring plasmids that have
been opened by a selective restriction enzyme are suitable as carrier DNA.
The DNA sequence produced for the gene transfer may, however, alternatively have an
annular structure (plasmid structure). Such plasmids comprise a DNA strand into which
the transforming DNA is integrated.
An example of such a plasmid is tlle frecly obtain.lble plasmid pUC~ (describe(l by
Messing, J. and J. Vieira, 1982). Also, fragments of na~urally occurring plasmids can be
used as carrier DNA. For example, the deletion mutant for gene Vl of the Glllliflower
Mosaic Virus can be used as carrier DNA.
According to the invention, the trau~sforming DNA may be either in the form of open or
closed rings (plasmid structure) or, preferably, in linearised forrn.

Æ0175'~
- 16-
The use of a mixture of linealised and super-coiled forms is preferred within the scope of
this invention.
Optical monitoring of thc cntirc microinjcction operalion is normally by microscopic
observation. Invcrse microscopes or stereomicroscopes having a combin;ltion of inci(lellt
li~ht and tr~msmitted light arc prefcrred. 't'he magnifica~ion chosen dcpends on thc size Of
the nlaterial to be injected and varies from l0x to 300x magnification~ A cokl ligllt sourcc
is preferably used us illumination in order to avoid the evolution of heat.
The process according to the invention C.lll thus be char~ctcrised by the following steps:
a) fixing the seedlings in position using a special holding device
b) injecting the DNA-containing solution into cells of the apical and/or axial meristem(s)
using the microinjection capillary
c) turning the seedlings and injecting them again.
After microinjection, the seedlings are transferred either directly into soil or, alternatively,
into a suitable culture medium.
Cultivation of the microinjected seedlings, which follows microinjection, eithercorresponds to a completely normal cultivation of seedlings in soil, as calried out as a
matter of routine in the field of plant breeding, or it is carried out in specific cultivation
media that are suitable for the further devclopment of the seedlings to the fully formed
plant.
Direct transfer of the microinjected seedlings into soil is especially preferred within the
scope of this invention.
The plantlets so fonned from the microinjected seedlings can then be further treated in a
greenhouse in the same manner as normal seedlings. Plants then develop that contain the
microinjected genetic material in only some of their cells and that are usually referred to
as chimaeras.

Z0175~1tL~;
For the production of pure-bred, homozygotic transforrnants, in panicular two altertlative
methods are available.
In the case of plants that ar~ amen.lble to self-pollination, that is to say, that have no
self-incompatibi~ es~ homo~y~otic lines caul be ob~ained by repeated self-pollinatioll of
primary trimsforrnants (chim.leras) ~nd the production of in-bred lines. In contrast to
homozygolic, diploid transformants in which the integrated gene is present on homologous
chromosomes and which obey Mendel's laws on the basis of the dominant heredity of this
gene, a selhng of the microinjected chimaeras leads to a heterozygotic situation in respect
of the integrated gene (monohybrid). Only in the course of the subsequent further
self-pollinations of the filial generations are homozygotic lines finally obtained in respect
of the inserted foreign gene.
The seeds obtained as a result of these crosses are in each case gerrninated on suitable
selection media in order to select the features inherited according to Mendel's laws.
These in-bred lines can then in turn be used to develop hybrids in so far as these primary
transformants contain transformed germ-line cells.
Plants that are not directly amenable to self-pollination, whether because the sexual organs
are on different plants or because the self-hybridisation for some reason does not lead to
the production of normal seeds, can also be used for the production of in-bred lines. In
that case, plants that have a high degree of relationship to one another are crossed with one
another, which can ultimately likewise lead to the desired goal, namely the provision of
in-bred lines for hybrid breedillg.
The processes given here as examples of lhe production of homozygotic plallts are known
to the expert in the field of plant breeding and are described in detail, for example, in the
following publication:
`'Breeding Field Crops, Third Edition, JM Poehlman, AVI Publishing Company, Inc.,
Westport, Colmecticut, 1987".
In the case of plants that are amenable to regeneration, for example by way of secondary
embryogenesis or adventitious shoot formation, it is possible to isolate transforrned

20175'~.
- 18-
individual cells or transforrned tissue from the heterogeneously transfomled plants and to
regenerate them _n vitro, under selection pressure, to pure transgenic plants.
In detail, the procedure may bc, for example, that, after microinjection, tlle treated apical
or axial meristem is cultivatcd in vitro in cultivation media that are suilable for the
regeneration of the cells or tissue to the fully developcd plant.
Regarding their composition, which is very well known to the expert in this fiel(l, thcse
media are distinguished especially by the fact that they contain in form of their
compounds especially the mineral substances or elements that are esscntial for the growth
and development of the plant cell (macroelements), such as, for example, nitrogen,
phosphorus, sulfur, potassium, calcium, magnesium and iron, in addition to the so-called
trace elements (microelements), such as, for example, sodium, zinc, copper, manganese,
boron, vanadium, selenium and molybdenum. While the macroelements are generally
present in amounts of from 10 mg/l to a few hundred mg/l, the concentration of the
microelements is generally a maximum of a few mg/l.
Further constituents of said media include a number of essential vitamins, such as
nicotinic acid, pyridoxine, thiamine, inositol, biotin, lipoic acid, riboflavin,Ca-pantothenate, etc., a suitable readily assimilable carbon source, such as, for example,
saccharose or glucose, and various growth regulators in the forrn of natural or synthetic
phytohormones from the class of auxins and cytokinins in a concentration range of from
0.01 mg/l to 20 mg/l in each case, preferably from 0.05 mgQ to 10 mg/l alld, very
especially preferred, fTom 0.1 mg/l to 1.2 mg/l.
According to the invention, special care should be taken that lhe composition of the media
and the concentration ratios of their individual components can be changed in accordance
with the stage of developmellt of the regenerallts. This applies especially to the ~rowth
regulators used which must be applied in a balanced ratio with respect to one another to
ensure, if desired, that the regeneration proceeds in a controlled manner.
It is not possible to give a general rule of thumb in this connection since both the
individual concentrations and the concentration ratios of auxin/cytokinin may vary very

