Genetic transformation and regeneration of mature

tissues of woody fruit plants bypassing the juvenile stage

MAGDALENA CERVERA, JOSEÂ JUAÂ REZ, ANTONIO NAVARRO,JOSEÂ A. PINA, NURIA DURAÂ N-VILA, LUIS NAVARRO andLEANDRO PENÄ A�Dpto ProteccioÂn Vegetal y BiotecnologõÂa, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado

Oficial, 46113-Moncada, Valencia, Spain (Fax: �34 6 1390240)

Received 15 April 1997; revised 18 August 1997; accepted 25 August 1997

Regeneration and transformation systems from mature plant material of woody fruit species have to be achieved as

a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of

resulting horticultural traits. We report here, for the first time, a procedure for genetic transformation and

regeneration of mature tissues of woody plants that overcomes the long juvenile periods and high heterozygosity

that are characteristic of most of these species. An improved regeneration frequency from mature explants was

obtained by invigoration of the plant material through grafting of mature buds on juvenile seedlings. Co-cultivation

of the explants in feederplates after inoculation with Agrobacterium tumefaciens resulted in enhanced

transformation frequencies. Furthermore, in vitro shoot-tip grafting of the regenerated mature shoots on seedling

rootstocks provided a rapid and efficient system for plant production. Citrus is the most extensively grown fruit

crop worldwide and sweet orange (Citrus sinensis L. Osbeck) accounts for approximately 70% of the Citrus total

production. Mature transgenic sweet orange plants have been obtained, which flowered and bore fruit in 14 months.

Keywords: sweet orange; Citrus; woody; transformation; Agrobacterium; mature

Introduction

Genetic engineering of crop plants is creating a new era inagriculture with the first generation of achievements inherbaceous plants already in commercial use and greaterprospects on the horizon. The potential of geneticimprovement is of great interest for growers of woodyfruit plants. Most commercial varieties are vegetativelypropagated and are hybrids of unknown origin orbudsports that have been selected by growers andessentially cannot be improved by traditional breedingdue to their high heterozygosity. Even when traditionalbreeding may be effective for crop improvement, anothermajor obstacle is the long period of time betweengenerations. Genetic engineering would allow the insertionof specific genes into the unknown genetic background ofcurrent varieties, theoretically adding desirable traitswithout affecting existing horticultural characteristics.

Improvement of woody fruit species through geneticengineering will have limited applications unless tissuefrom mature plants can be readily transformed. Transfor-mation and regeneration of these plants are usuallylimited to juvenile tissues derived from seeds or seedlingorgans such as zygotic embryos, hypocotyls or cotyledons(McGranahan et al., 1990; Mullins et al., 1990; Fitch etal., 1990, 1992, 1993; Hidaka et al., 1990; Mante et al.,1991; Smigocki and Hammerschlag, 1991; Uematsu etal., 1991; Laimer da CaÃmara Machado et al., 1992;Dandekar et al., 1994; Scorza et al., 1994; 1995a,b).Passage through a sexual stage results in a drasticreshuffling of the genome and hence in an alteration ofcultivar properties. In other cases, in which embryogeniccells of somatic origin, somatic embryos or vegetativelypropagated tissues have been used as starting material fortransformation, the explants were juvenile (Fitch et al.,1990; Vardi et al., 1990; Brasileiro et al., 1991; Ruginiet al., 1991; Uematsu et al., 1991; Moore et al., 1992;Janssen and Gardner, 1993; Le Gall et al., 1994;Manders et al., 1994; Martinelli and Mandolino, 1994;

Transgenic Research 7, 51±59 (1998)

0962±8819 # 1998 Chapman & Hall

�To whom correspondence should be addressed.

Kaneyoshi et al., 1994; Nakano et al., 1994; Cabrera-Ponce et al., 1995; da CaÃmara Machado et al., 1995;Krastanova et al., 1995; Mauro et al., 1995; PenÄa et al.,1995a,b; Kikkert et al., 1996; Perl et al., 1996) or wererejuvenated by successive in vitro micropropagation(James et al., 1989; Maheswaran et al., 1992; Atkinsonand Gardner, 1993; Norelli et al., 1994; Yao et al., 1995;De Bondt et al., 1996). Plants regenerated by theseprocedures have juvenile characters and several years ofcultivation are needed before horticultural and commer-cial traits of the transgenic plants can be evaluated.

Careful evaluation of the horticultural characteristics ofmature plants bearing fruits is a standard procedure forreleasing any new fruit variety which might be propagatedin millions of acres. Development of transformationprocedures that could bypass the juvenile phase couldgreatly reduce the time involved in improving andevaluating woody fruit trees by genetic engineering. Thiswould allow the recovery of transformed plants whoseevaluation could be performed rapidly, drastically reducingthe costs and minimizing the time required to becomeavailable for farmers and consumers.

In this report, Citrus has been chosen as a modelwoody fruit tree plant to develop a transformationprocedure for mature tissue of a selected cultivar.Juvenility greatly limits Citrus improvement since mostCitrus species require a long period to lose juvenilecharacters and to bring into production. In the case ofsweet oranges, up to 20 years may be needed to losethese juvenile characters.

We describe here a method that allows genetictransformation and regeneration of mature transgenicsweet orange plants.

Materials and methods

Source and preparation of plant material

Six- to twelve-month-old greenhouse-grown (17±28 8C)sweet orange (C. sinensis L. Osbeck cv. Pineapple)seedlings were used as source of juvenile tissue forregeneration and transformation experiments. In the caseof mature material, buds collected from C. sinensis L.Osbeck cv. Pineapple maintained in a screenhouse of thevirus-free Germplasm Bank Collection of the IVIA weregrafted on seedlings of C. volkameriana Ten & Pasq. atthe greenhouse (18±27 8C). Newly elongated matureshoots were allowed to develop one, two or threeflushes, that were used separately as the source ofmature tissues.

