Plant Cell Reports

Plant Cell Reports (1992) 11:25-29

9 Springer-Verlag 1992

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus Margit Laimer da Chmara Machado, Artur da Chmara Machado, Veronika Hanzer, Hans Weiss, Ferdinand Regner, Herta Steinkellner, Diethard Mattanovich, Regina Plail, Elisabeth Knapp, Birgit Kalthoff, and Hermann Katinger University of Agriculture and Forestry, Institute of Applied Microbiology, Nugdorfer L/inde 11, 1190 Vienna, Austria Received August 12, 1991/Revised version received October 11, 1991 - Communicated by H. L6rz

Summary. A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene B-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.

Abbreviations: GUS I~-glucuronidase, PPV Plum Pox Virus, BA 6-benzylaminopurine, NPTII neomycin phosphotransferase II, CP coat protein, CaMV Cauliflower Mosaic Virus, P35S 35S promoter, MS Murashige and Skoog, PCR polymerase chain reaction, P/C/I phenol/chloroform/isoamylalcohol, RNase ribonuclease, dNTP deoxyribonucleosidetriphosphate, DMSO dimethyl sulfoxide.

Introduction The family of potyviruses - the largest and most widely distributed group of plant viruses - is known for its ability to severely damage many important crop species (Hollings and Brunt 1981; Francki et al. 1985). This is also the case for Plum Pox Virus (PPV) with plums, apricots and peaches. Originally described in Bulgaria (Atanassov 1932), the Sharka disease has spread over great parts of Central and Southern Europe as well as over many mediterranean countries. An effective cure for Offprint requests to: M. Laimer da Cgtmara Machado

virus infected trees is not available. Furthermore the transmission of PPV through aphids renders the control of the virus even more difficult. Therefore, the necessity of resistant cultivars is evident. Improvement of fruit trees through traditional breeding methods is a long-term effort because of their long generation time. Thus, new approaches are needed to reach the envisaged breeding goals in a reasonable time. Genetic transformation is potentially useful because specific genetic changes can be made. In the last few years successful examples of resistance breeding against viruses from nine different plant virus families have been reported, using the coat proteinmediated cross protection approach (for review see: Beachy et al. 1990; Gadani et al. 1990). However, fruit trees have not been among these experiments due to difficulties in transformation protocols and lack of available genes. It is obvious that the main obstacle for transformation of fruit tree species is the regeneration of transformed plantlets. Attempts to improve crop plants by genetic engineering techniques will always depend very strongly on the availability of reliable protocols for transformation, selection and regeneration (Horsch et al. 1985; Mc Granahan et al. 19138; Laimer et al. 1989, 1990). Results, which we obtained in preliminary studies with mature embryos during the last few years, did not lead to sufficiently high regeneration rates to allow transformation experiments. Therefore we observed the development of the zygotic embryo of apricot in short time steps to encounter the appropriate time frame for shoot regeneration from cotyledons.

Material and methods Plant material.

Open pollinated immature embryos of Prunus

armeniaca cv. Kecskemeter were collected at day 49, 54, 61, 68, 76,

26 82, 89, 96, 103 and 111 after full bloom. Apricot fruits were washed

kanamycin, supplemented with 20uM acetosyringone and incubated at

under running tap water for about 5 minutes and disinfected with 25 %

28~

Domestos (commercial bleach) for 45 minutes. After 3 washes with

co-cultivation, the suspension was diluted 1:50 in MS culture medium.

with vigorous aeration in a reciprocally shaking water bath. For

sterile water explant preparation was started under sterile conditions. Apricot fruits were split open, the seeds gathered and the seed coats peeled off. Immature embryos were dissected away from the endosperm in the early stages. As soon as development o f the

CAC CAA GCT CCC ATG GCT

LB Pxss

Original Sequence Mutagoni-,ed Sequence

P~ A+

RB

embryos rendered it possible, cotyledons were split apart and the embryonic axis removed. Explants were placed on regeneration

PPV CP

medium A and B respectively with the abaxial surface in close contact

NPT II

(Kin R)

with the medium. The experiments were carded out with 10 cotyledons in each culture medium in 3 parallel assays: 1) untreated

pBinPPVm

control to observe the regeneration processes occuring during the development o f the immature embryo 2) transformation o f cotyledons with the GUS-gene 3) transformation o f cotyledons with the PPV-coat protein gene.

