Genetically transformed rose plants and methods for their production

Rose plant cells are transformed by incubation with Agrobacterium cells carrying an exogenous DNA sequence. The callus cells may be obtained from various tissue sources, including stamen filaments, leaf explants, and the like, and whole rose plants may be regenerated from the transformed callus cells. The exogenous DNA will be stably incorporated into the chromosomes of the regenerated rose plant which will be able to express gene(s) encoded by the DNA sequence.

The subject matter of the present invention is related to that of 
application serial number 07/542,841, filed Jun. 25, 1990, now abandoned, 
the full disclosure of which is incorporated herein by reference. 
BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to methods for genetically altering 
the cells of higher plants. More particularly, the invention relates to a 
method for genetically transforming cells from rose plants. 
The hybrid tea rose, Rosa hybrida, is one of the most popular of all 
cultivated plants. As with any valuable plant species, breeders have long 
been working to improve existing varieties and create new varieties using 
conventional cross-breeding techniques. Characteristics of particular 
interest include color, fragrance, morphology, herbicide resistance, 
pesticide resistance, environmental tolerance, vase life of the cut 
flower, and the like. While improvements and variations in most or all of 
these areas have been achieved, progress is slow because of the perennial 
nature of the plant and the high incidence of plant sterility caused by 
abnormal chromosome numbers. While rose tissue culture is now possible 
based on work described in co-pending application Ser. No. 542,841, 
referenced above, the natural genetic variation offered by tissue culture 
is random and still requires substantial effort to produce a particular 
genetic variation. 
For these reasons, it would be desirable to use recombinant DNA technology 
to produce new rose cultivars in a controlled and predictable manner. It 
would be particularly desirable to be able to genetically transform 
individual rose plant cells to introduce a desired characteristic and to 
be able to regenerate viable somatic embryos and rose plantlets from the 
modified cells. Such methods should be capable of introducing preselected 
exogenous genes to the rose plant cell and should permit selection of 
transformed cells which are capable of expressing the gene. The method 
should produce regenerated rose plants which have stably incorporated the 
gene(s). 
2. Description of the Background Art 
Abstract A203 (Noriega et al.) in Abstracts VIIth International Congress on 
Plant Tissue and Cell Culture, Amsterdam, Jun. 24-29, 1990, reports 
preliminary results on the production of calli from rose (Rosa hybrida) 
leaves. The reported results correspond to work described in related 
application U.S. Ser. No. 542,841, now abandoned previously incorporated 
herein by reference. 
Tissue culture methods involving Rosa hybrida and other rose species are 
described in Handbook of Plant Cell Culture, Ammirato et al. (eds.), 
Chapter 29, 716-743, McGraw-Hill (1990); Skirvin et al. (1979) Hort Sci., 
14:608-610; Hasegawa (1979) Hort Sci., 14:610-612; Khosh-Khui et al. 
(1982) J. Hort Sci., 57:315-319; Valles (1987) Acta Horticulturae, 
212:691-696; Lloyd et al. (1988) Euphytica, 37:31-36; Burger (1990) Plant 
Cell Tissue and Organ Culture, 21:147-152; Ishioka et al. (1990) Plant 
Cell, Tissue and Organ Culture, 22:197-199; Matthews et al. "A Protoplast 
to Plant System in Roses" 7th IAPTC Congress, Amsterdam; and de Wit et al. 
(1990) Plant Cell Reports, 9:456-458. 
The susceptibility of certain Rosa species to infection and tumor induction 
by Agrobacterium tumefaciens is described in De Cleene et al. (1976) The 
Botanical Review, 42:389-466. The susceptibility of certain Rosa species 
to infection and hairy root induction by Agrobacterium rhizogenes is 
described in De Cleene et al. (1981) The Botanical Review, 47:147-194. 
The transformation of embryogenic calli from Prunus persica (a member of 
the Rosaceae family) with Agrobacterium tumefaciens is reported in Scorza 
(1990) In Vitro Cell Dev. Biol., 26:829-834. No disclosure of transformed 
plant material beyond callus stage or of regeneration of whole plants is 
provided. The transformation of explant materials from other members of 
the Rosaceae family is described in James et al. (1989) Plant Cell 
Reports, 7:658-661, and Graham et al. (1990) Plant Cell, Tissue and Organ 
Culture, 20:35-39. 
The transformation of crushed tobacco callus with wild-type (virulent) 
Agrobacterium tumefaciens resulting in crown gall formation is reported in 
Muller et al. (1984) Biochem. and Biophys. Res. Comm., 123:458-462. 
SUMMARY OF THE INVENTION 
The present invention comprises methods for genetically transforming rose 
plant callus cells and, in the preferred embodiments, for regenerating the 
transformed callus cells into somatic embryos and ultimately back into 
viable rose plantlets. The callus cells are transformed by incubation with 
Agrobacterium cells carrying an exogenous DNA sequence which typically 
includes a selectable marker gene as well as one or more genes to be 
expressed. Transformed callus cells are selected, typically on a medium 
which inhibits growth in the absence of the marker, and may be regenerated 
into somatic embryos and plantlets which stably incorporate the DNA 
sequence(s). 
The present invention further comprises rose callus cells, somatic rose 
embryos, and rose plantlets which incorporate exogenous DNA sequences. 
Preferably, such transformed cells, embryos, and plantlets are obtained by 
the methods of the present invention. 
The methods of the present invention provide a particularly convenient 
technique for selectively breeding new rose cultivars in a predictable and 
expeditious manner. It is expected that a variety of traits, such as 
color, fragrance, morphology, herbicide resistance, pesticide resistance, 
flower vase life, environmental tolerance, other horticultural traits, and 
may be intentionally introduced into the callus cells and stably 
incorporated into the chromosomes of the regenerated embryos and plantlets 
.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
According to the present invention, genetically transformed rose plants, 
cells, and embryos are obtained by the selective introduction of exogenous 
DNA sequence(s) into the chromosomes of cultured rose callus cells. The 
methods require certain starting materials, including a source of rose 
plant material to produce callus cells, the DNA sequence(s) to be 
introduced, Agrobacterium cells to carry the DNA sequence(s) and mediate 
their transfer to the rose callus cells, and culture media suitable for 
the steps of callus induction, DNA transfer, and embryo and plantlet 
regeneration, as described in much greater detail hereinbelow. Each of the 
necessary starting materials will now be described. 
The following terms, as used in the specification and claims, are intended 
to have the following meanings. 
Somatic embryo: Structures similar to zygotic embryos which arise from 
somatic cells. 
Embryonic: Capable of becoming somatic embryos. In rose calli have surface 
structures (e.g., about 0.5 mm to 1 mm) which are capable of becoming 
embryos. 
Pre-embryogenic: Capable of becoming embryogenic. In rose, these calli are 
friable, whitish-creamish, granular. 
Callus: Undifferentiated cell mass produced usually by culture of different 
organs in vitro. It can be hard, soft, dispersible, compact, spongy, dry, 
watery, or etc. 
Callus Structures: See above. 
Somatic Cell: Any of the body of an organism except the germ cells (sexual 
reproductive cells). 