7S~
- 19-
widely depending on the plant material used. Thc procedure for deterrnining suitable
concentrations of growth-promoting substances and suitable concentration ratios of thc
individual growth-promoting substances with respect to one another are sllfhcicntly
known to the cxpert in this field.
Within the scope of this invcntion, the preferred concentration range of tllc
phytohormones from the class of auxins and cytokinins is from 0.01 mgJI ~o 2() mg/l in
each case, preferably from 0.05 mg/l to 1() mg/l and cspecially from 0.1 mg/l to 1.2 mg/l.
The culture media are, in addition, osmotically stabilised by thc addition of sugar alcohols
(for example mannitol), sugars (for example glucose) or salt ions and ~re adjusted to pH
values of from 4.5 to 7.5, preferably from 5.2 to 6.5.
Apart from the use of synthetic media having a precisely defined composition of various
individual components of known concentration, it is also possible to use complex nutrient
media that are composed entirely or at least partially of natural components. In this
connection, attention is drawn, for example, to potato extract, coconut milk and liquid
endosperm, which can be obtained by suction from the immature seed of plants.
Whereas the first cultivation steps, which follow immediately after microinjection, are
preferably carried out in liquid culture media, in the further course of regeneration
recourse may be had, as desired, eitller to liquid or to solid culture media which may be
prepared by the addition of one of the solidifying agcnts customarily uscd in
microbiological technology, or to a combin.ltion of solid and liqllid mcdia.
Suitable solidifying agents include, for example, agar, agarose, alginate, pectinate,
Gelrite(~, gelatine, etc.. Agar and ag;lrose, especially agar and agarose of the low-melting
type (LMT), are preferred
When using liquid media it may in some cases be advantageous to add to the medium an
osmotically neutral thickening agent so that the density of the medium increases without
its osmotic value being raised. As a result, the regenerants float on the medium and an
optimum gas exchange with the surrounding atmosphere is thereby ensured. Within the

~01~5
20 -
scope of this invention, synthetic polymers, for example, copolymers of saccharose and
epichlorohydrin having a molecular weight of from 70,000 to 400,000, are preferred, but
this does not constitule a limitation.
In a spccihc embodimcnt of the process a(:cordil1g to the invcntion, meristems
microinjccte(l in the manncr dcscribcd above are cxposcd immediately aftcr
microinjection and tr.msferrcd preferably onto commercial microcultivation plates, into or
onto me(lia that have a composition suitable for the tn vitro cultivation of this
meristematic tissue.
The cultivation of the microinjected meristems is preferably carried out on a solid medium
containing as additive a gelling agent selected from the group consisting of agar, agarose,
alginate, pectinate, Gelrite(~) and gelatine.
The cultivation density is preferably from 1 meristem/~ll to 50 meristems/~,ll of culture
medium, but the density very especially preferred is from 1 meristem/lll to 5 meristems/~
of culture medium. The cultivation density selected depends on the size and the stage of
development of the meristems used.
It has proved advantageous within the scope of this invention if the cultivation of the
microinjected apical andlor axial meristems is carried out at least for a time in a
2-compartment system that acts as a humidity chamber, ~he outer companmen~ bcingformed, for example, by water, culture medium or an aqucolls mannitol sohllion while the
meristems are in the inner compartment.
After a cultivation period of from 5 to 15 days, but preferably after approximatcly 1() days,
the developing apical and/or nxial meristems ure transferred onto a selective cultllrc
medium on which the isolated mcristems are subjected to a positive selection pressurc so
that only those cells which, as a result of thc foregoing transformation, are capablc of
resis~ing this selection pressure can survive and develop funher. All untransfonned cells,
on the other hand, die or are overgrown by the transformed cells.

2~S~
- 21 -
It is possible to use as selective media additives, for example, herbicides, insecticides,
fungicides and other cell-toxic compounds, such as, for example, various antibiotics or
othcr biocides. In addition, however, other parameters, such as, for example, salt
concentralion, 2 concentr-~tion, li~ht, pll, etc., can also act as a selective agent,
dependillg on the particular selective marker used and insened into the plant.
When the merixtems microcultivated in îhe previously described manner havc rcacllcd "
specific sta~e of development, generally after from 10 to 30 days, but especially llfter from
10 to 20 days, they are advantageously transferred to solid media which may optionally be
covered with a layer of liquid medium. These media are generally the culture media tllat
are normally used for the regeneration of plants and that give rise to increased adventitious
shoot regeneration or increased adventitious embryogenesis owing to their balanced
content of phytohormones, especially from the class of auxins and cytokinins. Especially
preferred are auxin and cytokinin concentrations of from 0.01 mg/l to 20 mg/l in each case
and, very especially preferred, from 0.1 mg/l to 1.0 mg/l, as are present, for example, in
the T3, T4 and TS media described in detail hereinafter.
After a total cultivation period of approximately from 4 to 8 weeks, depending on the plant
material used, the meristems can be transferred onIo solid media that have no or only a
low hormone concentration and can be further cultiva~ed here until small plantlets
(seedlings~ are formed.
In order to promote a rapid regeneration of the plants, it may be advantageolls to bring
about induction of root formation, for example by transfcmng the embryos to
root-inducing media known per se [cf., for examplc, mcdium T~].
The transgenic plants formed in this manner can then be planted in soil and further treated
in a greenhouse in the same manner as norrnal seedlings.
The previously described process for the production of transgenic plants using
microinjection technology, including all its steps, forms part of the present invention.

20P75
- 22 -
The proeess aeeording to the invention ean be applied universally to all plants that form
seeds and seedlings. It ineludes the mieroinjeetion both of natural DNA sequenees and of
hybrid gene eonstruetions produeed artifieially by means of recombinant DNA
technology,
The broad scope of the present inven~ion includes especially recombinant DNA molecules
thut contain DNA se(luences resulting in useful and desirable propenies in the
transformants.
Those moleeules are prefer;lbly recombinant DNA molecules containing one or more gene
sequences that code for a useful and desirable property and that are under the regulatory
eontrol of expression signals aetive in plants so that an expression of said gene sequences
in the transformed plant eell is ensured.
Suitable genes for use in the process aeeording to the inventton are therefore espeeially all
those that are expressed in the plant cell and impart to the plant a useful andlor desired
property, such as, for example, increased resistance towards pathogens (for example
towards phytopathogenic fungi, bacteria, viruses, etc.), a resistance towards chemicals [for
example towards herbicides (such as, for example, triazines, sulfonylureas, imida-
zolinones, triazole pyrimidines, Bialaphos, Glyphosate, etc.), insecticides or other
biocides~; resistance towards detrimental (endaphic or atmospheric) climatic influences
(for example heat, cold, wind, particular extreme soil conditions, humidity, dryness,
osmotic stress, ete.); or that lead to an increased or qualitatively improved formation of
reserve or stored substances in leaves, seeds, tubers, roots, stems, etc.. Desirable
substances that can be produeed by tr;msgenie plants inclu(le, for example, proteins,
st~urches, sugars, amino aeids, alkaloids, odoriferous substances, eolourin~ substances, fats,
etc..
It is also possible to use in the process according to the invention genes that eode for
pharmaceutically acceptable active substances, such as, for example, alkaloids, steroids,
hormones, immunomodulators, and other physiologically active substances.