Preparation of explants for regeneration and transfor-mation experiments was the same for both plantmaterials. Stem pieces (20 cm) were stripped of theirleaves and thorns, disinfected for 10 min in a 2% (v:v)sodium hypochlorite solution containing 0.1% (v:v)

Tween-20 and rinsed three times with sterile water.Internodal stem segments (1 cm) in a semi-hardenedstage were cut transversely from the stem pieces.

Regeneration of mature material

The regenerative potential of the three first flushes fromthe newly elongated mature plants as well as materialfrom juvenile plants was compared. Internodal stemsegments were cultured horizontally in MSB3 medium(MS salts (Murashige and Skoog, 1962), White vitamins(White, 1951), 3 mg lÿ1 benzylaminopurine (BAP), 3%sucrose and 1% agar, pH 5.7) (PenÄa et al., 1995a)maintained in the dark for 8 weeks at 26 8C and 60% RHand then exposed to a 16 h photoperiod under anillumination of 45 ìE mÿ2 sÿ1 with no changes in tem-perature and humidity conditions. Regeneration frequencywas evaluated three weeks later as the number ofregenerating explants per total number of culturedexplants.

Agrobacterium wild-type strains and inoculations

Susceptibility of Pineapple sweet orange seedlings to A.tumefaciens was investigated by inoculating 3 differentwild-type strains in the stem of 6-month-old greenhouse-grown seedlings. The following A. tumefaciens strainswere used: C58 (nopaline strain), Ach5 (octopine strain)and A281 (succinamopine strain). Inoculations wereperformed by using log-phase overnight bacterial cultureswith an estimated titer of 4 3 108 cells=ml (A660 � 0:3).

Each bacterial strain was inoculated on ten plants bypracticing three wounds per plant stem with a sterilescalpel and applying 10 ìl of bacterial culture per woundwith a micropipette. The wounds were covered withplastic strips for 15 days. Each wound was analysed as anindependent replicate. Susceptibility to A. tumefacienswas rated on the basis of tumour formation 2 and 5months after inoculation.

Bacterial strain and vector

A. tumefaciens EHA105 containing the binary plasmidp35SGUSINT (Vancanneyt et al., 1990) was used asvector system for transformation. A. tumefaciens EHA105is a disarmed derivative of A. tumefaciens A281 (Hood etal., 1993). Two gene cassettes in the T-DNA, 35S-uidA(GUSINT)-35S and NOS-NPTII-NOS, served respec-tively as reporter and selectable marker genes. The introninserted in the protein-coding region of the uidA geneprevents its expression in A. tumefaciens. Bacteria werecultured overnight in an orbital shaker at 28 8C and200 rpm in LB medium (Sambrook et al., 1989) contain-ing 25 mg lÿ1 kanamycin and 25 mg lÿ1 nalidixic acid.Bacterial cells were pelleted at 3500 rpm for 10 min,resuspended in liquid MSB1 medium (as MSB3, but

52 Cervera et al.

reducing BAP concentration to 1 mg lÿ1) and diluted to107 cells=ml in the same medium.

Transformation of juvenile material

Juvenile internodal stem segments were inoculated withbacteria in liquid MSB1 medium for 15 to 30 min, blotteddry with sterile filter paper and transferred to solid MSB1medium or to tomato (L. esculentum Mill.) feederplatesfor a 3-day co-cultivation period.

Tomato cell suspensions were maintained in TCS liquidmedium (MS salts (Murashige and Skoog, 1962) plustomato vitamins (DuraÂn-Vila et al., 1995), 3% sucrose,2 mg lÿ1 IAA, 1 mg lÿ1 2-ip, 2 mg lÿ1 2,4-D, pH 5.7) in ashaker at 100 rpm and 25 8C, and were subcultured tofresh medium every 15 days. Feederplates were preparedby pipetting 2 ml of 6±7-day-old tomato cell suspensionson the surface of 25 ml of TCS solid medium with asterile Whatman 5 paper on the top in 10 3 1:5 cm(diameter 3 height) plates.

Following cocultivation, the explants were blotted drywith sterile filter paper and transferred to MSB3 mediumcontaining 100 mg lÿ1 kanamycin for selection and250 mg lÿ1 cefotaxime and 250 mg lÿ1 vancomycin tocontrol bacterial growth. The plates were maintained inthe dark for 15 days at 26 8C and then transferred to a16 h photoperiod with 45 ìE mÿ2 sÿ1 of illumination,26 8C and 60% RH. The explants were assayed for GUSexpression after 6 weeks in selective medium to comparetransformation frequencies in both types of co-cultivationmethods. Transformation frequency was evaluated as thenumber of blue spots (transformed cell clusters) perinoculated explant.

Transformation of mature material

Only the first flush from mature plants was used in twoindependent transformation assays. Internodal stem seg-ments (1 cm) from these shoots were inoculated andcocultivated on feederplates as described previously forjuvenile material. Explants were subcultured to freshmedium every 4 weeks.