Rose plant tissue which is used for producing callus cells may be obtained 
from any species of the rose genus, Rosa. Exemplary species include Rosa 
damascena, Rosa multiflora, Rosa gallica, Rosa hybrida, and the like. Of 
particular interest are various cultivars of Rosa hybrida, such as 
Royalty, Frisco, Sonia, and the like. 
The plant tissue used for the production of callus cells may be mature or 
immature, preferably being mature somatic tissue. Suitable immature plant 
tissue can be obtained from in vitro plant tissue culture techniques, such 
as those described in Ammirato et al. (eds), Handbook of Plant Cell 
Culture, vol. 5, McGraw-Hill Publishing Co., New York, 1990, particularly 
at Chapter 29, pages 716-747, the disclosure of which is incorporated 
herein by reference. Callus cells obtained from tissue culture materials 
may be subjected to a "cell suspension" step prior to transformation as 
described below. Such cell suspension comprises suspending the cells in a 
liquid culture medium and shaking the suspension, typically at about 100 
to 500 rpm. In some cases, cell suspension may be useful to the production 
of embryonic cells. 
The preferred mature somatic plant tissues may be obtained from any part of 
the mature rose plant that is capable of producing calli. Suitable plant 
parts include stamen filaments, leaf explants, stem sections, shoot tips, 
petal, sepal, petiole, peduncle, and the like, with stamen filaments and 
leaf explants being particularly preferred. 
Generally, the mature plant tissue sources will be disinfected prior to 
introduction to the callus induction culture. A suitable disinfection step 
comprises an alcohol wash, e.g., with 75% ethanol for about one minute, 
followed by a wash with bleach (10%) and a suitable detergent, e.g., 0.1% 
Tween.RTM., for 20 minutes. The plant materials are then rinsed, usually 
two to three times for about five minutes each time, with sterile, 
deionized water prior to culturing. 
Suitable stamen filaments will have a length from about 0.5 to 1.5 cm, 
preferably being about 1 cm. The stem and leaf sections are preferably cut 
to a size below about 1 cm.times.1 cm, preferably being about 0.5 
cm.times.0.5 cm. Shoot tips will be cut to a length in the range from 
about 0.5 to 3 mm, preferably being about 1 mm in length. 
The exogenous DNA sequences to be introduced will usually carry at least 
one selectable marker gene to permit screening and selection of 
transformed callus cells (i.e., those cells which have incorporated the 
exogenous DNA into their chromosomes), as well as one or more "functional" 
genes which are chosen to provide, enhance, suppress, or otherwise modify 
expression of a desired trait or phenotype in the resulting plant. Such 
traits include color, fragrance, herbicide resistance, pesticide 
resistance, disease resistance, environmental tolerance, morphology, 
growth characteristics, and the like. 
The functional gene to be introduced may be a structural gene which encodes 
a polypeptide which imparts the desired phenotype. Alternatively, the 
functional gene may be a regulatory gene which might play a role in 
transcriptional and/or translational control to suppress, enhance, or 
otherwise modify the transcription and/or expression of an endogenous gene 
within the rose plant. It will be appreciated that control of gene 
expression can have a direct impact on the observable plant 
characteristics. Other functional "genes" include sense and anti-sense DNA 
sequences which may be prepared to suppress or otherwise modify the 
expression of endogenous genes. The use of anti-sense is described 
generally in van der Krol et al., (1990) Mol. Gen. Genet. 220:204-212, the 
disclosure of which is incorporated herein by reference. The use of sense 
DNA sequences is described in various references, including Napoli et al. 
(1990) Plant Cell, 2:279-289 and van der Krol et al. (1990) Plant Cell, 
2:291-299, the disclosures of which are incorporated herein by reference. 
Structural and regulatory genes to be inserted may be obtained from 
depositories, such as the American Type Culture Collection, Rockville, 
Maryland 20852, as well as by isolation from other organisms, typically by 
the screening of genomic or cDNA libraries using conventional 
hybridization techniques, such as those described in Maniatis et al., 
Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y. (1985). Screening may be performed by (1) nucleic 
acid hybridization using homologous genes from other organisms, (2) probes 
synthetically produced to hybridize to particular sequences coding for 
desired protein sequences, or (3) DNA sequencing and comparison to known 
sequences. Sequences for specific genes may be found in various computer 
databases, including GenBank, National Institutes of Health, as well as 
the database maintained by the United States Patent Office. 
The genes of interest may also be identified by antibody screening of 
expression libraries with antibodies made against homologous proteins to 
identify genes encoding for homologous functions. Transposon tagging can 
also be used to aid the isolation of a desired gene. Transposon tagging 
typically involves mutation of the target gene. A mutant gene is isolated 
in which a transposon has inserted into the target gene and altered the 
resulting phenotype. Using a probe for the transposon, the mutated gene 
can be isolated. Then, using the DNA adjacent to the transposon in the 
isolated, mutated gene as a probe, the normal wild-type allele of the 
target gene can be isolated. Such techniques are taught, for example, in 
McLaughlin and Walbot (1987) Genetics, 117:771-776; Dooner et al. (1985) 
Mol. Gen. Genetics, 200:240-246; and Federoff et al. (1984) Proc. Natl. 
Acad. Sci. USA, 81:3825-3829, the disclosures of which are incorporated 
herein by reference. 
Particular genes which may be incorporated into rose callus cells according 
to the method of the present invention include the chalcone synthase gene 
(Napoli et al. (1990) Plant Cell 2 279:289) and the insect resistance gene 
(Vaeck et al. (1987) Nature 328:33). 
The selectable marker gene on the DNA sequences to be inserted will usually 
encode a function which permits the survival of transformed callus cells 
in a selective medium. Usually, the selectable marker gene will encode 
antibiotic resistance, particularly kanamycin resistance, hygromycin 
resistance, streptomycin resistance, chlorosulfuron resistance, (herbicide 
resistance), or the like. The composition of a suitable selective medium 
is described hereinbelow. 
In addition to the "functional" gene and the selectable marker gene, the 
DNA sequences may also contain a reporter gene which facilitates screening 
of the transformed callus cells and, plant material for the presence and 
expression of the exogenous DNA sequences. Exemplary reporter genes 
include .beta.-glucuronidase and luciferase, as described in more detail 
hereinafter. 
The exogenous DNA sequences will be introduced to the callus cells by 
incubation with Agrobacterium cells which carry the sequences to be 
transferred within a transfer DNA (T-DNA) region found on a suitable 
plasmid, typically the Ti plasmid. Ti plasmids contain two regions 
essential for the transformation of plant cells. One of these, the T-DNA 
region, is transferred to the plant nuclei and induces tumor formation. 
The other, referred to as the virulence (vir) region, is essential for the 
transfer of the T-DNA but is not itself transferred. By inserting the DNA 
sequence to be transferred into the T-DNA region, introduction of the DNA 
sequences to the plant genome can be effected. Usually, the Ti plasmid 
will be modified to delete or inactivate the tumor-causing genes so that 
they are suitable for use as vector for the transfer of the gene 
constructs of the present invention. Other plasmids may be utilized in 
conjunction with Agrobacterium for transferring the DNA sequences of the 
present invention to callus cells. 