;~0~L~75~LI~
- 23 -
A resistance towards cytotoxins can be achieved, for example, by transferring a gene tl-.lt
is capable, on expression in the plant cell, of making an enzyme available tllat detoxifies
the cytotoxin, such as, for example, typc 11 neomycin phosphotransferase or type IV
aminoglycoside phosphotransferase, whicll contribute to a detoxification of ka~ mycin,
hygromycin an(l other aminoglycoside an~ibiotics, or a glutatllione-S-lransfcr.lse,
cytochrotne P-450 or other catabolically .ICliVC enzymes that are known to detoxify
tria~ines, sulfonylureas or other herbicidcs, A resis~.lnce ~owards cy~o~oxins can also be
mediated by a gene that expresses in a plant a specific form of a "~arget en~ytne" (point of
attack of cytotoxin activity) that is resistant to the activity of the cytotoxin, such as, for
example, a variant of the hydroxyacetic aeid synthase that is insensitive to the inhibitory
action of sulfonylureas, imidazolinones or o~her herbicides that in~eract wi~h this specific
metabolic step; or a variant of EPSP synthase that has proved to be insensitive to the
inhibitory action of Glyphosate. It may be advantageous to express these modified target
enzymes in a form that permits their transport into the correct cellular compartment, such
as, for example, in the above case, into the chloroplasts.
In certain eases, it may be advantageous to direct the gene products into the rnitochondria,
the vacuoles, the endoplasmatic reticulum or into other eell regions, possibly even into the
intercellular cavities (apoplasts).
A resistance towards certain classes of fungi can be achieved, for example, by inserting a
gene that expresses chitinase in the plant tissues. Numerous phytopathogellic fungi
contain chitin as an integral eomponent of their hyphae and spore structures, for example
the basidiomycetes (smut and rust fungi), ascomycetes and Fun~i ~ (inclu(ling
Alternaria and Bipolaris, Exerophilum turcicllm, Colletotrieum, Gleocercosl-or.l and
Cercospora), Chitinase is capable of inhibiting the myeelium growttl of cert.lin p.l~hogens
in vitro. A plant leaf or a root that expresses chitinase eonsti~utively or as a response to
the penetration of a pathogen should thus be protected against at~ack from a large number
of different fungi. Depending on the situation, a constitutive expression may beadvantageous as eompared with an indueible expression, which occurs in many plants as
the normal reaction to a pathogen attack, since the chitinase is immediately present in high
concentration without having to wait for a lag-phase for the new synthesis.

~0175~
- 24-
A resistance towards insects can be transferred, for example, by a gene coding for a
polypeptide that is toxic towar~s insects and/or the larvae thcrcof, such as, for examplc,
the crystalline protein of Bacillus thuringiensis [Barton et ah, 1987; Viaeck et al., 19X71. A
second class of protcins thal mediate resistance towards insects is thc protease inhihilors.
Protease inhibitors fotrn a normal constituent of plant storage structures (Ryall, 1973). It
has bcen demonstrated that a Bowman-Birk protease inhibitor isolated and purificd from
soybeans inhibits the intestinal protease of Tencbrio larvae (Birk et ak, 1963). l`he gene
that codes for the trypsin inhibitor from the cow-pea is described by Hilder _ al. (1987).
A gene that codes for a protease inhibitor can, in a sui~able vector, be brought under the
control of a plant promoter, especially a constitutive promoter, such as, for example, the
CaMV 35S promoter [which is described by Odell et ah, (1985)]. The gene, for ex~mple
the coding sequence of the Bowman-Birk protease inhibitor from the soybean, can be
obtained by means of the cDNA cloning method described by Hammond et ah (1984).
A further method of producing a protease inhibitor consists in the synthetic production
thereof, in so far as it has less than 100 amino acids, such as, for example, the trypsin
inhibitor of thc Lima bean. The coding sequence can be predicted by back-translation of
the amino acid sequence. In addition, restriction cleavage sites are incorporated at both
ends, which sites are suitable for the particular vector desired. The synthetic gene is
produced by synthesising overlapping oligonucleotide fragments of from 30 to 60 base
pairs by first subjecting them to a kinase reaction, then linldng them to one anothcr
(Maniatis et al., 1982) and f~lnally cloning them in a suitablc vcctor. Using DNA
sequencing, a clone having the insert in a correct orienîation can thcn bc identihed.
Isolated plasmid DNA is preferably uscd for insertion into the mcristcms.
Also included in the present invention are genes that code for pharmaceutic.llly active
constitltents, for example alkaloids, steroids, hormones and othcr physiologically active
substances, and also flavins, vitamins and colouring substances. Suitable genes within the
scope of this invention therefore include, without being limited thereto, plant-specific
genes, such as, for example, the zein gene (Wienand et ah, 1981), mammal-specific genes,
such as the insulin gene, the somatostatin gene, the interleukin genes, the t-PA gene
(Per,nica et al., 1983) etc., or genes of microbial origin, such as the NPT II gene, and

~017~
synthetic genes, such as the insulin gene (Itakura _ ak, 1975).
Apart from naturally occurring genes that code for a useful and desirable property, withi
the scope of this invention it is also possible to use genes that have been modificd
previously in a specific manner using chetnical or L~ene~ic en~ineering m~hOdS.
Thc brosld concept of the present invention also inchJdes genes that arc pr~luce(l cntir~ly
by chemical synthesis.
Also suitable for use in the process according to the invention are other useful DNA
sequences, especially non-coding DNA sequences that have regul~tory functions. These
non-coding DNA sequences are capable, for example, of regulating the transcription of an
associated DNA sequence in plant tissues.
There may be mentioned in this connection, for example, the promoter of the hps70 gene
which can be induced by a heat shock (Spena et aL, 1985); the 5'-flanking sequence of the
nucleus-coded sequence of the small subunit of the ribulose-1,5-biphosphate carboxylase
(RUBISCO) gene [Morelli et al., 1985] or of the chlorophyll alb binding protein, which
can be induced by light; or the 5'-flanking region of the alcohol dehydrogenase (adhl-s)
gene from maize which, under anaerobic conditions, induces the transcription of an
associated reporter gene [for example chloramphenicolacetyltransferase gene (CAT)]
(Howard_ah, 1977).
A further group of regulatable DNA sequences are chemically rcgulatable scqucllces
which are present, for example, in the PR ("pathogenesis related protein") protcin gencs of
tobacco nnd can be induced with îhe aid of chemical regulators.
The regulatable DNA sequences mentioned above by way of example m.ly be either of
naîural or of synthetic origin or they may consist of a mixturc of natur.ll al-d synthetic
DNA sequences.
Genes or DNA sequences îhat are suitable for use within the scope of îhe presentinvention are therefore both homologous and heterologous gene(s) or DNA and synthetic