Regenerated shoots were harvested from the stemsegments and excised in two pieces. The shoot basalends were assayed for GUS activity and the remainingportions were shoot-tip grafted in vitro onto Troyercitrange (C. sinensis L. Osbeck 3 P: trifoliata L. Raf.)seedlings, as described previously (PenÄa et al., 1995a,b).Briefly, Troyer citrange seedlings were decapitatedleaving 1±1.5 cm of the epicotyl. The roots wereshortened to 4±6 cm and the cotyledons and theiraxillary buds were removed. Then, the regenerated shootapical ends were placed on the top cut surfaces of thedecapitated citrange epicotyls, in contact with thevascular ring (Navarro, 1992). After three weeks, thein vitro grafted plantlets were again grafted in the

greenhouse onto vigorous 5-month-old seedlings ofRough lemon (C. jambhiri Lush).

PCR, GUS and NPTII assays

Standard PCR techniques were used to detect NPTII anduidA gene sequences. The uidA primers were 59-GGTGGGAAAGCGCGTTACAAG-39 and 59-TGGATC-CCGGCATAGTTAAA-39, and the NPTII primers were59-GACGAGGCAGCGCGGCTAT-39 and 59-AAGAAG-GCGATAGAAGGCGA-39. Reactions were performedusing 35 cycles of 1 min at 94 8C, 1 min at 55 8C and1.5 min at 74 8C for uidA gene detections and 35 cycles of0.5 min at 94 8C, 0.5 min at 60 8C and 0.7 min at 72 8C forNPTII gene detections. Stem segments, shoots, leaves,flowers and immature fruits were tested for histochemicalGUS activity using X-Gluc as substrate (Jefferson, 1987).Assayed tissues were observed under a stereo microscopeafter overnight incubation at 37 8C. GUS activity in leafsamples was also assessed by measuring the fluorescenceemitted at 445 nm in the hydrolysis of 4-MUG to 4-MU(Jefferson, 1987). NPTII activity in leaf samples wasquantitated by ELISA, using a commercial kit (5Prime! 3 Prime, Inc.).

Ploidy determination and DNA analysis

Ploidy determination of the mature transgenic plants wasperformed on leaves. The plant tissue was processed usinga high resolution DNA kit (PARTEC) and analysed byflow cytometry, using a ploidy analyser (PARTEC PA).DNA was isolated from leaves according to Dellaporta etal. (1983). For Southern analysis, 15 ìg of Eco RI,Hind III or Pst I-digested DNA samples were separated on0.8% agarose gels, blotted to nylon membranes (Hybond-N�, Amersham) and probed with DIG (Boehringer-Mannheim) labelled coding regions of uidA or NPTIIgenes following the supplier instructions.

Results

In vitro regeneration of mature sweet orange plants

The regenerative potential of explants from mature sweetorange plants was assessed prior to the application ofgenetic transformation.

Stem segments from the first, second and third flushesof mature plants were evaluated for their regenerationcapacity in comparison to stem segments from juvenileplants. Results indicated that explants from the first andsecond flushes produced similar regeneration frequencies,significantly higher than that of the explants from thethird flush. As expected, stem segments from juvenileplants produced the highest regeneration frequency (Table1). The first flush of the adult plants was selected as thesource of tissue for genetic transformation experiments.

Genetic transformation of mature woody fruit plants 53

Optimal conditions for transformation of sweet orangeplants

In order to choose a proper A. tumefaciens strain fortransformation, inoculations in stems of greenhouse-grownsweet orange seedlings were performed with three differ-ent wild-type strains. As shown in Table 2, A. tumefaciensA281 produced the earliest and the highest frequency oftumour formation. Therefore, A. tumefaciens EHA105, anon-oncogenic derivative of this strain, was used in thefollowing transformation experiments.

The transformation procedure was first established for

juvenile plant materials and then applied to adultmaterials. Assays were conducted to determine theinfluence of different bacterial concentrations (107 and108 cells=ml) and co-cultivation periods (2, 3 and 5 days)in the transformation frequency. Histochemical GUSanalysis of the stem segments, six weeks after co-cultivation, showed no significant differences in thenumber of stable transformation events (blue spots onthe cut edges) per explant among treatments. The datavaried around 36% of explants showing blue sectors in anaverage number of 2.5 blue spots per explant (treatmentMSB1 on Table 3). Extending the cocultivation time to 5days and=or using the highest Agrobacterium concentra-tion usually resulted in a overgrowth of the bacteria thatwas difficult to control. In the following experiments, abacterial concentration of 107 cells per ml and a 3-dayco-cultivation period were used as standard conditions.

Other assays were performed with juvenile material tocompare the effect of the 3-day co-cultivation period onMSB1 medium or on tomato feederplates. After sixweeks on selection medium, the explants co-cultivated onfeederplates showed the highest transformation frequency,estimated as the number of blue spots per explant and therate of explants with blue spots (Table 3). Therefore, co-cultivation on feederplates was used in further transfor-mation experiments with mature plant material.

Transformation and regeneration of mature sweet orangeplants

After co-cultivation on feederplates, the explants weretransferred onto the selection medium and maintained for15 days in darkness, to stimulate regeneration ofadventitious shoots, before exposure to light. Shootregeneration was observed after 2 to 5 months on selectivemedium. Regenerated shoots 0.2±0.5 cm high (Fig. 1A)were excised from the explants and cut transversely in twopieces. The basal portion of the shoots was assayed forGUS activity (Fig. 1B) and the remaining apical part wasin vitro grafted on Troyer citrange seedlings (Fig. 1C).