The construction of recombinant Ti plasmids may be accomplished using 
conventional recombinant DNA techniques, such as those described in 
Maniatis et al., supra. Frequently, the plasmids will include additional 
selective marker genes which permit manipulation and construction of the 
plasmid in suitable hosts, typically bacterial hosts other than 
Agrobacterium, such as E. coli. Suitable selective marker genes include 
tetracycline resistance, kanamycin resistance, ampillcilin resistance, and 
the like. 
The genes within the DNA sequences will typically be linked to appropriate 
transcriptional and translational control sequences which are suitable for 
the rose plant host. For example, the gene will typically be situated at a 
distance from a promoter corresponding to the distance at which the 
promoter is normally effective in order to ensure transcriptional 
activity. Usually, a polyadenylation site and transcription termination 
sites will be provided at the 3'-end of the gene coding sequence. 
Frequently, the necessary control functions can be obtained together with 
the structural gene when it is isolated from a target plant of other host. 
Such intact genes will usually include coding sequences, intron(s), a 
promoter, enhancers, and all other regulatory elements either upstream 
(5') or downstream (3') of the coding sequences. 
Optionally, a binary vector system may be used to introduce the DNA 
sequences according to the present invention. A first plasmid vector 
strain would carry the T-DNA sequence while a second plasmid vector would 
carry a virulence (vir) region. By incubating Agrobacterium cells carrying 
both plasmids with the callus cells, infection of the callus cells can be 
achieved. See, Hoekema et al. (1983) Nature 303:179-180, the disclosure of 
which is incorporated herein by reference. 
Suitable Agrobacterium strains include Agrobacterium tumefaciens and 
Agrobacterium rhizogenes. While the wild-type Agrobacterium rhizogenes may 
be used, the Agrobacterium tumefaciens should be "disarmed," i.e., have 
its tumor-inducing activity removed, prior to use. Preferred Agrobacterium 
tumefaciens strains include LBA4404, as described by Hoekema et al. (1983) 
Nature, 303:179-180, and EHA101 (Hood et al. (1986) J. Bacteriol., 
168:1291-1301. A preferred Agrobacterium rhizogenes strain is 15834, as 
described by Birot et al. (1987) Plant Physiol. Biochem., 25:323-325. 
After the Agrobacterium strain(s) carrying the desired exogenous DNA 
sequence(s) have been prepared, they will usually be cultured for a period 
of time prior to incubation with the rose callus cells. Initially, the 
Agrobacterium may be cultured on a solid media including nutrients, an 
energy source, and a gelling agent. Suitable nutrients include salts, 
tryptone, and yeast extracts, while most sugars are suitable as the energy 
source and the gelling agent can be agar, Gel-rite.RTM., or the like. A 
preferred medium is L-Broth, which is described in detail in the 
Experimental section hereinafter. Usually, medium will include an 
antibiotic to select for Agrobacterium carrying the plasmid DNA sequences. 
The Agrobacterium cells are typically cultured for about one to three days, 
preferably in the dark at about 28.degree. C., and are collected while 
still a white-creamish color, i.e., before browning, typically by being 
scraped off the solid medium. The cells are then suspended in a liquid 
medium, e.g., L-broth, or more preferably in an induction broth containing 
the following components: 
______________________________________ 
Broad Range Preferred 
______________________________________ 
Ammonium chloride 
0.5-3 g/l 1 g/l 
Magnesium sulfate 
0.5-3 g/l 1 g/l 
Potassium chloride 
0.05-2 g/l 0.15 g/l 
Calcium 2-20 mg/l 10 mg/l 
Ferrus sulfate 0.5-10 mg/l 2.5 mg/l 
Phosphate monobasic 
50-1000 mg/l 272 mg/l 
MES 1000-10,000 
mg/l 3904 mg/l 
Glucose 2-30 g/l 5 g/l 
Acetosyringone 10-200 .mu.M 100 .mu.M 
Sucrose 10-30 g/l 20 g/l 
pH 5-7 5.5 
______________________________________ 
The Agrobacterium cells are cultured in the L-broth or induction broth for 
about one to ten hours, preferably from about two to three hours, while 
being agitated, preferably at moderate temperatures from about 20.degree. 
C. to 30.degree. C. 
Rose callus cells which may be transformed according to the method of the 
present invention may be produced as described in copending application 
Ser. No. 542,841, the disclosure of which has previously been incorporated 
herein by reference. Rose tissue is obtained from any of the plant parts 
described above and placed in a callus induction medium including suitable 
nutrients, an energy source, growth regulators, and the like, selected to 
induce callus formation in the plant material. A variety of basal nutrient 
media are known which provide adequate supplies of nitrogen and salts to 
support callus growth, such as White's, B5, N6 and MS medium. Any sugar 
may be employed as energy source. Among the appropriate choices are 
glucose, maltose, sucrose, or lactose, or sucrose in combination with any 
of the named sugars, or mannose. A preferred sugar for this purpose is 
sucrose, at a level of about 10-50 g/l, but molar equivalents of other 
sugars may also be employed. 
Callus induction medium preferably contains at least one auxin and at least 
one cytokinin. The auxins may be any auxin, natural or synthetic, for 
example, indole acetic acid (IAA), naphthalene acetic acid (NAA), 
2,4-dichlorophenoxy acetic acid (2,4-D), picloram, and dicamba. The 
cytokinin may be selected from any of the known cytokinins, natural or 
synthetic, for example 6-benzyladenine (6-BA), zeatin (ZEA), kinetin 
(KIN), and isopentyladenosine (iP). Callus may be induced in the presence 
of several combinations of auxin and cytokinin. However, superior results 
are observed on an induction medium comprising 2,4-D and zeatin. An 
alternate useful combination is NAA with kinetin. Generally, an auxin will 
be present in an amount of about 0.1 to 10 mg/ml, and cytokinin in an 
amount of about 0.2 to 15.0 mg/ml. When the auxin is NAA, the 
concentration in the medium is preferably from about 0.5 to 2.5 mg/l, and 
most preferably about 2.0 mg/l. When 2,4-D is used, the amount is 
preferably from about 0.5 to 10.0 mg/l and most preferably about 2.5 
mg/l. When the cytokinin is kinetin, the concentration in the medium is 
preferably from about 0.5 to 5 mg/l and most preferably about 0.5 mg/l. 
When zeatin is used, the concentration is preferably from about 0.2 to 
12.5 mg/l and most preferably about 1.5 mg/l. Other nonessential 
components may also be added to the medium to optimize callus induction. 
For example, amino acids, such as glycine, may be employed as a nitrogen 
source. In certain embodiments, use of additional growth regulators may be 
helpful in promoting callus induction. For example, addition of abscisic 
acid (ABA), in the amount of about 0.1 to 0.2 mg/l may be useful in callus 
induction, particularly to promote a more globular callus, which leads to 
embryogenic tissue. ABA may be used with all explant sources, but has been 
especially useful with the culture of in vitro leaf explants. 
Those skilled in the art will recognize that other components which are 
frequently employed in plant tissue culture may also be incorporated in 
the callus induction medium. Addition of various vitamins, e.g., MS 
vitamins, White vitamins, nicotinic acid, inositol, pyridoxine or thiamine 
is common. Similarly, for solid media, an appropriate amount of 
solidifying agent, such as agar or Gel-rite.RTM., is also added to the 
mixture. 