;~175'~
- 26 -
gene(s) or DNA according to the definition given within the scope of the presentinvention.
The DNA sequence can be constructed exclusively from gcnomic DNA, from cDNA or
from synthetic DNA. Another possibility is the construction of a hybrid DNA se(lllence
consisting of both cDNA and genomic DNA and/or synthetic DNA.
In that case the cDNA may originate from the same gene as the genomic DNA or both the
cDNA and the genomic DNA may originate from different genes. In any case, however,
the genomic DNA and/or the cDNA may each be prepared individually from the same
gene or from different genes.
If the DNA sequence contains portions of more than one gene, these genes may originate
from one and the same organism, from several organisms belonging to various strains or
varieties of the sarne species or various species of the same genus, or from organisms
belonging to more than one genus of the same or a different taxonomic unit (kingdom).
In order to ensure the expression of said structural genes in the plant cell, it is
advantageous if the coding gene sequences are first linked in operable manner toexpression sequences capable of functioning in plant cells.
The hybrid gene constructions within the scope of the present invention thus contain, in
addition to the structural gene(s), expression signals that also include both promoter and
terminator sequences and other regulatory sequenccs of thc 3' and 5' untranslated rcgions.
Any promoter and any tenninator that is capable of bringing about an inductioll of Ihe
expression of a coding DNA sequcncc (structur;ll gcne) can bc used as a consti~llent of ~he
hybrid gene sequence. Especi;llly suitablc ;ure exprcssion signals th;lt origin;ltc from gCllCS
of plants or plant viruscs. Exatnplcs of suitable promoters and ~ermina~ors are thosc of ~hc
nopaline synthase genes (nos), of the octopine synthasc genes (ocs) and of the C~uliflower
Mosaic Virus genes (CaMV), or homologous DNA sequences that still have the
characteristic properties of the mentioned expression signals.

;~O~75~
Preferre~l within thc scopc of this invention are the 35S and 19S expression signals of the
CaMV genome or their homologu~s which can be isolated from said genomc using
molecular biolo~ical mctllo~ls, as described, for example, by Maniatis et al., 1982, and
linked to the coding DNA s~qucnce.
Within the scope of this invention, homologues of the 35S and 19S c~prcssion si~ ls
shall be understood as being sequences which, in spite of slight differences ilt SeqllellCC,
~re essentially homologous to the starting sequences and still fulhl the s~me function as
those starting sequences.
According to the invention, there may be used as starting material for the 35S transcription
control sequences, for example, the ScaI fragment of the CaMV strain "S" which includes
the nucleotides 6808-7632 of the gene map (Frank G et ah, 1980).
The 19S promoter and 5' untranslated region is on a genome fragment between the PstI
site (position 5386) and the HindIII site (position 5850) of the CaMV gene map (Hohn et
ah, 1982). The corresponding terminator and 3' untranslated region lies on an
EcoRV/BglII fragment between positions 7342 and 7643 of the CaMV genome.
Also preferred within the scope of this invention are the expression signals of the CaMV
strain CM 1841 whose complete nucleotide sequence is described by Gardner RC et ah,
1981.
A further effective example of a plant promotcr that can be used is an ovcr-producillg
plant promoter. This type of promoter should, if linked in opcrable manner to a gene
sequence that codes for a desired ~ene prodllct, bc capable of mcdiating the expression of
said gene sequence.
Over-producing plant promoters that may be used within the scope of the present
invention include the promoter of the small subunit (ss) of the ribulose-1,5-biphosphate
carboxylase from soybeans [Berry-Lowe _ ah, J. Molecular and App. Gen., 1: 483-498
(1982)] and the promoter of the chlorophyll-a/b-binding protein. These two promoters are
known for the fact that they are induced by light in eukaryotic plant cells [see, for

~175~
example, Genetic En~ineering o_nts, an A~ricultural Perspective, A Cashmore,
Plenum, New York 1983, pages 29-38; Coru~zi, a et ah, The Journal of Biolo~ical
Chemistrv, 258: 1399 (1983) and Dunsmuir, P ct al., Journal of Moleculamand Applied
Genctics, 2: 285 (1')83)J.
'rhc various sections of l~NA sequence can be linked to one another to form a complete
coding DNA sequencc by methods known ~ se. Suitable methods inclu(le, for exatllple,
the tn vtvo recombination of DNA sequences ~hat have homologous sections anci thc in
vitro linking of restriction fra~ments.
There are generally used as cloning vectors plasmid or virus (bacteriophage) vectors
having replication and control sequences originating from species that are compatible with
the host cell.
The cloning vector generally carries an origin of replication, and also specific genes that
result in phenotypic selectivn features in the transformed host cell, especially resistances
towards antibiotics or towards specific herbicides. After transformation, the transformed
vectors can be selected in a host cell on the basis of these phenotypic markers.
Selectable phenotypic markers that may be used within the scope of this invention include,
for example, without this limiting the subject of the invention, resistances towards
ampicillin, tetracycline, hygromycin, kanamycin, metotrexate, G418 and neomycin.
Within the scope of this invention, suitable host cells are prokaryotes, including bacteri,ll
hosts, such as, for example, A. tumef,lciens, E. coli, S. tvphimurium and Serratia
marcescens, and also cyanobactcria. Eukaryotic hosts, such as ycasts, mycelium forming
fungi and plant cells, can also be used within the scope of this invention.
Splicing of the hybrid gene construction according to the invention into a suitable cloning
vector is carried out using standard methods, such as those described, for example, by
Maniatis et ah, 1982.

20175~
- 2g -
~s a rule, the vector and the DNA sequence to be spliced in are first cleaved with suitable
restriction enzymes. Suitable restriction enzymes are, for example, those that yi~ld
fragments having blunt ends, such as, for example, Smal, Hpal and EcoRV, or enzyl-lcs
that form cohesive ends, such as, for example, EcoRI, SacI and Bam~ll.
Both fragments having blunt ends .uld those having cohesive cnds that are complemcllt;lry
to one ant~ther c1n be linketl again using suitable VNA ligases ~o form a continuolls
mliform DNA molecule.
Blunt ends call also be procluced by treating DNA fragments that have projecling collesive
ends with the Klenow fragment of the E. coli DNA polymerase by filling tlle gaps with the
corresponding complementary nucleotides.
On the other hand, cohesive ends can also be produced artificially, for example by adding
complementary homopolymeric tails to the ends of a desired DNA sequence and of the
cleaved vector molecule using a terminal deoxynucleotidyl transferase or by adding
synthetic oligonucleotide sequences (linkers) that carry a restriction cleavage site and then
cleaving with the appropriate enzyme.
The cloning vector and the host cell transformed by that vector are generally used to
increase the number of copies of the vector. With an increased number of copies, it is
possible to isolate the vector that carries the hybrid gene construction and to use it, for
example, for inserting the chimaeric gene sequence into the plant cell.
In a further process step, these plasmids are used to insert the structur;ll gCllCS co(ling for a
desired gene product, or non-coding DNA se(lllences having a regulatory fullctioll~ into the
plant cell and to inîegrate them in the plan( genome.
The broad concept of this invention also includes transgenic plants that have bcen
transformed by means of the previously described process according to the invention, that
is to say, that demonstrably contain and express the inserted genetic material, and also the
asexual and/or sexual progeny thereof that still contains the inserted genetic material and
therefore, as a rule, has the novel and desirable property or properties resulting from the