--[] RK2 ori

Culture media. The regeneration media used were basically Murashige

t m R Bact

and Skoog (MS) media (1962) supplemented with 100 mg.1-1 myoinositoi, 20 g.1-1 sucrose and 0.8% purified agar for microbiology

FIGURE

(Merck, no. 1614).

constructed to express the Plum Pox Virus coat protein c-DNA. An

Regeneration

medium

A

additionally

contained

a

1.

Diagramatic representation o f the chimeric gene

hormone

oligonucleotide was designed to engineer a translational start codon

combination used for the apricot cultivar 'Royal'(Pieterse 1989): 1.0

ATG and a NcoI restriction site at the beginning of the coat protein

uM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 uM BA.

gene (change indicated).

Regeneration medium B contained 2.5uM IH-indole-3-butyric acid (IBA) and 7.5uM (1,65 rag/l) N-phenyl-N'-l,2,3,-thidiazol-5-ylurea

Cocultivation with Agrobacterium. Twentyfour hours after culture

(TDZ, Thidiazuron) as growth regulators (Mante et al. 1989). Media

initiation immature embryos were immersed for 5-10 seconds in the

were adjusted to a pH o f 5.6-5.7 with 1 N KOH or 1N HCI and

Agrobacterium suspension and incubated for 48 h on regeneration

autoclaved for 20 minutes. Aliquots of 30 ml were dispensed into 60

medium. Explants were rinsed with sterile half strength MS-medium,

rnm Petri dishes. The cultivation medium C was based on macro- and

blotted dry on sterile filter paper and placed on regeneration medium

microelements of Lloyd and Mc Cown (1981) supplemented with 100

A and B, containing additionally 250 ug/ml carbenicillin to inhibit

mg.1-1 inositol, 20 g.1-1 sucrose and 0,8% agar. The growth

further bacterial growth.

regulators added were 6 uM

2-isopentenyladenine (2iP) and

2.2 uM BA. Aliquots o f 12 ml were dispensed into glass tubes

Analysis of GUS activity. This analysis was performed essentially as

(20xl50mm). The explants were cultured under I00 umol m-2s-1

described (Jefferson et al. 1987). After 3 and 21 days respectively

provided by cool white fluorescent tubes at 24 + / - 2~

with 16 h

tissue samples were harvested and prepared for the histological

photoperiod. Regenerating shoots were excised and subcultured at

enzyme assays. They were exposed to 5-bromo-4-chloro-3-indolyl-B-

intervals o f 3 weeks.

D-glucuronide (X-Glu) for 24

h at 37~

and observed for

development o f indigo dye under the microscope.

Agrobacterium strains and plasmids. Agrobacterium tumefaciens strain LBA 4404 (Hoekema et al. 1983) containing pBinGUSint (Vancanneyt

Plant DNA isolation, PCR and detection by electrophoresis. To show

et al. 1990), carrying the marker gene B-glucuronidase (GUS) and

the integration of a foreign gene in the plant genome, PCR is a

pBinPPVm, carrying the coat protein gene o f Plum Pox Virus (PPV)

powerful technique (Hamill et al. 1990). To detect the coat protein

was used for transformation experiments.

gene of PPV in transgenic stone fruit plants we amplified the gene by

For construction o f the

plant expression vector pBinPPVm the coat protein gene of PPV

PCR using specific primers within the cp-gene and visualized the

(Mattanovich et al. 1988,1991) has been mutagenized in vitro using

product in an agarose gel electrophoresis with ethidium bromide at

the Amersham oligonucleotide directed mutagenesis system (Fig. 1).