The rose tissue is cultured on the callus induction medium for a time 
sufficient to produce at least one callus which serves as a source of 
dispersed callus cells for transformation according to the present 
invention. Typically, tissue may be maintained in the callus induction 
medium for from about three to thirteen weeks, usually from about seven to 
ten weeks, and preferably for about eight weeks, to yield a fast growing 
callus. Initially, callus morphology may be hard, spongy, watery, sandy, 
or globular, and may have a white, cream, or yellow color, depending on 
the particular composition of the medium. The preferred morphology for use 
in the transformation methods of the present invention occurs after from 
about seven to ten weeks, usually at about eight weeks, when the calli 
become highly friable or dispersable with a whitish-creamish color and a 
granular consistency. While cells from calli having these characteristics 
have been found to be most suitable, cells from calli which are hard and 
compact may also be used for transformation by cutting into small 
sections, typically having dimensions of about 2 to 3 mm. 
Calli cultured as just described may be used directly as the source of 
callus cells for transformation or may be subcultured prior to use as a 
starting material. Subculturing allows the continuing maintenance of 
callus cells as a source of starting materials for the method of the 
present invention. 
In order to achieve the desired transformation, the callus material 
described above is incubated with the Agrobacterium cells carrying the 
exogenous DNA sequence to be transferred, typically for about one to four 
days. Incubation is achieved in a cocultivation medium which includes 
nutrients, an energy source, and an induction compound which is selected 
to induce the virulence (vir) region of Agrobacterium to enhance 
transformation efficiency. The induction compound can be any phenolic 
compound which is known to induce such virulence, preferably being 
acetosyringone (AS) present at from about 10 to 200 .mu.M, preferably at 
about 100 .mu.M. Suitable phenolic compounds are described in Bolton et 
al. (1986) Science 232:983-985. 
The preferred cocultivation medium includes sucrose (20 g/l) as the energy 
source, 2,4-D (5 mg/l) as the auxin, and zeatin (1 mg/l) as the cytokinin. 
Gibberellic acid (1 mg/l) is also preferably present as a growth 
regulator. A preferred formulation for the cocultivation medium is N12AS 
set forth in the Experimental section hereinafter. 
Callus cells are combined with the Agrobacterium cells in the cocultivation 
medium at a moderate temperature, typically in the range from about 
20.degree. to 28.degree. C., preferably at about 24.degree. C., from about 
one to four days, usually from about one to two days. The medium is 
preferably kept in the dark, and the cocultivation continued until the 
Agrobacterium have grown sufficiently so that colonies are observable on 
the calli, either directly or through a microscope. 
The Agrobacterium cells are present at a concentration from about 10.sup.7 
to 10.sup.10 cells/ml, preferably at about 10.sup.9 cells/ml. The callus 
cells are present at a ratio of from about 1:1 to about 10:1 (callus 
cells:Agrobacterium cells), preferably at about 3:1, on a volume basis. 
Usually, a total of about 1 to 100 ml of callus material is used, 
preferably about 10 ml, in a total culture volume of about 1 to 100 ml, 
preferably about 10 to 12 ml. Preferably, the callus cells and 
Agrobacterium cells are placed on a filter paper matrix, such as Whatman 
#1, on the cocultivation medium. 
After transformation is completed, the callus cells are washed from the 
Agrobacterium cells with water or a culture medium containing nutrients, 
an energy source, growth regulators, and the like. For smaller callus 
structures, typically in the range from about 0.2 to 0.3 mm in size, use 
of N12 medium (see the Experimental section hereinafter) is particularly 
suitable. For larger callus structures, typically from about 0.4 to 0.7 mm 
in size, use of M53 medium is particularly suitable. 
The transformed calli are mixed with the wash medium, typically at a volume 
ratio of from about 1:3 to about 1:30 (calli:liquid), preferably at about 
1:10, and centrifuged, preferably at 500 rpm for about 5 minutes. The 
resulting liquid fraction containing most of the bacteria is removed, 
while the denser fraction containing the calli is saved. The wash is 
repeated, typically from two to six times, with antibiotics being used in 
at least the later washes in order to kill any remaining Agrobacterium 
cells. Any antibiotic capable of killing Agrobacterium may be used, with 
carbenicillin (200 to 1000 mg/l), vancomycin (100 to 500 mg/l), 
cloxacillin (200 to 1000 mg/l) cefotaxin (200 to 1000 mg/l), and 
erythromycin (200 to 1000 mg/l), being preferred. 
After washing, the calli are placed on a suitable selection medium 
including a plant selection agent which permits identification of 
transformed calli based on the presence of the marker introduced as part 
of the exogenous DNA. Conveniently, the selective media is placed in a 
petri dish with portions of the calli, typically about 100 mg each. The 
selection medium is a general growth medium, such as N12 or M53 (as 
described in the Experimental section hereinafter) supplemented with the 
plant selection agent, and usually including the anti-Agrobacterium 
antibiotic. Suitable plant selection agents include the following. 
______________________________________ 
Concentration of 
Antibiotic Resistance 
Antibiotic Selection Medium 
______________________________________ 
kanamycin 200-500 mg/l 
hygromycin 20-80 mg/l 
spectinomycin 20-80 mg/l 
streptomycin 100-500 mg/l 
chlorsulfuron 0.001-0.05 mg/l 
______________________________________ 
Preferred selection media are N12 and M53 (see Experimental section 
hereinafter) containing no cytokinin or auxins, but having abscisic acid 
added at from about 0.5 to 4 mg/l, preferably at about 2 mg/l. M53 (see 
Experimental section hereinafter) is particularly preferred when the 
callus structures are sized from about 0.4 to 0.7 mm. When kanamycin 
resistance is the selectable marker, N12CK and M53CK (see Experimental 
section hereinafter) are particularly suitable. 
The selection culture will be maintained for a time sufficient to permit 
transformed callus cells to grow and produce white-cream colored calli, 
while the non-transformed callus cells turn brown and die. Typically, the 
selection culture will last from about 25 to 50 days, depending primarily 
on the concentration of the plant selective agent. For example, thirty 
days is generally sufficient for kanamycin at 300 mg/l, while fifty days 
is suitable for kanamycin at 200 mg/l. The primary criterion in ending the 
selection culture, however, is a clear distinction between proliferating 
cells which have been transformed and non-proliferating cells which have 
not been transformed. 
While viability is indicative that the callus cells have been transformed, 
it is usually desirable to confirm transformation using a standard assay 
procedure, such as Southern blotting, Northern blotting, restriction 
enzyme digestion, polymerase chain reaction (PCR) assays, or through the 
use of reporter genes. Suitable reporter genes and assays include 
.beta.-glucuronidase (GUS) assays as described by Jefferson, GUS Gene 
Fusion Systems User's Manual, Cambridge, England (1987) and luciferase 
assays as described by Ow (1986) Science 234:856-859. It will be 
appreciated that these assays can be performed immediately following the 
transformation procedures or at any subsequent point during the 
regeneration of the transformed plant materials according to the present 
invention. 
Following transformation, the calli are transferred to a maintenance medium 
for generation of somatic embryos. This medium contains as its principle 
elements an auxin, a cytokinin, an energy source, and an appropriate 
nutrient medium such as White's or B5 media. The maintenance medium will 
also include an anti-Agrobacterium antibiotic and, usually, ABA or 
gibberellic acid. 