~3L75'~
- 30-
transformation of the parent plant, and also plant propagation material in gener~l.
The expression "ascxual and/or sexual progeny of trans~enic plants"t as defllled ~vithin the
scope of this invention~ therefore also covers all mutants and variants ~h.lt still col~aill thc
inser~ed ~enelic material and therefore, .IS .I rule, have the characteristic propcrlies of thc
transforme(l startin6 plant, and all hybridis.ltion and fusion products cont.linillg thc
trallsformed plant material.
The expression "plant propag.ltion material" as defincd within the scope of this invcntio
should be understood as covering, for example, plant protoplasts, cclls, cell clones, cell
agglomerates, callus cultures, tissue cultures and/or organ cultures and also seeds, pollen,
ovules, zygotes, embryos or other propagation material originatin~ from germ-line cells;
this list, which is given only by way of example, is not intended to limit the subject of the
invention.
This invention also relates to parts of plants, such as, for example, blossoms, stems, fruit,
leaves and roots, that originate from transgenic plants or the progeny thereof that have
been transformed previously by means of the process according to the invention and are
therefore at least partially made up of transgenic cells.
The process according to the invention is suitable for the transformatioll of all pl.mts,
especially those of the systematic groups Angiospermae and Gvmnospermae
Of the Gymnospermae, the plants from the Coniferac ChlSS arc of particlll;lr intcrcst.
Of particular interest amon~ the An iosperm.lc an~. in addition to dcciduolls trcc~ an(l
sllrllbs, plallts of the families Solanaceae, Cmcifcr.le, Compositae, Liliaceae, Villlce.le,
Chellopodiaceae, Rutace.le, A!liaceae, Am,~rvllid.lceae, Aspara~aceae, Orchidaceae,
_allllae, romeliaceae, Rubi,lceae, Theaceae, Musace,le, Malvaceae or Gramineae an(l of
the order Le~utninosae and, of these, especially of the family Papilionaceae.
Representatives of the Solanaceae, Cruciferae, Le~uminosae, Malvaceae and Gramineae
are preferred.

~0~75'~"-
- 31 -
The target crops within the scope of the present invcntion also include, for example, those
selected from the series: Fragaria, Lotus, Medica~o~ Onobrvchis, Trifolium, Tri~ollcll.l,
Vigna, Citrus, Linum, Geranium, Manillot, Dallcus, Arabidop~sis, Bra_C;t, R~ alllls,
~pls, AtroQa, Capsicum, Datura, ~ )sc~, Lvcoper~sicon~ Nicoti;lll;l, Sol;llllltm,
Pe~Jnia, ~, Majoransl, Cichorium, Heliantlllls, l~c~uca, Bromus, Aspar,l~lls,
Antirrhinum, Hemerocallis, ~Nemesi l, Pe~o m, Panicllm, Pe etl!m, Ranuncllllls,
Senecio, ~lossis, Cucumis, Browslllisl, Glvcine, Lolium, Zea, rriticum and ~or~hum,
Gossypium, Ipomoea, Passiflora, Cvclamen, M.JIIIS, Prunus, Rosa, Rublls, Popullls,
Santalum, Allium, Lilium, Narcissus, Anan.ls, Arachis, Phaseollls and Pisutn
Especially preferred are representatives of the Gramineae family, such as, for exatllple,
plants that are cultivated over a large area and produce high yields. Examples that may be
mentioned are: maize, rice, wheat, barley, rye, oats, millet and sorghum and also pasture
grasses
Other target crops that are especially preferred for use in the process according to the
invention are, for example, plants of the genera Allium, Avena, Hordeum, Orvza,
Panicum, Saccharum, Secale, Setaria, Sor~hum, Triticum, Zea, Musa, Cocos, Phoenix and
Elaeis.
Successful transformation can be verified in a manner known Per se, for example by
molecular biological investigations which include, especially, Southern blot an;llysis.
In that method of analysis, the extractcd DNA is first t~atcd with restricti()ll cnzymes,
then subjected to electrophoresis in a 0.8 % to I % ag;lrose ~el, trallsferred onto a
nitrocellulose membrane [Southern, EM, J. Mol. Biol. 9~, 503-517 (1975~] all(l hybridised
with the DNA to be detected, which has prcviously undergone a nick trallsl;ltion [Rigby,
WJ, Dieckmann, M, Rhodes, C and P Berg, J. Mol. Biol. 113, 237-251~ (DNA-specific
activities of from 5 x 108 to 10 x 108 c.pml./~g). The filters are washed three times for one
hour each time with an aqueous solution of 0.03 M sodium citrate and 0.3 M sodium
chloride at 65C. The hybridised DNA is rendered visible by blackening an X-ray film for
from 24 to 48 hours.

~O~7
- 32 -
To illustrate the rather general description, and for a better underslanding of the present
invention, reference will now be made to specific Examples which are not of a limiting
nature unless thcre is a spccific indication to thc contrary. The same applies to all Iists
given by way of cxample in the abovc (lescription,
Non-lim ~x ~:
Ex.lm~: Construction of plasmid pABDI
-
'rhc freely accessible plasmids pkm21 and pkm244 [Beck, E, et ak, Gcne 19, 327-336
(1982)] are cleaved with the restriction endonuclease Pstl. The plasmid fragmcnls, whicll
are used for recombination, are purified by electrophoresis in 0.8 % agarose gel. Plasmid
pkm21244, obtained by joining the fragments, contains a combination of the 5'- and
3'-Bal31 deletions of the NPT-II gene, as described by Beck et al. in Gene, 19, 327-336
(1982). In order to join the promoter signal of the Cauliflower Mosaic Virus to tlle HindIII
fragment of plasrnid pkm21244, coupling plasmid pJPAX is constructed. Coupling
plasmid pJPAX is obtained from plasmids pUC8 and pUC9 [Messing, J and J Vieira,
Gene 19, 269-276 (1982)]. 10 base pairs of the linker sequence of plasmid pUC9 are
separated by cleaving at the HindIII and the SalI sites and subsequently making up the
cohesive ends using the polymerase I Klenow fragment [Jacobsen, H, et al., Eur. J.
Biochem. 45, 623, (1974)] and joining the polynucleotide chain, as a result of which the
HindlII site is produced again. A synthetic linker element of 8 base pairs (Xhol) is
inserted at the SmaI site of this separated linker sequence. The recombinalion of the
suitable XorI and HindlII fragments of plasmid pUC8 and of modificd plasltli(l pUC9
produces plasmid pJPAX having a p;wti;llly asymmctric linkcr sc(lllcn~:e with ~he
following successive restriction sites: EcoRI, Smal, B,~mtll, Sall, Ps~l, llindlll, Bam~ll,
XhoI and EcoRI. The CaMV gene Vl promoter region, which is linkcd to the NPT-II
structural gene, originates from the genome of CaMV strain CM 4-184, a variant of the
CaMV strain CM 1841, the complete nucleotide sequellce of which is described by
Gardner, RC et al., 1981. The CaMV promoter region is cloned in cosmid pHC79, which
comprises 6 kBp (kBp: 1000 base pairs) and which is a derivative of the E. coli plasmid
pBR322 [Hohn, B and Collins, J, 19801, the CaMV genome and cosmid pHC79 being
cleaved with BstII and the resulting fragments being linked to one another. The S'
expression signal of the Cauliflower Mosaic Virus gene VI and the HindIII fragment of