In two different experiments, 211 shoots were recov-ered from 294 inoculated mature explants. Thirteenshoots (6.1%) were GUS-positive and three shoots

Table 1. Effect of the flush on the regeneration frequency from

Citrus sinensis mature internodal explants and comparison to

juvenile explants

Regeneration

frequencya Mean of shoots

Material 3 100 per explant � SEc

Juvenile 53 (39)b 4.3 � 0.4 x

Mature 1st flush 23 (38) 2.1 � 0.4 y

2nd flush 18 (38) 1.4 � 0.6 y

3rd flush 5 (39) 1.7 � 1.3 xy

aRegeneration frequency defined as the number of regenerating explants pertotal number of cultured explants.bFigures in brackets represent the total number of evaluated explants afterdiscarding contaminated cultures.cDifferent letters (x, y) indicate significant differences with a P � 0:5%.

Table 2. Comparison of virulence of three different strains of A.

tumefaciens on sweet orange seedlings

Number of Number of

Number of tumours tumours

Strain inoculationsa in 2 months in 5 months

A281 30 20 24

Ach5 30 0 0

C58 30 2 7

Control 15 0 0

aThree inoculations per plant were performed on 10 sweet orange seedlings.The thirty inoculations were considered independent replicates since nosignificant differences at P e 0:05 were observed among upper, inter-mediate and lower inoculations.

Table 3. Effect of tomato feederplates during co-cultivation on the transformation

frequencies

% Explants Maximum=minimum Average number

with blue number of blue of blue spots per

Treatmenta spotsb spots per explant explant � SEc

MSB1 36% 24=0 2.5 � 0.5 x

Feederplates 56% 42=0 4.2 � 1.2 y

aJuvenile stem segments were inoculated with the bacteria for 15±30 minutes and thentransferred to solid MSB1 medium or feederplates for three days.bThese data correspond to the summary of three experiments. Forty explants were analysed ineach experiment. The three experiments were comparable at a confidence level of P e 0:05.cDifferent letters (x, y) indicate significant differences with a P e 0:05.

54 Cervera et al.

Fig. 1. (A) A shoot regenerated from a mature sweet orange explant with the optimum size to be GUS-analysed and micrografted. The

scale bar on this picture represents 1 mm. (B) Histochemical GUS analysis of the basal portions of transgenic regenerated shoots (left) and

control regenerated shoots (right). (C) Three stages of development of transgenic shoots grafted in vitro on Troyer rootstocks. At the right,

the plantlet would be ready for being grafted on a vigorous seedling at the greenhouse.

Fig. 2. (A) Comparison of thorniness among flushes of: a juvenile seedling (up), a transgenic mature plant (middle) and a source mature

plant from which the shoots were taken for transformation experiments (down). (B) Histochemical GUS analysis of flowers from a control

(left) and a transgenic sweet orange plants (right). (C) Histochemical GUS analysis of horizontal sections of flowers from a control (left)

and a transgenic sweet orange plant (right). (D) Histochemical GUS analysis of transversal sections of immature fruits (and ovules) of a

control (left) and a transgenic plant (right). Scale bar on each picture represents 2 mm.

Genetic transformation of mature woody fruit plants 55

showed a sectorial chimaeric GUS expression. Transfor-mation frequencies were comparable in both experiments.

Two weeks after shoot-tip grafting, all 16 putativelytransgenic scions had developed several expanded leaves(Fig. 1C). One of the leaves was then removed for PCRanalysis in order to test for the presence of uidA andNPTII genes in the plantlet's genome. A predictedinternal fragment of either uidA or NPTII genes wasamplified in all of the samples from the 13 GUS-positiveand the 3 chimaeric plantlets (data not shown).

Three weeks after shoot-tip grafting, all of theputatively transgenic and control plantlets were againgrafted at the greenhouse on vigorous 5-month-old Roughlemon seedlings.

GUS expression and phenotype of the mature transgenicplants

In Citrus, the juvenile phase is mainly characterized by aprominent thorniness and vigorous growth. In this stage,Citrus plants never flower. On the contrary, mature plantsare less vigorous, almost thornless and set flowers andfruits. The putative mature transgenic sweet orange plantsshowed morphology and growth habits of an adult plant,as compared to control mature plants. Whereas juvenileplants showed a pronounced thorniness, transgenic matureplants were almost thornless, similar to the mature plantsfrom which the explants were taken for transformation(Fig. 2A). Monthly GUS assays during more than a yearfrom samples of different tissues resulted in uniform bluestainings, except for the chimaeric plants, which alwaysshowed a sectorial staining pattern. No loss or change inGUS expression was observed during this period in any ofthe transgenic plants.

After 14 months in the greenhouse, the transgenic andcontrol plants started to flower, confirming their maturenature. No phenotypic differences were observed amongthe flowers or developing fruits of transgenic and non-transgenic mature plants (Fig. 2B,D).

The flowers from the transgenic plants showed a darkblue colour in sepals, petals, pistil and anthers afterovernight incubation in X-Gluc, but little or no stainingwas seen in peduncle and filaments of stamens (Fig. 2B).To overcome a possible substrate penetration problem,flower horizontal sections were immersed in an X-Glucsolution. GUS activity was also observed in peduncle,receptacle and entire stamens (Fig. 2C). As illustrated ina transversal section of a transgenic immature fruit, oneweek post-anthesis, ovules, as well as the rest of theovary, appeared to be strongly blue stained (Fig. 2D).

Molecular analyses of mature transgenic plants

In order to demonstrate stable integration and expressionof NPTII and uidA gene cassettes, analyses of genomicDNA and protein activities were performed. The results offluorimetric GUS activity and immunological detection of

NPTII protein are shown in Fig. 3. Transgenic plantsexpressed both GUS and NPTII activities. Expressionlevels varied from 6 to 13 ng NPTII per mg total proteinand from 40 to 240 pmol MU per min and ìg total proteinin the analysed transgenic lines.

Flow cytometric analysis showed that the transgenicplants were diploid as well as the source mature plantsfrom which the shoot material was taken for transforma-tion.