0.3ug.m1-1 . Preparation of DNA was performed by grinding the plant

This mutated gene was cloned into pRT 103 (T6pfer et al. 1987) to

material with sand. The homogenized powder was mixed with buffer

connect it to the 35S promoter and terminator from CaMV. Finally the

(15mM EDTA, 50mM Tris/Cl pH 8, I% SDS) and then extracted

entire recombinant gene was cloned into Bin 19 (Bevan 1984). Then

with P/C/I (25:24:1) several times until there was no interphase

we introduced Bin PPVm into Agrobacterium tumefaciens LBA 4404

visible. After RNAse digestion and another P/C/I-extraction DNA was

by electroporation (Mattanovich et al. 1989).

precipitated with ethanol. The primers for the PPV coat protein gene were designed for the position 8958 - 8975 and 8623 - 8640; the

Culture media and growth of bacteria. The Agrobacterium strains

sequences are 5'AGC TCT CGT GTT TGA CAA 3' and 3'GTC AGC

were inoculated from an overnight culture and grown to a density of

CAT ACT GAC CTC 5'respectively.

OD600 =

0.6 in LB medium in the presence of 50 mg.m1-1

27

Results and discussion

(data not shown). Cotyledons taken into culture during the early stages of embryo development (day 49-61) produced shoots not only in reduced number, but also showing a slower development (Fig. 2). For Sundrop apricot 57 days were considered to be the optimum (Lane and Cossio 1986), which differs considerably from our results and might be explained by different regeneration capacity of different genotypes. Use of medium B showed better results than medium A, not only if the number of cotyledons forming shoots was considered but also if the nuber of shoots regenerationg from each cotyledon was compared. This was also reflected in the further development of established shoot primordia. On the other hand from explants between day 68 and 89 on one cotyledon as many as 21 shoot primordia were identified after 3 weeks in culture (Fig. 3). These primordia developed into plantlets, which had to be isolated and subeultured soon after they emerged from the epidermal layers of the cotyledons to avoid their loss due to competition.

Regeneration of apricots from cotyledons of immature embryos

Transformation of apricots with the fl-glucuronidase (GUS) gene.

The observations of cotyledons after 3 and 6 weeks gave the results presented in Table 1. During the development of the zygotic embryo there exists a time frame, where explants might be induced to undergo dedifferentiation and subsequently redifferentiation, which results in a direct shoot regeneration.

The GUS experiment was carried out to gain indications about transformation efficiency, behaviour of the tissue during the co-cultivation with Agrobacterium tumefaciens and the following selection procedures. The obtained data served as guidelines for the handling of presumptive transformants with the PPV-coat protein gene. The first GUS-assay after three days showed a satisfactory transformation of the tissue. The blue dye was well distributed in spots over the whole cotyledonary surface. The observations after 21 days (Fig. 4) revealed a transformation rate of 1-3 primordia per cotyledon, which does not seem to correlate with the originally observed transformation efficiency. Therefore, we assume that untransformed cells are at an advantage compared to transformed tissues, which occurred in even smaller amount. Preliminary experiments had shown the high sensitivity of the Prunus tissues towards kanamycin, which leads to an inhibition of regeneration, if applied from the beginning, even at very low concentrations. Therefore we decided to favour the transformed tissues just at the stage of plant primordia from day 21 on, 'by applying a low dose of kanamycin (35 mg.l-1), which we expected to slow down development of non transformed shoots and consequently decrease competition between shoots on the cotyledons. In order to avoid background problems with GUSactivity caused by agrobacteria persisting in the tissues due to incomplete elimination by the antibiotic treatment (Martin et al. 1990), we decided to use a chimeric gene