~17S~
the NPT-II gene are joined in plasmid pJPAX by inserting the promoter region of the
Cauliflower Mosaic Virus gene Vl between the Pstl site and the HindIII site. Thercsulting plastnid is cleaved at the sin~le HindllI site and the Hindlll fragment of plasmid
pkm21244 is inserte(l into this cleavage site in both directions of orientation, resulting itl
plasmids pJPAXCakm~ an~ pJPAXGIkm~. 111 or~Jer to create an Ecol~V sequcllce in thc
vicini~y of the 3' tcrmination sigllal of lhe NPT-ll hybrid gene, a BamHI fra~lncllt of
pln~mi~l pJPAXCakm~ is insèrtetl into the BamHI site of plasmid pBR327 [Soberon, ~ ct
al., Gene ~), 287-3()5 (Ig8())]. Plasmid pBK327Cakm is obtained. The Ecol~V fragment of
plasmi~l pB1~327(:akm, which contains the new DNA construction, is used to replace tl-e
EcoRV region of the Cauliflower Mosaic Virus gene Vl ~hat has been cloned at the Sall
site in plasmid pUC8, as a result of which the protein-coding DNA sequence of the NP ï'-lI
gene is placed under the control of the S' and 3' expression signals of the Cauliflower
Mosaic Virus gene Vl. The resulting plasmids are designated pABDI and pABDII.
Example 2: Construction of plasmid pDH51
Plasmid pDHS1 is described by Pietrzak ah, 1986. This plasrnid contains the 35S
promoter region and terminator region of CaMV which are separated by an insertedpolylinker region containing numerous cloning sites.
The starting material used for the 35S promoter/terminator region is a Scal fragment of
CaMV "S" which includes the nucleo~ides 6808-7632 of the gene map (Frank, G et ak,
Cell, 21: 285-294, 1980) and which is spliced into the Smal site of pUC 18 (Norrander, J et
al., Gene, 26: 101-106, 1983) to form plasmid pDB21. This is then digested with the
restriction cnzyme Hphl. The cohesive ends are removed by tre;ltment with T4 DNApolymerase.
A fragment containing the 35S promoter region 1PUCI 8 POS. 21 (Hp}~ 412 (Smal) plus
CaMV pos. 6808-7437 (Hphl)] is linked to Kpnl linkers, cleaved with NcoI (pos. 6909
CaMV), the cohesive ends are made up using the Klenow fragment of DNA polymerase,
linked to EcoRI linkers and fin;llly digested with EcoRI and Kpnl. The resultingEcoRVKpnl fragment, which contains the CaMV promoter sequence, is then spliced into
plasmid pUC18 between the KpnI and EcoRI restriction cleavage sites. In this manner,
plasmid pDG2 is obtained.

2~175
- 34-
A second fragment carrying the termin.ation signals ICaMV pos. 7439 ~phI)-7632 plus
pUC18 pos. 413-1550 (~Iplll)] is cle.lved with EcoRI, the cohesive ends are made up USillg
Klenow, provided with Hindlll linkcrs and digcsted with Hindlll and Sphl~
The resullin~ Sphl-~lindlll fra~ment, which con~ains the CaMV terminator sequence, is
then spliced intO the ~lindlll and Sphl restriction cle.avage sites of plasmid pDG2.
Example 3: Construction of plasrnid p~lP23
Thc construction of plasmid pHP23 is effected by inserting an EcoRV fragment of
plasmid pABDI, which contains the APH(3')II structural gene and also a p~rt of the 19S
RNA promoter sequence of CaMV, into the SmaI cleavage site of plasmid pDH51.
Example 4: Preparation and microiniection of tobacco seedlin s
4.1. Plant material
Seeds of Nicotiana tabacum c.v. Petite Havanna SR1 plants are germinated on non-sterile
soil and grown to seedlings. Illumination is from one side, from above, while the sides
and the base are protected from irradiation by applying a black covering. The shoot axis is
thereby kept short. The germination period depends on the germination activity of the
seeds used. It is generally from 3 to 6 days.
4.2. PrepaMtion of the seedlin~s for microin3ection
When the cotyledons have reached a size of approximately 1.5 mm, the seedlings are
removed from the soil and the growing cones are exposed. Eilher one or both cotyle(lons
is/are removed or the cotylcdons are pulled apart by means of a speci31 holding device to
the extent that the apical meristem becomes visible.
4.3 Microiniection of pHP23 plasmid DNA into tobacco seedlin~s
4.3.1. Apparatus
The microinjection into the shoot growing cone of tobacco seedlings is carried out under
microscopic observation using an inverse rnicroscope or a stereomicroscope with a

~0~7~
combination of ineident an~l transmitted light. A eold light souree is used as illumin"tion
in or(ler to avoi(l the evolution of hcat. 'I'he magnifieation depends on the size of ~he
see~llings and varies from l()x to 3()()x magnifieation.
'I'he manipulalion of the secdlin~s is carried out usin~ micromanipulators whicll can be
fnstelted to the microscope stand (motor-driven maniplllators) or stand at the sidc of the
microscope (mech.lnic.ll manipulators). 'I`he mieromanipulators are eqllipped witl
capilhlry hoklers.
'I'here are used as microinjeetion eapillaries mieroeleetrcx~e capillaries that have seale(l
glass filaments of borosilieate and have an outsi(le diameter of from 1.00 mm to 1.50 mm.
In order to fix the seedlings in position during the injeetion operation, elamping deviees
produeed especially for that purpose are used. On the elamping device, the eotyledons are
seeurely clamped on a speeial support, for exarnple a metal holding deviee, and thus fixed
in position; the supports are movable with respect to one another so that the cotyledons
ean be pulled apart to the extent that the growing cone beeomes visible and is therefore
aeeessible for subsequent microinjeetion. The injeetion eapillary is drawn using a
eommereial eapillary drawing apparatus so that suitable injection eapillaries (tip diameter
approximately 51 ~,lm) are produeed.
It is, of eourse, also possible to use any other suitable proeess for exposing the growing
eone.
The DNA solutioll is injeeted using a mieroinjeetor. This mieroinjce~or is .a pncllm.ltically
driven pressure deviee whieh permits a very aecuratcly metcred injcetion intO cclls of thc
growing eone. The injeetion volume v;~ries in aeeord.lllce with the eell size from I pl to
100 pl. Suitable mieroinjeetors eall be obtained, for example, from Eppell(lorf, PRG or
Baellhofer, FRG.
4.3.2. DNA solution
The mieroinjected DNA solution is super-coiled or linearised pHP23 DNA or a mixture of
super-eoiled and linearised pHP23 DNA in a Tris-HCI buffer consisting of 50 mM of
Tris-HCI, pH 7-7.8, and 50 mM of NaCI.