Southern blot analysis confirmed the presence of stableintegrated uidA and NPTII gene cassettes into the plant'sgenome (Fig. 4). Digestions with either Hind III or Pst Iresulted in the production of respective internal fragments ofuidA and NPTII cassettes (Fig. 4C) with the expected sizesof 2.8 and 1.6 kb (Fig. 4A,B; lanes 2, 4 and 6). Thecorresponding non-transgenic control sample showed nohybridization signals (Fig. 4A,B; lanes 8). Digestions ofgenomic DNA with Eco RI, that has a unique restriction siteat the end of the uidA cassette next to the left T-DNA border,revealed different integration patterns on the transgenicplants (Fig. 4A,B; lanes 1, 3 and 5): a unique copy of uidAgene and two copies of NPTII gene (lanes 1), two integrationloci for uidA gene and one for NPTII gene (lanes 3) or onlyone full copy of the T-DNA (lanes 5). Additional bands onlanes 6 in Fig. 4A,B might have resulted from partialdigestion of this DNA sample, since only one copy of uidAand NPTII cassettes were detected (lanes 5).

Discussion

Maturation and aging seem to be responsible for theexplant regenerative potential decline found in plant tissueculture of most woody species (Durzan, 1990). For thisreason, juvenile material has been extensively used forgenetic transformation of woody species. When maturematerial with cultivar properties was used for transforma-tion, in vitro micropropagation of the source materialresulted in rejuvenation of the regenerated plants (Jameset al., 1989; Maheswaran et al., 1992; Atkinson andGardner, 1993; Norelli et al., 1994; Yao et al., 1995; DeBondt et al., 1996; James et al., 1996).

We have directly transformed and regenerated maturetissues, bypassing the juvenile stage. This process greatlyshortens the period of time until flowering and bearingfruits and decreases the time to achieve horticulturallyacceptable characteristics by years. Optimization oftransformation and regeneration conditions have led tothe production of transgenic Citrus plants flowering andbearing fruit in 14 months after transferring to thegreenhouse. This is the standard period of time requiredfor mature plants to achieve the adequate size and vigourto flower and set fruits.

The use of a proper Agrobacterium strain as well as co-cultivation on feederplates resulted in increased transfor-mation frequencies. The inclusion of suspension cell

56 Cervera et al.

feederlayers during co-cultivation has been reported toimprove transformation frequencies in some species(Mullins et al., 1990; Janssen and Gardner, 1993; Horschet al., 1985; Stachel et al., 1985; McCormick et al., 1986;Fillatti et al., 1987a,b; Newell et al., 1991), possibly bysupplying A. tumefaciens vir-inducing substances asacetosyringone and other phenolic compounds, by redu-cing the effects of bacterial induced stress or by providinga moist environment beneficial for the explants. We haveachieved a transformation frequency of 6.1% working withmature material, approximately threefold lower than thatobtained for juvenile material following our improvedprocedure (results not shown). This is probably due to thefact that ageing also decreases the susceptibility of plantcells to Agrobacterium infection.

In our laboratory, shoot-tip grafting of transgenicregenerated shoots is being currently used for rapid andefficient production of Citrus transgenic plants (PenÄa etal., 1995a,b), as an alternative to shoot elongation androoting, which is a critical step in in vitro regeneration ofcitrus plants. More than 100 juvenile transgenic plants ofdifferent citrus species have already been produced andtransferred to the greenhouse using this technique.Furthermore, it could be applicable to plant productionfrom transgenic shoots in other woody species, likeapple, plum, apricot, avocado, cherry, peach, pine,Sequoia, etc., in which shoots or plantlets are difficultto elongate and=or root and shoot-tip grafting has beensuccessfully performed (Navarro, 1990).

Improvement of Citrus by conventional breedingmethods is constrained by genetic crossing barriers, suchas self and cross incompatibility, partial apomixis andsterility, high heterozygosity and long juvenile periods

(Soost and Cameron, 1975). The application of biotech-nology and, particularly, genetic transformation to im-provement programmes could circumvent some of theselimitations. We have shown here, for the first time, areliable method for the production of mature transgenicCitrus plants via Agrobacterium. The establishment of asystem that allows high transformation frequencies andhigh rates of production of whole transgenic plants is acrucial step for the rapid and efficient development oftransgenic mature woody plants. We are already usingthis transformation and regeneration system to introducegenes able to confer to Citrus cultivars agronomicallyimportant characters like resistance to citrus tristeza virus(CTV), tolerance to Phytophtora or reduced tree size.Furthermore, we are trying to adapt the developed systemto other woody fruit species.

25

20

15

10

5

0C 1 2 3 4 5 6

250

200

150

100

50

0

NP

TII

activ

ity (

ng N

PT

II/m

g to

tal p

rote

in)

GU

S a

ctiv

ity (

pmol

MU

/min

/µg

tota

l pro

tein

)

Fig. 3. NPTII j and GUS u activity in leaf samples from 6

different Citrus mature transgenic lines (1±6) growing at the

greenhouse. Leaf samples from a non-transgenic plant (C), a

regenerated plant from a mature explant, served as control.