Due to the power of the PCR technique it is important to include proper controls in plant transformation experiments, because a very low level of agrobacterial contamination persisting in cultures could give false positive results (tfamill et al. 1990). To assure that the results would not be artefacts produced by endogenous agrobacteria carrying the plasmid pBinPPVm, we constructed primers for the amplification of the bacterial kanamycin resistance gene, which is located outside the T-DNA border regions of pBinl9. The positions of the primers of the bacterial kanamycin resistance gene were 362-381 and 690-709 respectively; the sequences were 5'ATC GGC TCC GTC GAT ACT AT 3' and 3'TG GTG GAT ACT ACA CCT TGC 5'. For each sample we incubated 0.5 ug genomic plant DNA in lx TAQ buffer (10x TAQ buffer: 500 mM KCI, 100 mM Tris-Cl, pH 8.3 (at R.T.) 15 raM MgCI2, 0.1% (w/v) gelatine), 200uM each dNTP, 10pM each primer, 5% DMSO (Pomp, Medrano) with 2 U TAQ DNA Polymerase (USB) and amplified with 30 cycles. A temperature programme with 1' 90~

1' 40~

and 1.5' 72~ was used to allow

DNA replication to occur.

;!i;i:

\ A

\

%

o

3

a

3

2

2

o

B

o 49

TABLE

1.

Plant

54

61

regeneration

68

76

82

from apricot

89

96

cotyledons.

103

11

The

regeneration of shoots from apricot cotyledons taken into culture between days 49 and 111 after full bloom on media A and B were compared. Medium B gave better results than medium A.

The regeneration rate was highest between day 68 and 89 after full bloom, not only concerning the number of cotyledons forming shoots, but also the number of shoots which could be regenerated per cotyledon. Similar results were obtained with Prunus domestica

28 construct which automatically would appearance of false positives. Therefore work with pBinGUSint, which contains (Vancanneyt et al. 1990), thus restricting of the gene to plant cells.

eliminate the we decided to a plant intron the expression

and due to careful selection procedures, it was possible to isolate 41 candidates after 7 months, which we expected to carry the PPV-coat protein gene. Difficulties in the transformation of fruit trees are not only the development of efficient regeneration protocols, but also the establishment of sensitive detection methods for the desired transformation procedure, especially considering the small quantity of

FIGURE 2. Regeneration of shoots from cotyledons during the early stage of development (days 49 to 61).

FIGURE 4. Regeneration of shoots on a cotyledon transformed with pBinGUSint and assayed for GUS after 21 days. The optical evaluation o f the transformation experiment with the GUS-gene allows to distinguish clearly the transformed (stained blue) from the untransformed (pale) tissue segments.

FIGURE 3. Regeneration of shoots from cotyledons during the optimal stage of development (days 68 to 89).

Transformation of apricots with the PPV cp-gene. Due to the high regeneration rate obtained between days 68 and 89, which gave rise to 254 shoots on medium A and 462 shoots on medium B in the control experiment,

FIGURE 5. Transgenic plants of P r u n u s armeniaca carrying the coat protein gene of Plum Pox Virus.

cv. K e c s k e m e t e r

29 tissue ameanable to analysis after transformation and regeneration compared to herbaceous hosts. Therefore we decided to use PCR as a first test method to verify the introduction of PPV coat protein gene into apricot plants (Fig. 5). Figure 6 presents data of four transformants. Plants 1, 3 and 4 show a clear band corresponding to the relevant sequence within the CP gene. The same is evident for the positive control, the plasmid preparation (P) and a transgenic herbaceous plant, Nicotiana benthamiana, carrying the PPV-CP gene. In plant 2 apparently no integration of the gene has occurred. We decided to include a further control (A) by spraying apricot shoots with Agrobacterium tumefaciens LBA4404 carrying pBinPPVm and by preparing DNA in the same manner as we did for the other plant samples. The control using primers for the bacterial kanamycin resistance gene confirmed that there is no DNA from posibly contaminating agrobacteria found by PCR assay.