~017
- 36 -
4.3.3. Microiniection
The microinjection of the previously described DNA-containillg solution into cells of the
apical meristem of tobacco seedlings i~ carried out an~logou~ly to the metho(l describcd
by Neuhaus et ah, 1~84; 1()86.
Eacll seedling is first secured by both or only onc cotyledon(s) to a metal holding dcvice,
there being one holding device for each cotyledon. Since thesc metal holding devices can
be moved with respect to one anotker, the cotyledon(s) can be carcfully pulled apart ulltil
the apical meristem is exposed to the extent that the subsequent microinjection can be
carried out without difficulty.
Each seedling is injected from 4x to 60x, depending on the size of the exposed apical
meristem, and is turned between the individual injection operations so that as many cells
as possible are microinjected. The injected seedlings are then transferred back to soil and
cultivated furiher.
4.4. Cultivation of the microiniected tobacco seedlin~s and rowth of whole tobacco
plants
After rnicroinjection, the seedlings are transferred back to soil in plastics containers
having a diameter of 9 cm and a depth of 5 cm and are there cultivated at 25 to 28C with
a lightldark regime of 16/8 hours (4000 lux, cold white light) with a humidity of from 70
% to 80 % and at normal pressure (atmospheric pressure, approximately 760 mb,lr) until
the plantlets have reached a size of 3 cm. This has generally occurrcd aftcr from 2 to 5
days; the plants are then placed in a greenhouse, tr.msferred to soil alld ~rown in thc satllc
manner as normal tobacco scedlings until the plants mature.
The plants obtained in the manner described above are heterogeneous tr;msfonn;lnts the
chimaeric nature of wllich can be investigated using the methods described under sections
7 and 8.

~0~75~
Example 5: Production and microinjection of isolated apical meristems of tobaccoseedlin~s
5.1. Plant material
Seeds of Nicoliana ta um c.v. Petilc l-lavann.l SR I plants are surface-sterilised. For th.lt
purpose, ~he plants are firs~ washcd bricfly (for a few scconds) in 70 % ethanol and thcn
immediately rinsed with stcrile distilled water, They are subsequcntly treated for 30
minutes in a 1.5 % calcium hypochlorite solution. The plants are then washcd three times
itl previously sterilised dislilled water and dried Approximately 5Q seeds are then placed
in a petri dish (10 cm diameter) containing a customary nutrient medium (Tl) and caused
to germinate, Illumination is from one side, from above, while the sides and the base are
protected against irradiation by applying a black covering. The result of these measures is
that the shoot axes of the seedlings are kept short, which is advantageous for the
microinjection which follows.
5.2. Microiniection of pHP23 plasmid DNA into tobacco seedlin~s
The preparation of the seedlings for microinjection and the microinjection itself are
carried out analogously to the methods described in sections 4.2 and 4.3.
5.3. Isolation of the microinjected ~rowin~ cone
After microinjection, both cotyledons, as well as other rudimentary leaves which may still
be present, are removed from the seedlings under a stereomicroscope. The exposed shoot
tip is separated from the rest of the shoot approximately 0.5 mm to 2 mm, but prcferably
1 mm, below the tip USillg a scalpel and further cultivatcd on a suitable culture mcdium
(T2). Terrasaki plates tNunc, Denmark) arc gcncrally uscd for tllc cultivation of the
isolated shoot tips, whicll are cultivatcd on frolll lO ~,~l to 20 I,ll of T2 me(lium.
After approximately from 5 to lS days, but prefer.lbly after lO days, the developillg apic;ll
meristems are transferred to a selective culturc medium (T3). Costar wells (24 wclls;
16 mm diameter, manufactured by Tissue Culture Cluster, Cambridge, Mass.) containing a
T2 medium are gener.llly used, the medium containing as selective additive the antibiotic
kanamycin at a concentration of from 20 llg/ml to 200 ~,lg/ml, but preferably 50 llg/ml
(T3). Under those selection conditions, the only cells that survive are those which contain

~0175~
the microinjected resistance gene incorporated in their genome and also express it, since
only those cells are resistant to the alllibiotic. After a further cultivation period of from 1()
to 20 days, preferably 15 d.lys, the growing ccll masses are transferrcd to another culture
mcdium (T4) which also contains the antibiotic kanamycin at thc salne concentr;ltioll but
which stimulates the cclls to increased adventitious shoot regener.ltion as a result of thc
hormone concentration that has becn selected. In this case ~oo, only those shoots will
develop whicll have the injected resistance gene inte~rated in their gcnome since, as
before, the medium exerts a selection pressure.
Finally, the regenerated shoots are caused to root by being transferred to suitable media
(TS), are transferred into soil and finally grown in a greenhouse until the seeds ripen.
Example 6: Detection of the NPT~ ene in the plant enotYpe
Leaf tissue of the regenerated and transformed plants is homogenised at 0C in 15 %
saccharose solution containing 50 mmol/l of ethylenediamine-N,N,N',N'-tetraacetic acid
(EDTA), 0.25 mol/l of sodium chloride and 50 mmol/l of
a,a,a-Tris-(hydroxymethyl)-methylamine hydrochloride (TRIS-HCI) at pH 8Ø The cell
nuclei are roughly separated off by centrifuging the homogenate for 5 minutes at 1000 g.
The cell nuclei are resuspended in 15 ~o saccharose solution containing 50 mmolll of
EDTA and 50 mmol/l of TRIS-HCl at pH 8.0; sodium dodecylsulfate (SDS) is added until
a final concentration of 0.2 % is reached, and the whole is heated for 10 minutes at 70C.
After the mixture has been cooled to from 20 to 25C, potassium acctatc is added until a
concentration of 0.5 mol/l is reached. This mixture is incubated for I hollr at 0C. Thc
resulting precipitate is separa(ed by centrifug;ltioll (15 millutes at 4C in a
Microcentrifuge). The DNA is precipitntcd from the supcrnatant liquid at from 20 to 25C
by adding 2.5 times the volume of ethanol. The scp;urated DNA is dissolved in a solution
of 10 mM TRIS-HCI containing 10 ~Ig/ml of ribonuclease A and incubaled for 10 minutes at
37C; proteinase K is added until a concentration of 250 llg/ml is reached, and Ihe whole
is incubated for a further hour at 37C. The proteinase K is removed by phenol and
chloroform/isoamyl alcohol extraction processes. The DNA is precipitated from the
aqueous phase by the addition of 0.6 part by volume of a 0.6 M solution of sodium acetate
in isopropanol, and dissolved in 50 ,LI of a solution containing 10 rnmol/l of TRIS-HCI and
5 mmolll of EDTA at pH 7.5. By means of this preparation, DNA sequences, most of