Fig. 4. Southern analysis of DNA isolated from mature sweet

orange plants. Total genomic DNA (15 ìg) was digested with (A)

Eco RI or Hind III and (B) Eco RI or Pst I, subjected to

electrophoresis, blotted and hybridized with a DIG-labelled probe

specific for a fragment of the coding region of the (A) uidA gene

and (B) NPTII gene. (A) Lanes 1, 3, 5 and 7: Eco RI digests, and

lanes 2, 4, 6 and 8: Hind III digests, (B) Lanes 1, 3, 5 and 7:

Eco RI digests, and lanes 2, 4, 6 and 8: Pst I digests. DNA from:

(lanes 1±2) plant 1, (lanes 3±4) plant 2 (lanes 5±6) plant 3 and

(lanes 7±8) a non-transformed regenerated mature plant. ë cut

with Hind III was used as size marker (given in kb). (C) Schematic

representation of the T-DNA of p35SGUSINT, showing the

restriction sites for Eco RI, Hind III and Pst I, the relative sizes

of the uidA and NPTII probes and the expected hybridization

products.

NPT IIprobe

uidAprobeRB

Nos-pro NPT II Nos-ter 35S-pro GUSINT pA

1.6 Kb

Pst I

Hind IIIP

st I

2.8 Kb

Hind III

.5 Kb

EcoR

IP

stI

LB

C

Genetic transformation of mature woody fruit plants 57

Acknowledgements

We thank Dr I.K. Vasil (University of Florida, Gaines-ville), Dr W.O. Dawson (CREC, University of Florida,Lake Alfred) and Dr P. Moreno (IVIA, Spain) forstimulating suggestions and critical reading of the manu-script; Mr M.D. Chilton (Ciba-Geigy) for providing A.tumefaciens strain A281; Dr M.M. LoÂpez for helping withthe A. tumefaciens wild-type inoculations and for provid-ing A. tumefaciens strains C58 and Ach5; Carmen Ortegafor excellent technical assistance and F. Bimbo and N.MunÄoz for illustrations. This research was supported by agrant from the Instituto Nacional de InvestigacionesAgrarias. No. SC97±102 and a grant from the GeneralitatValenciana No. GV-D-AG-01-161-96.

References

Atkinson, R.G. and Gardner, R.C. (1993) Regeneration of

transgenic tamarillo plants. Plant Cell Rep. 12, 347±51.

Brasileiro, A.C.M., Leple, J.C., Muzzin, J., Ounnoughi, D., Michel,

M.F. and Jouanin, L. (1991) An alternative approach for gene

transfer in trees using wild-type Agrobacterium strains. Plant

Mol. Biol. 17, 441±52.

Cabrera-Ponce, J.L., Vegas-Garcia, A. and Herrera-Estrella, L.

(1995) Herbicide resistant transgenic papaya plants produced

by an efficient particle bombardment transformation methods.

Plant Cell Rep. 15, 1±7.

da CaÃmara Machado, A., Puschmann, M., PuÈhringer, H., Kremen,

R., Katinger, H. and Laimer da CaÃmara Machado, M. (1995)

Somatic embryogenesis of Prunus subhirtella autumno rosa

and regeneration of transgenic plants after Agrobacterium-

mediated transformation. Plant Cell Rep. 14, 335±40.

Dandekar, A.M., McGranahan, G.H., Vail, P.V., Uratsu, S.L.,

Leslie, C. and Tebbets, J.S. (1994) Low levels of expression

of wild type Bacillus thuringiensis var. kurstaki cryIA(c)

sequences in transgenic walnut somatic embryos. Plant Sci.

96, 151±62.

De Bondt, A., Eggermont, K., Penninckx, I., Goderis, I. and

Broekaert, W.F. (1996) Agrobacterium-mediated transforma-

tion of apple (Malus 3 domestica Borkh.): an assessment of

factors affecting regeneration of transgenic plants. Plant Cell

Rep. 15, 549±54.

Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983) A plant DNA

minipreparation: Version II. Plant Mol. Biol. Rep. 4, 19±21.

DuraÂn-Vila, N., Carbonell, E.A., PeÂrez Boada, S. and Semancik,

J.S. (1995) Growth of healthy and viroid-infected tomato cells

in vitro. Plant Sci. 105, 111±20.

Durzan, D. (1990) Adult vs. juvenile explants: directed totipotency.

In RodrõÂguez, R., SaÂnchez-TameÂs, R. and Durzan, D.J. eds.,

Plant Aging. Basic and Applied Approaches, NATO ASI

Series. Series A: Life Sciences. Vol. 186. pp. 19±25.

Fillatti, J.J., Kiser, J., Rose, R. and Comai, L. (1987a) Efficient transfer

of a glyphosate tolerance gene into tomato using a binary

Agrobacterium tumefaciens vector. Bio=Technology 5, 726±30.

Fillatti, J.J., Sellmer, J., McCown, B., Haissig, B. and Comai, L.

(1987b) Agrobacterium-mediated transformation and regenera-

tion of Populus. Mol. Gen. Genet. 206, 192±9.

Fitch, M.M.M., Manshardt, R.M., Gonsalves, D. and Slightom, J.L.

(1990) Stable transformation of papaya via microprojectile

bombardment. Plant Cell Rep. 9, 189±94.

Fitch, M.M.M., Manshardt, R.M., Gonsalves, D., Slightom, J.L. and

Sanford, J.C. (1992) Virus resistant papaya plants derived

from tissues bombarded with the coat protein gene of papaya

ringspot virus. Bio=Technology 10, 1466±72.

Fitch, M.M.M., Manshardt, R.M., Gonsalves, D. and Slightom, J.L.

(1993) Transgenic papaya plants from Agrobacterium-

mediated transformation of somatic embryos. Plant Cell

Rep. 12, 245±9.

Hidaka, T., Omura, M., Ugaki, M., Tomiyama, M., Kato, A.,

Ohshima, M. and Motoyoshi, F. (1990) Agrobacterium-

mediated transformation and regeneration of Citrus spp. from

suspension cells. Japan. J. Breed. 40, 199±207.