FIGURE 6. Ethidium bromide stained electrophoresis of PCRproducts to show the integration of the Plum Pox Virus-coat protein gene into apricot plant genome. Primers for samples lett of standard (ST) are within the PPV coat protein gene; primers for samples right of the ST are within the bacterial kanamycin resistance gene, which is located outside the border regions of the T-DNA and should help to detect contamination of agrobacteria. Besides +/- controls, a preparation of the plasmid pBinPPVm (P) was used as a further positive control. Transgenic Nicotiana benthamiana (N) carrying the PPV-coat protein gene was used as a positive control, to detect possible differences between woody and herbaceous tissues following this procedure. We decided to include a further control by spraying tissue of apricot (A) with Agrobacterium LBA 4404 carrying pBinPPVm. Sample preparation was the same as for the transgenic plants. This control, following this preparation procedure, makes sure that any amplification of agrobacterial DNA is avoided. Plants 1, 3 and 4 show a band corresponding to the relevant sequence within the coat protein gene. In plant 2 no integration of coat protein gene has occurred.

References Atanassov D (1932) Yearbook Univ. Sofia, Agronom. Faculty. 11:49 Beachy RN, Loesch-Fries LS, Turner NE (1990) Annual Rev. of Phytopath. 451-474 Bevan M (1984) Nucl.Acids Res. 12:8711-8721 Francki RI, Milne RG, Hatta T (1985) In: Atlas of Plant Viruses Vol. II. CRC Press, Boca Ratoon, Florida, pp 183-217 Gadani F, Mansky l.aM, Medici R, Miller WA, Hill JH (1990) Arch. Virol. 115:1-21 Hamill JD, Rounsley S, Spencer A, Todd G, Rhodes MJC (1990) In: Nijkamp,H.J., van der Plas L, van Aartrijk J (eds.) Progress in plant cellular and molecular biology. Kluwer Acad. Publ., The Netherlands, pp 183-188 Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Nature 303:179-180 Hollings ML, Brunt AA (1981) In: Kurstak E (ed.), Handbook of plant Virus Infections: Comparative Diagnosis. Elsevier, The Netherlands, pp 731-807 Horsch RB, Fry JE, Hoffmann NI, Eichholtz D, Rogers SG, Fraley RRT (1985) Science 227:1229-1231 Jefferson RA, Burgess SM, Hirsh D (1987) EMBO J. 6 (13):39013907 Laimer M, da C~mara Machado A, Hanzer V, Himrnler G, Mattanovich D, Katinger HWD (1989) Acta Hort. 235:85-92 Laimer M, da C~mara Machado A, Mattanovich D, Regner F, Hanzer V, Steinkellner H, Durniok B, Himmler G, Katinger H (1990) Acta Hort. 280:577-580 Lane WD, Cossio F (1986) Can. J. Plant Sci. 66:953-959 Lloyd G, McCown B (1981) Comb. Int. Plant. Prop. Soc. (1980) 30:421-427 Mante S, Scorza R, Cordts JM (1989) Plant Cell, Tissue and Organ Culture 19:1-11 Martin GC, Miller AN, Castle LA, Morris JW, Dandekar AM (1990) J. Amer. Soc. Hort. Sci. 115(4):686-691 Mattanovich D, Himmler G, Laimer M, Maiss E, Regner F, da CSmara Machado A, Hanzer V, Casper R, Katinger H (1988) Virus Genes 2: l 19-127 Mattanovich D, Riiker F, da CSmara Machado A, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H (1989) Nucleic Acids Res. 17:6747 Mattanovich D, Laimer da C~mara Machado M, Regner F, da C~mara Machado A, Himmler G, Hanzer V, Kalthoff B, Steinkelner H, Katinger H (1991) Bio-Engineering 7/2:44-47 McGranahan GH, Leslie CA, Uratsu Sl, Martin LA, Dandekar AM (1988) Bio/Technology 6:800-804 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Pieterse RE (1989) Plant Cell, Tissue and Organ Culture 19:175-179 T6pfer R, Matzeit V, Gronenborn B, Schell J, Steinbiss H-H (1987) Nucl. Acids Res. 15:5890 Vancanneyt G, Schmidt R, O'Connor Sanchez A, Willmitzer L, Rocha Sosa M (1990) Mol. Gen. Genet. 220:245-250

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus.

A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus arme...
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