The formulation of the maintenance medium may be adjusted depending on the 
source of somatic tissue. If the mature somatic tissue was obtained from a 
stamen filament or cell suspension culture, the ratio of auxin to 
cytokinin may be decreased by a factor of at least two and up to as much 
as 15 relative to the ratio of auxin to cytokinin present in callus 
induction medium and/or the source of the auxin and cytokinin in the 
regeneration will differ from the source of the auxin and cytokinin in the 
callus induction medium. In a preferred embodiment, a weaker cytokinin and 
auxin is used in the regeneration media than in the induction media and 
selection medium. Specifically, 2,4-D is a stronger auxin, i.e., has a 
greater effect on growth regulation than NAA and zeatin is a stronger 
cytokinin than kinetin. As an example, regeneration of filaments can occur 
in a medium comprising 2,4-D/zeatin at a ratio of 1.3, compared with 
NAA/kinetin at a ratio of 4.0 in callus induction medium. 
If the mature somatic tissue was obtained from a leaf explant, the ratio of 
auxin to cytokinin may be increased relative to the ratio of auxin to 
cytokinin present in callus induction medium and/or the source of the 
auxin and cytokinin in the regeneration medium will differ from the source 
of the auxin and cytokinin in the callus induction medium. As an example, 
regeneration of leaf explants can occur in a maintenance medium comprising 
NAA/KIN at a ratio of 2.0 compared with 2,4-D/zeatin at a ratio of 1.3. 
Preferred maintenance media are M53C (particularly if N12CK was the 
selection medium) and M20C (particularly if M53CK was the selection 
medium). 
The period on maintenance medium for regeneration generally takes about 20 
to 60 days, usually about 30 days. Globular to heart-shaped embryos will 
usually be apparent on the surface of the culture after this time. In many 
cases, the embryos so formed are capable, upon subculture, to give rise on 
their outer surface to secondary embryos. If this secondary embryo 
production is specifically desired, the globular embryos can be 
transferred to fresh regeneration media and cultured from 3 to 6 weeks. 
The somatic embryos produced on the maintenance medium as just described 
can be repeatedly subcultured in order to provide for an increased number 
of transformed embryos. In order to reproduce whole plant material, 
however, it is desirable that the somatic embryos be subjected to a 
maturation process. 
Maturation of somatic embryos is accomplished by transfer of globular 
embryos to a medium comprising nutrients, an energy source, and a growth 
regulator which may include but is not limited to an auxin, a cytokinin, 
abscisic acid, and gibberellic acid. The auxins may be any auxin, natural 
or synthetic, for example, IAA, NAA, 2,4-D, and picloram. The auxin will 
be present in an amount of about 0.1 to 10 mg/ml. The cytokinin may be 
selected from any of the known cytokinins, natural or synthetic, for 
example, 6-BA, ZEA, KIN, and iP. A cytokinin may be present in an amount 
of about 0.2 to 15.0 mg/ml. Abscisic acid may be present in the amount of 
about 0.2 to 2 mg/l. Gibberellic acid may be present in the amount of 
about 0.5 to 5 mg/l. A preferred maturation medium is M20 (see 
Experimental section hereinafter). 
Callus cells are held on the maturation medium with subculturing preferably 
about every 30 days, until mature somatic embryos are obtained. The period 
of maturation generally takes about three to six weeks. Globular embryos 
will appear on the surface of the maturation medium, with many embryos 
giving rise on their outer surface to secondary embryos. If such secondary 
embryo production is desired, the globular embryos can be transferred to 
fresh maintenance medium (as described above) and can be subcultured 
repeatedly in order to provide a greater number of embryos. Such 
subculturing is preferably performed on M20 medium. 
The mature somatic embryos produced as described above are next transferred 
to a germination medium in order to produce germinated embryos. The 
germination medium comprises nutrients and an energy source. The medium 
may further comprise a growth regulator which may include but is not 
limited to a cytokinin, abscisic acid, and gibberellic acid. The cytokinin 
may be present at a concentration of about 0.1 to 1.0 mg/l. Abscisic acid 
may be present in the amount of about 0.2 to 2 mg/l. Gibberellic acid may 
be present in the amount of about 0.5 to 5 mg/l. The germination media 
may also further comprise coconut water at about 5 to 15%, v/v. A 
preferred germination medium is M13. The somatic embryos are held on the 
germination medium for from about 1 to 45 days, usually about 24 days, to 
yield germinated embryos. 
Early stages of embryo germination are characterized by hypocotyl 
elongation, cotyledonary leaves and chlorophyll development. In late 
stages of germination, cotyledonary leaves enlarge, the hypocotyl 
elongates, and a tap root develops. The differentiated embryos may be 
cultured on germination media for about 1 to 4 weeks. The result is 
somatic embryos with shoots 1 to 4 mm in length having from 2 to 4 leaves. 
Optionally, the germinated embryos may be transferred to a shoot elongation 
medium to produce elongated shoots. The medium will include nutrients, an 
energy source, and growth regulators, generally as described above, but 
will have a reduced salt concentration (up to 50% lower) and a reduced 
growth regulator content, preferably BA at 1 to 6 mg/l and IAA at 0.1 to 1 
mg/l. A preferred shoot elongation medium is M13-8 (see Experimental 
section hereinafter). The embryos are maintained in the elongation medium 
until the shoots are about 10 to 20mm in length and develop three to five 
fully green and elongated leaves and stems, typically requiring three to 
four weeks. 
The germinated (and optionally shoot elongated) embryos are subsequently 
transferred to a propagation (or shoot multiplication) medium which 
comprises appropriate nutrients, an energy source, an auxin, and a 
cytokinin. The auxin may be any auxin, natural or synthetic, for example, 
IAA, NAA, 2,4-D and picloram. The auxin will be present in an amount of 
about 0.1 to 10 mg/l. The cytokinin may be selected from any of the known 
cytokinins, natural or synthetic, for example, 6-BA, ZEA, KIN, and iP. A 
cytokinin may be present in an amount of about 0.2 to 15.0 mg/l. In a 
preferred embodiment, the auxin is IAA, present at a concentration of 
about 0.3 mg/l and the cytokinin is 6-BA, present at a concentration of 
about 3.0 mg/l. A preferred propagation or shoot multiplication medium is 
M13 (see Experimental section hereinafter). 
The germinated embryo may be cultured in propagation medium for about 20 to 
200 days, preferably about 30 days. Well developed plantlets may be 
obtained and can be transferred to, for example, artificial soil for root 
regeneration. In one embodiment, multiple shoots can be isolated from one 
single plantlet before transferring to soil. 
Well developed shoots, typically having a length in the range from about 10 
to 40 mm and preferably having from about 5 to 10 leaves, are selected for 
root regeneration. The preferred method for root regeneration is to 
transfer the shoots to be rooted into small pots containing an artificial 
soil, typically saturated with a medium containing root inducing hormones. 
A suitable root induction contains nutrients but is deprived of sugar and 
other energy sources. The medium may further contain thiamine, preferably 
in the form of thiamine-HCl at about 0.5 to 2 mg/l, and an auxin, such as 
IAA at about 1 to 4 mg/l. A preferred root regeneration medium N3-4 (see 
Experimental section hereinafter). While in the pots, the shoots may be 
placed in a container, such as a magenta GA-7 culture container and 
incubated in a growth chamber preferably under a regime of 16 hours light 
per 24 hour period. 