Hood, E.E., Gelvin, S.B., Melchers, L.S. and Hoekema, A. (1993)

New Agrobacterium helper plasmids for gene transfer to

plants. Transgenic Res. 2, 208±18.

Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eighholtz, D., Rogers,

S.G. and Fraley, R.T. (1985) Transferring genes into plants.

Science 227, 1229±31.

James, D.J., Passey, A.J., Barbara, D.J. and Bevan, M. (1989)

Genetic transformation of apple (Malus pumila Mill.) using a

disarmed Ti-binary vector. Plant Cell Rep. 7, 658±61.

James, D.J., Passey, A.J., Baker, S.A. and Wilson, F.M. (1996)

Transgenes display stable patterns of expression in apple fruit

and mendelian segregation in the progeny. Bio=Technology 14,

56±60.

Janssen, B.J. and Gardner, R.C. (1993) The use of transient GUS

expression to develop an Agrobacterium-mediated gene

transfer system for kiwifruit. Plant Cell Rep. 13, 28±31.

Jefferson, R.A. (1987) Assaying chimeric genes in plants: the GUS

gene fusion system. Plant Mol. Biol. Rep. 5, 387±405.

Kaneyoshi, J., Kobayashi, S., Nakamura, Y., Shigemoto, N. and

Doi, Y. (1994) A simple and efficient gene transfer system of

trifoliate orange. Plant Cell Rep. 13, 541±5.

Kikkert, J.R., Hebert-Soule, D., Wallace, P.G., Striem, M.J. and

Reisch, B.I. (1996) Transgenic plantlets of `Chancellor'

grapevine (Vitis sp.) from biolistic transformation of embryo-

genic cell suspensions. Plant Cell Rep. 15, 311±6.

Krastanova, S., Perrin, M., Barbier, P., Demangeat, G., Cornuet, P.,

Bardonnet, N., Otten, L., Pinck, L. and Walter, B. (1995)

Transformation of grapevine rootstocks with the coat protein

gene of grapevine fanleaf nepovirus. Plant Cell Rep. 14, 550±4.

Laimer da CaÃmara Machado, M., da CaÃmara Machado, A., Hanzer,

V., Weiss, H., Regner, F., Steinkellner, H., Mattanovich, D.,

Plail, R., Knapp, E., Kalthoff, B. and Katinger, H. (1992)

Regeneration of transgenic plants of Prunus armaniaca

containing the coat protein gene of plum pox virus. Plant

Cell Rep. 11, 25±9.

Le Gall, O., Torregrosa, L., Danglot, Y., Candresse, T. and

Bouquet, A. (1994) Agrobacterium-mediated genetic transfor-

mation of grapevine somatic embryos and regeneration of

transgenic plants expressing the coat protein of grapevine

chrome mosaic nepovirus (GCMV). Plant Sci. 102, 161±70.

Maheswaran, G., Welander, M., Hutchinson, J.F., Graham, M.W. and

Richards, D. (1992) Transformation of apple rootstock M26

with Agrobacterium tumefaciens. J. Plant Phys. 139, 560±8.

58 Cervera et al.

Manders, G., Otoni, W.C., d'Utra Vaz, F.B., Blackhall, N.W.,

Power, J.B. and Davey, M.R. (1994) Transformation of

passionfruit (Passiflora edulis fv flavicarpa Degener.) using

Agrobacterium tumefaciens. Plant Cell Rep. 13, 697±702.

Mante, S., Morgens, P.H., Scorza, R., Cordts, J.M. and Callahan,

A.M. (1991) Agrobacterium-mediated transformation of plum

(Prunus domestica L.) hypocotyl slices and regeneration of

transgenic plants. Bio=Technology 9, 853±7.

Martinelli, L. and Mandolino, G. (1994) Genetic transformation

and regeneration of transgenic plants in grapevine (Vitis

rupestris S.). Theor. Appl. Gen. 88, 621±8.

Mauro, M.C., Toutain, S., Walter, B., Pinck, L., Otten, L., Coutos-

Thevenot, P., Deloire, A. and Barbier, P. (1995) High

efficiency regeneration of grapevine plants transformed with

the GFLV coat protein gene. Plant Sci. 112, 97±106.

McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R.

and Fraley, R. (1986) Leaf disc transformation of cultivated

tomato (L. esculentum) using Agrobacterium tumefaciens.

Plant Cell Rep. 5, 81±4.

McGranahan, G.H., Leslie, C.A., Uratsu, S.L. and Dandekar, A.M.

(1990) Improved efficiency of the walnut somatic embryo

gene transfer system. Plant Cell Rep. 8, 512±6.

McGranahan, G.H., Leslie, C.A., Dandekar, A.M., Uratsu, S.L. and

Yates, I.E. (1993) Transformation of pecan and regeneration of

transgenic plants. Plant Cell Rep. 12, 634±8.

Moore, G.A., Jacono, C.C., Neidigh, J.L., Lawrence, S.D. and

Cline, K. (1992) Agrobacterium-mediated transformation of

citrus stem segments and regeneration of transgenic plants.

Plant Cell. Rep. 11, 238±42.

Mullins, M.G., Tang, F.C.A. and Facciotti, D. (1990) Agrobacter-

ium-mediated genetic transformation of grapevines: transgenic

plants of Vitis rupestris Scheele and buds of Vitis vinifera L.

Bio=Technology 8, 1041±5.

Murashige, T. and Skoog, F. (1962) A revised medium for rapid

growth and bioassays with tobacco tissue cultures. Physiol.

Plant. 15, 473±9.