;~0175
- 39 -
which contain more than 50,000 base pairs, are obtained. Restriction of this DNA with
EcoRV endonuclease, hybridisation of the fragments with radioactively labelled ~lindlll
fragments of the NPT-II gene and comparison with plasmid p~BDI dcmonstrates, in a
Southern blot analysis, the presence of the NPT-II gene in the cell nuclells DNA of thc
transforme(l plant cclls.
Exam~le ?,: ~ot-blot all.llYsls
1() ~lg of ~enomic DNA ,ure di~cstc(l ovemight wi~h EcoRI and ~hcn applied in ~he form of
lots, without previous electrophoretic separation, directly on~o a nitrocellulosc mcmb~ e
(see procedural instructions of Schleicher and SchUII GmbH, Dassel, FRG).
The hybridisation reaction and the subsequent auloradiography are carried out in a mallner
analo~ous to the Southern blot method.
Example 8: Southern blot analysis
The Southern blot analysis of the plant DNA is carried out in accordance with a method
described by Paszkowski and Saul, 1983.
Approximately 5 llg of genomic DNA are cleaved either with EcoRV or with HindlIIrestriction enzymes, as desired, and the resulting fragments are separated by
electrophoresis in a 0.8 % to I % standard agarose gel (,Mania~is, T et ah, 1982).
The nick translation is replaced by thc ral)dol1l r)rimcr mc~}l(xl dcscribcd by Feinbcr~ all(l
Vogelsteill~ 1983.
Ex.mlple 9: Investi~.!tioll of the ;Imino~c ~hnspholr;~nsft:r;!sc cll~o~me v~f
tr.msformed pl~mts
Pieces of leaf (100 to 200 mg) are llomogenised in 20 111 of extraction buffer in an
Eppendorf centrifugation tube. The buffer is a modification of the buffcr use(l by
Herrera-Estrella, L, DeBlock, M, Messens, E, Hernalsteens, J-P, Van Montagu, M and J
Scllell, EMBO J. 2, 987-995 (1983) in which bovine serum albumin is replaced by 0.1 M
saccharose. The extracts are centrifuged for 5 minutes at 12,000 g and bromophenol blue
is added to the supernatant phase until a final concentration of 0.004 % is obtained. The

%0~75~
- 40 -
proteins are separated from 35 ,LI of the supernatant phase by electrophoresis in a 10 %
non-denaturing polyacrylamide gel. A layer of agarose gel containing kanamycin and
~32P-labelled ATP is placed over the gel, incubation is carried out and the phosphoryl.lte(l
reaction products are transferred onto Whatman p81 phosphocellulosc paper. The papcr is
was~ed six times with deionised water of 90C and then subjec~ed to autoradiogr.lphy.
Reslllts:
It was possible to effect re~eneration of whole transgenic plants from microinjec~ed apic;ll
meristems vi direct growth of In vitro cultivated plants under selection pressure in the
case of tobacco, the rates of regeneration achieved being very high. In the case of in vitro
cultures with selection, the regenerants are used to determine the transformation
(expression) frequency.
The following method is used for all other plants obtainable directly from the
microinjected seedlings: after self-fertilisation, the sæds of these plants are caused to
gerrninate and this generation of plants is used to demonstrate the transformation and
expression of the gene. The injected NPT-II gene could still be detected, by itsexpression, 6 weeks after rnicroinjection. The expression of the integrated gene is
demonstrated by means of the enzyme assay described in Example 9 (investigation of the
aminoglycoside phosphotransferase enzyme activity). The expression frequencies
determined in this manner are in a range of from 5 % to 35 %. It is possible to demonstrate
the stable integration of the complete microinjected genes into the high-molecul3r-weight
DNA of the Fl generation, which originates from the primary regenerants, using Southern
blot analysis of the genomic DNA of said Fl generation plants.

175
- 41 -
Plilsmid p}-lP23 used within the scope of the prcscnt invell~ion was dcposited at thc
"l~eutschc Sammlung von Mikroor~anismen" (DSM), in Brunswick, l cdcral Republic of
Gcrm~ny, which is reco~nised as an Internatioll.ll Dcpository~ in accord.lncc with the
requirements of the 13udapest Tre.lty on the ln~ernational Rccogllilion of the Deposit of
Microorgtlllisms for the Purposes of Patent Procedure. A cleclaration rc~ar(ling the
viubility of the depositcd samples was issued by the said Intcmation.ll Dcpository.
microorganism deposition deposit date of vi~bility
date number* cenificate
pHP23 (Escher-
DHl transfor- 21.12.1987 DSM 4323 21.12.1987
med with pHP23
plasmid DNA
*issued by the above-indicated International Depository

20175f~t
- 42 -
Media
m~ Tl T2 T3 T4 TS
.
ca(No3)2 (4~120) 1()00
KNO3 250 1010 1010 1010 19()0
Kl12PO4 25() 136 136 136 170
NH4NO3 800 800 800 1650
CaCl2-2H2O 440 440 440 440
M~SO4-7H2O 250 740 740 740 370
NH4-succinate 50 50 50
Na2EDTA 74.6 74.6 74.6 74.6
FeCI3 6H2O 27.0 27.0 27.0 27.0
Fe-EDTA 6.4
H3BO3 3 3 3 6.2
KI 0.75 0.75 0.75 0.83
MnSO4 H2o 10 10 10 16 9
ZnSO4 7H2O 2 2 2 8.6
CuSO4 SH2O 0.025 0.025 0.025 0.025
Na2MoO4 2H2O 0.25 0.25 0.25 0.25
CoCI2 6H2O 0.025 0.025 0.025
CoS04 7H2 o.o~s
m-inositol 1()0 100 100 100
pyridoxine-HCI I 1 1
thiamine-HCI 10 10 10 0.()4
nicotinic acid 1 1 1
saccharose 30 g/l 30 g/l 20 g/l 30 g/l
mannitol 30 g/l

20175't~
- 43 -
mg/lTl T2 T3 T4 T5
a~ar 8()00 600()6()00600()
S~a Plaque(B) a~arose 6()00
NAA (naphthyl-1- 0.1 0.1 0.1
acctic acid)
BAP (ben~ylamino- 1 I0.25
purine)
IBA (3-indolebut-
yric acid)

5~
- 44 -
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