An alternate regeneration method is to dip the shoots in a suitable 
root-inducing hormone, such as RooTone.TM.. The shoots are then placed 
directly in the soil in the greenhouse, preferably being maintained under 
a plastic cover to maintain a high relative humidity. The cover can be 
gradually removed over a period of days in order to cause hardening of the 
shoots. 
With either of the above approaches, roots are typically obtained in about 
7 to 35 days. The rooted shoots can then be transplanted within the 
greenhouse or elsewhere in a conventional manner for tissue culture 
plantlets. 
Transformation of the resulting plantlets can be confirmed by assaying the 
plant material for any of the phenotypes which have been introduced by the 
exogenous DNA. In particular, suitable assays exist for determining the 
presence of certain reporter genes, such as .beta.-glucuronidase and/or 
luciferase, as described hereinabove. Other procedures, such as PCR, 
restriction enzyme digestion, Southern blot hybridization, and Northern 
blot hybridization may also be used. 
The following examples are offered by way of illustration, not by way of 
limitation. 
______________________________________ 
EXPERIMENTAL 
MATERIALS 
Abbreviations/Names 
Source/Reference 
______________________________________ 
ABA; Abscisic Acid 
Sigma Chemical Co., St. Louis, MO, 
USA 
Acetosyringone Aldrich Chemical Co., Milwaukee, 
WI, USA 
Agar; TC Agar Hazleton Biologics, Inc., Lenexa, 
KS, USA 
As; Acetosyringone 
Aldrich Chemical Co., Milwaukee, 
WI, USA 
B-5 Salts Gamborg et al. (1968) Exp. Cell 
Res. 50:151-158 
BA; Benzyl Adenine 
Sigma Chemical co., St. Louis, MO, 
USA 
Bactogar Difco, 
Carbenicillin Geopen, 
2,4-D; 2,4-Dichloro- 
Sigma Chemical Co., St. Louis, 
phenoxyacetic Acid 
MO, USA 
Dropp, a cotton 
Nor-Am Chemical Co., Wilmington, 
defoliant whose 
DE, USA 
active ingredient 
is thidauzuron 
GA.sub.3 ; Gibberellic Acid 
Sigma Chemical Co., St. Louis, MO, 
USA 
G418; Geneticin 
Sigma Chemical Co., St. Louis, MO, 
USA 
Gel-rite .RTM. Scott Lab. Inc., Warwick, RI, USA 
GUS; .beta.-glucuronidase 
IAA; Indole-3-Acetic 
Sigma Chemical Co., St. Louis, 
Acid MO, USA 
IBA; Indole Butyric 
Sigma Chemical Co., St. Louis, 
Acid MO, USA 
Insolitol Sigma Chemical Co., St. Louis, 
MO, USA 
Jiffy Mix Ball Jiffy, Chicago, IL, USA 
Jiffy Pots Ball Jiffy, Chicago, IL, USA 
Kanamycin, Kanamycin 
Sigma Chemical Co., St. Louis, 
Sulfate MO, USA 
KIN, Kinetin Sigma Chemical Co., St. Louis, 
MO, USA 
LUC, Luciferase 
Analytical Luminescence Lab, San 
Diego, CA, USA 
Luciferin, D-Luciferin- 
Analytical Luminescence Lab, 
sodium San Diego, CA, USA 
MES, 2-N Morpholino- 
Sigma Chemical Co., St. Louis, 
ethanesulfonic Acid 
MO, USA 
MS Salts JRH Bioscience, Lenexa, KS, USA 
MS Vitamins Murashige, et al., Physiol. Plant 
(1962) 15:473-97 
N.sub.6 Salts Chu, et al., Scientia Sinica 
(1975) 18:659-668 
NAA, Naphthalene 
Sigma Chemical Co., St. Louis, 
Acetic Acid MO, USA 
Nicotinic Acid Sigma Chemical Co., St. Louis, 
MO, USA 
NPT, Neomycinphospho- 
transferase 
Pyridoxine Sigma Chemical Co., St. Louis, 
MO, USA 
RooTone .TM. Cooke Lab Products, Portland, OR, 
USA 
TDZ, Thidiazuron 
Purified from Dropp by dissolving 
in dimethylsulfoxide and passing 
through a 0.2 .mu.m nylon filter. 
Tetracycline Sigma Chemical Co., St. Louis, MO, 
USA 
Thiamine-HCl Sigma Chemical Co., St. Louis, MO, 
USA 
Triton, TritonX-100 
Sigma Chemical Co., St. Louis, 
MO, USA 
Tryptone Difco-Lab, Detroit, MI, USA 
Tween .RTM. ICI United States, Inc., 
Wilmington, DE, USA 
Vancomycin Sigma Chemical Co., St. Louis, MO, 
USA 
Vitamins Sigma Chemical Co., St. Louis, MO, 
USA 
X-GUS, 5-Bromo-4- 
Diagnostic Chem. Ltd., Monroe, 
chloro-3-Indolyl- 
CT, USA 
.beta.-D-Glucuronide 
Yeast Extract Difco-Lab, Detroit, MI, USA 
Zeatin Sigma Chemical Co., St. Louis, MO, 
USA 
______________________________________ 
______________________________________ 
MEDIA COMPOSITIONS 
______________________________________ 
M13 
MS Salts 1x 
Thiamine HCl 0.5 mg/l 
Inositol 100.0 mg/l 
Pyridoxine 0.5 mg/l 
Nicotine Acid 0.5 mg/l 
Glycine 2.0 mg/l 
BA 3.0 mg/l 
IAA 0.3 mg/l 
Agar 6.0 g/l 
Sucrose 30 g/l 
pH 5.8 
M13-8 
Same except: MS Salts 3/4x 
Pyridoxine 1.5 mg/l 
Nicotinic Acid 
1.5 mg/l 
M20 (alternatively 
M134-20) 
MS Salts 1x 
Thiamine HCl 5 mg/l 
Inositol 100.0 mg/l 
Pyridoxine 1.5 mg/l 
Nicotinic Acid 1.5 mg/l 
Glycine 2.0 mg/l 
GA.sub.3 1.0 mg/l 
ABA 0.2 mg/l 
KAO Vitamins* 1x 
Coconut Water** 10% v/v 
Sucrose 20 g/l 
Gel-rite .RTM. 2.4 g/l 
pH 5.5 
______________________________________ 
*Kao et al., 1975, Planta 126:105 
**Not essential 
M20C 
M20 plus carbenicillin 500 mg/l 
M20K200C 
M20C plus kanamycin 200 mg/l 
M53 
MS Salts 1x 
Thiamine HCl 5 mg/l 
Inositol 20.1 g/l 
Pyridoxine 1.5 mg/l 
Nicotinic Acid 1.5 mg/l 
Glycine 2.0 mg/l 
GA.sub.3 1.0 mg/l 
ABA 2.0 mg/l 
Sucrose 30 g/l 
Gel-rite .RTM. 2.4 g/l1 
pH 5.5 
M53AS 
M53 plus As 100 .mu.M 
M53C 
M53 plus carbenicillin 500 mg/l 
M53CK 
M53C plus kanamycin 300 mg/l 
M130-3 
MS salts 1x 
MS vitamins 1x 
Glycine 2 mg/l 
KIN 0.5 mg/l 
NAA 2 mg/l 
Sucrose 30 g/l 
Gel-rite .RTM. 2.4 g/l 
pH 5.7 
M134-1 
MS Salts 1x 
Thiamine-HCl 5 mg/l 
Inositol 100 mg/l 
Pyridoxine 1.5 mg/l 
Nicotinic Acid 1.5 mg/l 
Glycine 2 mg/l 
Zeatin 1.5 mg/l 
NAA 0.025 mg/l 
GA.sub.3 1 mg/l 
Sucrose 20 g/l 
Gel-rite .RTM. 2.4 g/l 
pH 5.7 
M139 
B-5 salts 1x 
Ammonia Sulfate 329 mg/l 
Thiamine-HCl 5 g/l 
Inositol 100 mg/l 
Pyridoxine 1.5 mg/l 
Nicotinic Acid 1.5 mg/l 
Glycine 2 mg/l 
2,4-D 1.55 mg/l 
Sucrose 30 g/l 
Gel-rite .RTM. 2.4 g/l 
pH 5.6 
M139-2 
M139 modified as follows: 
2,4-D 2.0 mg/l 
Zeatin 1.5 mg/l 
N3-1 
N.sub.6 salts 1/2x 
Thiamine HCl 1.0 mg/l 
Sucrose 20 g/l 
Gel-rite .RTM. 2.2 g/l 
pH 5.6 
N3-4 
N3-1 modified as follows: 
NAA without sucrose 2.0 mg/l 
and Gel-rite .RTM. 