Nakano, M., Hoshino, Y. and Mii, M. (1994) Regeneration of

transgenic plants of grapevine (Vitis vinifera L.) via

Agrobacterium rhizogenes-mediated transformation of embryo-

genic calli. J. Exp. Bot. 45, 649±56.

Navarro, L. (1990) Shoot-tip grafting in vitro of woody species and

its influence on plant age. In RodrõÂguez, R., SaÂnchez-TameÂs,

R. and Durzan, D.J. eds., Plant Aging. Basic and Applied

Approaches, NATO ASI Series. Series A: Life Sciences. Vol.

186. pp. 117±23.

Navarro, L. (1992) Citrus shoot-tip grafting in vitro. In Y.P.S. Bajaj

ed., Biotechnology in Agriculture and Forestry, Springer-

Verlag. Vol. 18. pp. 328±38.

Newell, C.A., Rozman, R., Hinchee, M.A., Lawson, E.C., Haley,

L., Sanders, P., Kaniewski, W., Tumer, N.E., Horsch, R.B. and

Fraley, R.T. (1991) Agrobacterium-mediated transformation of

Solanum tuberosum L. cv. Russet Burbank. Plant Cell Rep.

10, 30±4.

Norelli, J.L., Aldwinckle, H.S., DesteÂfano-Beltran, L. and Jaynes,

J.M. (1994) Transgenic `Malling 26' apple expressing the

attacin E gene has increased resistance to Erwinia amylovora.

Euphytica 77, 123±8.

PenÄa, L., Cervera, M., JuaÂrez, J., Navarro, A., Pina, J.A., DuraÂn-

Vila, N. and Navarro, L. (1995a) Agrobacterium-mediated

transformation of sweet orange and regeneration of transgenic

plants. Plant Cell Rep. 14, 616±9.

PenÄa, L., Cervera, M., JuaÂrez, J., Ortega, C., Pina, J.A., DuraÂn-Vila,

N. and Navarro, L. (1995b) High efficiency Agrobacterium-

mediated transformation and regeneration of citrus. Plant Sci.

104, 183±91.

Perl, A., Lotan, O., Abu-Abied, M. and Holland, D. (1996)

Establishment of an Agrobacterium-mediated transformation

system for grape (Vitis vinifera L.): The role of antioxidants

during grape-Agrobacterium interactions. Nature Biotechnol-

ogy 14, 624±8.

Rugini, E., Pellegrineschi, A., Mencuccini, M. and Mariotti, D.

(1991) Increase of rooting ability in the woody species kiwi

(Actinidia deliciosa A. Chev.) by transformation with

Agrobacterium rhizogenes rol genes. Plant Cell Rep. 10,

291±5.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular

Cloning: a Laboratory Manual, 2nd edition. Cold Spring

Habor, NY, USA: Cold Spring Habor Laboratory Press.

Scorza, R., Ravelonandro, M., Callahan, A.M., Cordts, J.M., Fuchs,

M., Dunez, J. and Gonsalves, D. (1994) Transgenic plums

(Prunus domestica L.) express the plum pox virus coat protein

gene. Plant Cell Rep. 14, 18±22.

Scorza, R., Cordts, J.M., Ramming, D.W. and Emershad, R.L.

(1995a) Transformation of grape (Vitis vinifera L.) zygotic-

derived somatic embryos and regeneration of transgenic

plants. Plant Cell Rep. 14, 589±92.

Scorza, R., Levy, L., Damsteegt, V., Yepes, L.M., Cordts, J.,

Hadidi, A., Slightom, J. and Gonsalves, D. (1995b) Transfor-

mation of plum with the papaya ringspot virus coat protein

gene and reaction of transgenic plants to plum pox virus.

J. Am. Soc. Hort. Sci. 120, 943±52.

Smigocki, A.C. and Hammerschlag, F.A. (1991) Regeneration of

plants from peach embryo cells infected with a shooty mutant

strain of Agrobacterium. J. Am. Soc. Hort. Sci. 116, 1092±7.

Soost, R.K. and Cameron, J.W. (1975) Citrus. In Janick, J. and

Moore, J.N. eds., Advances in Fruit Breeding, West Lafaye-

tete, IN, USA: Purdue University, pp. 507±40.

Stachel, S.E., Messens, E., van Montagu, M. and Zambryski, P.

(1985) Identification of the signals molecules produced by

wounded plant cells that activate T-DNA transfer in

Agrobacterium tumefaciens. Nature 318, 624±9.

Uematsu, C., Murase, M., Ichikawa, H. and Imamura, J. (1991)

Agrobacterium-mediated transformation and regeneration of

kiwi fruit. Plant Cell Rep. 10, 286±90.

Vancanneyt, G., Schmidt, R., O'Connor-SaÂnchez, A., Willmitzer, L.

and Rocha-Sosa, M. (1990) Construction of an intron-

containing marker gene: splicing of the intron in transgenic

plants and its use in monitoring early events in Agrobacter-

ium-mediated plant transformation. Mol. Gen. Genet. 220,

245±50.

Vardi, A., Bleichman, S. and Aviv, D. (1990) Genetic transforma-

tion of citrus protoplasts and regeneration of transgenic plants.

Plant Sci. 69, 199±206.

White, P.R. (1951) Nutritional requirements of isolated plant

tissues and organs. Ann. Rev. Plant Physiol. 2, 231.

Yao, J.L., Cohen, D., Atkinson, R., Richardson, K. and Morris, B.

(1995) Regeneration of transgenic plants from the commercial

apple cultivar Royal Gala. Plant Cell Rep. 14, 407±12.

Genetic transformation of mature woody fruit plants 59