N12 
N.sub.6 salts 1x 
Thiamine HCl 5 mg/l 
Inositol 100.0 mg/l 
Pyridoxine 1.5 mg/l 
Nicotinic Acid 1.5 mg/l 
Glycine 2.0 mg/l 
2,4-D: 5.0 mg/l 
Zeatin 1.0 mg/l 
GA.sub.3 1.0 g/l 
KAO Vitamins 1x 
Sucrose 20 g/l 
Gel-rite .RTM. 2.4 g/l 
pH 5.5 
N12AS 
N12 plus As 100 .mu.M 
N12C 
N12 plus cabenicillin 500 mg/l 
N12CK 
N12C plus kanamycin 300 mg/l 
MinA 
KH.sub.2 PO.sub.4 10.5 g/l 
(NH.sub.4).sub.2 SO.sub.4 
1.0 g/l 
Sodium citrate.2H.sub.2 O 
0.5 g/l 
Agar 15 g/l 
L-Broth* 
Tryptone 10 g/l 
Yeast Extract 5 g/l 
NaCl 5 g/l 
Glucose 1 g/l 
Agar 15 g/l 
______________________________________ 
*pH adjusted to 7.0 to 7.2 using 0.1-5N NaOH, before adding agar; dispens 
at 25 ml/plate. 
METHODS AND RESULTS 
Example 1 
Agrobacterium rhizogenes transformation of rose 
1. Culture tissue on callus induction medium to yield calli. 
Stamen filaments of Rosa hybrida L. var. Royalty (obtained from DeVore 
Nurseries, Watsonville, Calif.) were excised from flower buds of ca. 1.5 
cm long, after a cold pretreatment at 2.degree. C. during 14 days. Buds 
were disinfected with clorox (10%)/Tween.RTM.-20 (0.1%) for 20 mins., 
rinsed three times with sterile deionized water and placed in callus 
induction medium (M130-3). All media were autoclaved for 20 min. at 
24.degree. C. and 15 psi after pH adjustment. Cultures in petri dishes 
were sealed with Parafilm and kept in the dark at 24.degree. C. A 
fast-growing, semi-hard, yellow callus was obtained from filament explants 
after 3 weeks in M130-3. After subculture in this medium, the callus 
changed to a drier appearance. 
The callus was placed in maintenance medium M139. M139 medium improved 
callus quality preventing oxidation and leading to a less compact callus. 
2. Pre-embryogenic callus induction medium and their 
maintenance. 
M139 medium with modified growth regulators 2,4-D (2.0 mg/l) and zeatin 
(1.5 mg/l), was used as pre-embryogenic friable callus induction medium 
(M139-2). Early stages of pre-embryogenic calli were observed after 8 
weeks of callus culture on M139-2 at a frequency of 1.43%. Globular 
structures were subcultured on a proliferation medium, M134. KM-8P 
vitamins (Kao and Michayluk (1975) Planta, 126:105-110) and growth 
regulators were filter sterilized and added into the autoclaved portion of 
proliferation medium. After 3 weeks, a very fast-growing friable, and 
white embryonic tissue with the presence of globular structures was 
produced. Periodic subculture of this tissue on medium maintained its 
capacity to proliferate and to produce globular structures. Such tissue 
was able to be maintained on N12 medium for 8 months. 
3. Agrobacterium rhizogenes culture and preparation. 
Agrobacterium rhizogenes wild-type strain 15834 (Birot et al. (1987) Plant 
Physiol. Biochem. 25:323-325) containing the binary vector pJJ3499 was 
used for transformation. pJJ3499 contains the nopaline synthase promoter 
and neomycin phosphotransferase II (NPT II) gene which confers kanamycin 
resistance as well as the cauliflower mosaic virus 35S promoter. The 
.beta.-glucuronidase gene (Jefferson (1986) Proc. Natl. Acad. Sci. USA 
83:8447-8451) is present as a reporter gene. Strain 15834 alone was used 
as a control inoculum. Bacteria were maintained on L-broth medium 
solidified with 1.5% Bactoagar containing 10 mg/l tetracycline. Bacteria 
were scraped off the solid medium using a loop and suspended in "Induction 
Broth" medium (Winans et al. (1989) J. Bact. 171:1616-1622) containing 100 
.mu.M acetosyringone, and cultured on a shaker (120 rpm) at 28.degree. C. 
for 3 hours. 
4. Cocultivation on cocultivation medium. 
Agrobacterium cells were mixed at the volume ratio of 3:1 (plant 
cell:Agrobacterium cell) with the friable calli selected after 6 months. 
Calli and Agrobacterium were placed on 7.0 cm sterile Whatman #1 filter 
paper circles on the top of cocultivation medium N12 supplemented with 100 
.mu.M acetosyringone. Plates were placed in a 24.degree. C. controlled 
environment incubator in the dark for 48 hours. 
5. Wash. 
Calli were washed from Agrobacterium with the liquid medium N12 
supplemented with 500 mg/l carbenicillin. Calli were mixed well with the 
medium at a volume ratio of 1:10 (calli:medium), centrifuged (500 rpm for 
5 min.), and the supernatant was discarded. Washing was repeated 4 times. 
6. Selection medium. 
After washing, 10-12 chunks (about 100 mg each) of calli were placed and 
spread on selection medium N12CK containing 300 mg/l kanamycin sulfate for 
selection and 500 mg/l carbenicillin to kill off the residual 
Agrobacterium. Tissues remained on this medium for 30 days. At the end of 
the 30 day culture period, most parts of the calli turned brown, however 
one to a few sections of each callus started growing to produce 
white-cream colored calli. 75 out of 81 inoculated calli produced 
kanamycin-resistant calli (Table 1). 
TABLE 1 
______________________________________ 
Recovery of Kanamycin-Resistant Calli on N12 Medium 
Number of 
Chunks Plated 
on Selection 
Number of 
Kanamycin Medium After 
Calli Growing 
Treatment Level (mg/l) 
Cocultivation 
1 month later 
______________________________________ 
Inoculated 
300 81 75 
with 15834 
0 23 23 
(Example 1) 
Inoculated 
300 33 25 
with LBA 4404 
0 12 12 
(Example 2) 
Uninoculated 
300 25 0 
Control 0 15 15 
______________________________________ 
7. Culture on maintenance medium to yield somatic embryos. 
White-cream colored callus tissues were then transferred to N12C medium 
containing 500 mg/l carbenicillin (but no kanamycin) or M53C for 23 days. 
The tissue on N12C was then transferred to medium M53 for three weeks. 
Calli proliferated further on these media and produced larger globular 
structures. 
8. Culture on maturation medium to yield mature somatic embryos. 
The callus tissue from part 7 was subsequently cultured in maturation 
medium M20 for either 8 or 11 weeks. On this medium, mature embryos were 
obtained starting after four weeks and continuing afterwards. Mature 
embryos appeared on structures with wide cotyledons (usually 2 and 
occasional 3 or 4) and very short hypocotyl and radical. The embryos were 
white. The same results were obtained for both the 8 week and 11 week 
culture period. 
9. Culture on germination medium. 
Germination of the matured embryonic tissue was accomplished on M13 medium 
after 2 weeks. Under 16 hr/day light illumination (around 1500 lux) 
tissues became green, cotyledons expanded 5-10 times, and embryos enlarged 
in size 3-5 times and produced 1-5 green shoots. 
10. Culture on shoot multiplication medium. 
Germinated embryos were subcultured on fresh M13 medium. On this medium 
shoots multiplied further, and after 4 weeks, ten to 30 shoots per 
original embryo were produced. 
11. Culture on shoot elongation medium. 
Sections of the shoot clusters were cut off and transferred to M13-8 medium 
with 4-6 shoots per cluster. Shoots elongated to 10-15 cm in size within 
3-4 weeks. 
12. Culture on artificial soil for root regeneration. 
Shoots were cultivated in Jiffy Mix saturated with N3-4 medium. After 6 
weeks, well developed shoots were obtained and were in condition for 
transfer to artificial soil. 
13. Culturing shoots in soil for root regeneration. 
Shoots were dipped in RooTone.TM. and planted in a mix soil (3:1 Super 
Soil: Perlett, Rod McLellan Co., So. San Francisco, Calif., USA) in 
greenhouse and watered as needed. After 3 weeks roots were regenerated and 
complete transgenic plants were obtained. Plants were covered with a 
plastic sheet which was gradually (within 2 weeks) removed to harden off 
the plants. 
14. Results and demonstration of transformation. 
Transformation was confirmed by several means: 1) transformed calli 
transferred onto M20K200C were able to continue their growth, whereas 
nontransformed control calli stopped growth on the medium, turned brown 
and eventually died (Table 2); 2) transformed calli, somatic embryos, and 
leaf sections from transformed shoots all tested positive and 
nontransformed controls tested negative in the GUS assays (Table 3) 
(transformants stained blue and nontransformed tissues did not stain 
blue). 
Leaf callus assays were performed on five transgenic shoots. The medium 
contained 50 mg/l kanamycin to verify that the tissues had been 
transformed. All transformants formed calli in the presence of the 
kanamycin, thus confirming transformation. 
TABLE 2 
______________________________________ 
Assay for Kanamycin Resistance 
of Embryogenic Calli on M20 Medium 
Embryonic Kanamycin # Resistant 
Calli # Calli Level (mg/l) 
Surviving Calli 
______________________________________ 
Putative 
15834- 43 200 43 
Transformed 
40 0 40 
Calli 
(Example 1) 
Putative 
LBA4404- 23 200 23 
Transformed 
10 0 10 
Calli 
(Example 2) 
Untransformed 
25 200 0 
Controls 25 0 25 
______________________________________ 
TABLE 3 
______________________________________ 
GUS Assays.sup.1 
Tissue No. No. Percent 
Materials Tested Positive Positive 
______________________________________ 
Friable cells 
65 65 100 
Embryonic 38 38 100 
Calli 
Somatic 41 40 98 
Embryos 
Shoots 16 16 100 
Plants 2 2 100 
______________________________________ 
.sup.1 Assays performed as described in Jefferson (1987), supra. 
Example 2 
Agrobacterium tumefaciens transformation of rose 
1. Culture tissues on callus induction medium to yield calli. 
Same as Example 1. 
2. Pre-embryogenic callus induction medium and their maintenance. 
Same as Example 1. 
3. Agrobacterium tumefaciens culture and preparation. 
Same as Example 1 except Agrobacterium tumefaciens strain LBA4404 (Hoekema 
et al. (1983), supra.) containing the binary vector pJJ3931 (FIG. 2) was 
used for transformation. pJJ3931 is same as pJJ3499 except that it carries 
the luciferase (LUC) gene (Ow et al. (1986), supra.) instead of GUS, under 
the control of 35S promoter, used as a reporter gene. 
4. Cocultivation on cocultivation medium. 
Same as Example 1. 
5. Wash. 
Same as Example 1. 
6. Selection medium. 
Same as Example 1 except that 25 out of 33 inoculated calli produced 
kanamycin-resistant calli (Table 1). 
7. Culture on maintenance medium to yield somatic embryos. 
Same as Example 1. 
8. Culture on maturation medium to yield mature somatic embryos. 
Same as Example 1. 
9. Culture on germination medium. 
Same as Example 1. 
10. Culture on shoot multiplication medium. 
Same as Example 1. 
11. Culture on shoot elongation medium. 
Same as Example 1. 
12. Culture on artificial soil for root regeneration. 
Same as Example 1, except shoots were cultured in Jiffy Pots saturated with 
N3-4 medium. After four weeks, complete plants were transferred to soil. 
13. Transfer to soil. 
Complete plants were transferred to soil and incubated in a growth chamber 
(16 hr/day light, 16.degree. C. night, 24.degree. C. day temperature) for 
2 weeks. Plants were covered with plastic which was gradually removed over 
2 weeks to harden off the plants. 
14. Results and demonstration of transformation. 
Transformation was confirmed by several means: 1) transformed calli were 
able to continue growth on M20 K200C medium (Table 2) and 2) most 
transformed calli tested positive and non-transformed calli tested 
negative in a LUC assay (Table 4 and FIG. 3) 
TABLE 4 
______________________________________ 
LUC Assay.sup.1 
Tissue 
Materials # Tested % Positive 
% Positive 
______________________________________ 
Friable 15 14 93 
Calli 
Embryogenic 
13 13 100 
Calli 
______________________________________ 
.sup.1 Assays performed as described in Ow (1986), supra. 
Although the foregoing invention has been described in detail for purposes 
of clarity of understanding, it will be obvious that certain modifications 
may be practiced within the scope of the appended claims.