13. Genetic Engineering of Plum (Prunusdomestica L.) for Plant Improvement andGenomics Research in Rosaceae

Cesar Petri, Ralph Scorza, and Chris Dardick

1 Introduction

Fruit trees are among the most recalcitrant of plants to regenerate adventitiousshoots. In most woody fruit species, transformation and regeneration are difficultand often limited to a few genotypes or to seedlings (Petri and Burgos, 2005). Thisfeature is the major limiting factor preventing the development of gene transfertechnologies for fruit trees (Petri and Scorza, 2008). Such barriers slow the abil-

ity to relate the wealth of Prunus structural data, namely from peach. directly to

testable questions of agricultural relevance. However, plumhas been one of themore successful rosaceaous fruits to regenerate and transform, especially among

Prunus species. A number of regeneration protocols have been reported from dif-

ferent tissues of European plum (Prunus (jomestica L.), such as leaf explants (Bassi

and Cossio. 1991: Csányi et al., 1999; Escalettes and Dosba, 1993; Mikhailov andDolgov, 2007; Nowak et al., 2004), or seed-derived tissues (Mante et al., 1989, 1991)

(Table I).Agrobacterwin tumefaciens-mcdiated transformation has been the principal tech-

nique applied to plum. One publication reported the use of Agrobacterifill rhi-

zogenes, but no transgenic shoots were recovered (Escalettes ci al.. 1994). Thereare few reports of regeneration of transformed plum shoots from clonal explantsin European plum (Table 2). In these reports, transformation efficiencies are lowand the varieties used as explant sources are generally of limited or local impor-tance. Furthermore, in most cases, only marker genes were introduced into theplant genOnle. with few reports of modification of agronomically important traits(Escalettes et al.. 1994: Mikhailov and Dolgov, 2007; Mikhailov et al., 2007;

Yancheva ci al., 2002).The most successful transformation/regeneration protocol reported in plum has

been developed in the authors laboratory (USDA. Kearneysville), using embryonic

R. Scone ()Department of Genetic Improvement of Fruit Crops. USDA-ARS-Appalachian Fruit Research Sta-tion. 2217 Wiltshire Road. Kearneysville, WV 25430, USA

K.M. Folta, S.E. Gardiner (eds.), Genetic.v and Geno,nic.s of Rosaceae. Plant Genetics 277

and Genomics: Crops and Models n. ON fl. 1007/978-0- 3)87-7 1 49 1-6-

C Springer Science+Bitsiness Medii. 1.1 .( :fls 5)

278 C. Petri ci at.

Table I Adventitious shoot regeneration in plum (Prunus do,n.sticu)

Regeneration Shoots perVariety/clone Explain (%) explant Rctrence

Besztercei Leaves 32 - Csanyi ci at. (1999)Bluefrec Susina di Dro Leaves 25 17 2.0 2.3 Bassi and Cossin (1991)P.1869 Leaves 12 - Escalettes and Dosha (1993)Startovaya Leaves 80 - Mikliailov and Do!gov (2007)Wegierka Zwykla Leaves 65 10.0 Nowak ci at. (2004)Stanley I lypocotyls 63 6.8 Manic ci at. (1991)B70173 Cotyledons 85 18.7 Mante eta]. (1989)

When not indicated it was non-specified by authors.

hypocotyl slices as the source of explants (Mante et al.. 1991) (Fig. I a). This pro-tocol has been employed successfully for the introduction of agronornically usefulgenes into this species (Callahan and Scorza 2007; Gonzalez-Padilla et at.. 2003;Hily et at.. 2007; Scorza et at.. 1994, 1995: Nagel et al., 2008: Petri et at., 2008).Although transformation of seed-derived material is not an ideal system for improv-ing vegetatively propagated plum scion cultivars, it could have an impact on thedevelopment of new seed-propagated rootstock varieties and the introduction ofnovel genes into the plum germplasm. The high efficiency of the system will alsoenable functional genomics studies.

2 The Plum System

2.1 Description of Methodology

Mante et al. ( 199 1) developed an Agrohacteriu,n-mediated transformation proto-col in plum hypocotyls. This protocol was enhanced by improving the selection180 mg 1 kanamycin (kill) was added Just after co-cultivation] as well as rootingand acclimatization steps and reached 4.2% transformation efficiency (Gonzalez-Padilla et at., 2003). Recently, the addition of 2,4-D during co-cultivation and theoptimization of the timing of each step in the protocol has allowed transformationefficiencies up to 42% and enabled the production of self-rooted transgenic plantsin the greenhouse in approximately 6 months (Petri et at.. 2008).

Figure 1 represents a schematic of the Petri et at. (2008) procedure. Briefly,after the endocarp had been removed with a nutcracker, the seeds were surface-disinfected by immersion for 30 ruin in a 1% sodium hypochiorite solution con-taining approximately 20 p.1 Tween-20 per 100 ml solution and rinsed three timeswith sterile distilled water in a laminar flow bench. Disinfected seeds were soakedin sterile water overnight at room temperaturetemperature and then the seed coats were removedwith the aid of a scalpel. The radicle and the epicotyl were discarded, and thehypocotyl was sliced into three cross sections (0.5—I mm), which were used for

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Fig. I Regeneration ofiransgenic plums. (a) Plum hypocotyl slices, where I. 2 and 3 represents theexplants used in this study. Epicotyl (F) and radicle (R) were not used. Vertical bar indicates 1 mm.(b) Explants co-cultivated with .4çrobacteriuni /uoiefedjeJi,s in regeneration medium (SRM) slip-plemented 2 mg/I 2.4-f) for 3 days (C) Adventitious regeneration from hypocotyl slices in selectivemedium. (d) Putative transgenic shoot cluster after cultivation in shoot growing medium (SGM)supplemented with tinlentin and kanamycin. (e) Shoots rooted in selective rooting medium. (f)Plant introduced in a plastic bag provided with zipper seal. (g) Rooted plantlet maintained in theculture room after the plastic hag is fully opened. (h) Transgenic plants grown in a greenhouse(Figure from Petri Ct al. (2008))

regeneration/transforniation (Fig. In). Media used were described by Gonzalez-Padilla et al. (2003). They were MS medium-based with the following growth reg-ulators: shoot regeneration medium (SRM) contained 7.5 tiM thidiazuron (TDZ)and 0.25 IIM indole butyric acid (IBA), shoot growing medium (SGM) contained3.0 irM benzylaminopurine (BAP) and rooting medium (RM) contained 0.1 1iMkinetin (K) and 5.0 IIM a-naphthalencacetic acid (NAA).

Immediately following the 3 day co-cultivation of hypocotyl segments with A.tuinefaciens on SRM without antibiotics (Fig. I b), the hypocotyl slices were trans-ferred onto SRM with 300 mg l tirnentin (tim) and 80 mg l km. After 5-8 weeks,when shoots began to appear (Fig. Ic), explants were placed onto SGM supple-mented with 300 mg 1' tim and 80 ing I_i km. Following three weeks on SGM,vigorous green shoot clusters were collected, labeled and placed onto fresh media(Fig. Id). Shoot clusters were sub-cultured every 3 weeks on fresh SGM. Whenshoots reached 2-3 cm long they were separated from the cluster and transferredto RM supplemented with 40 mg 1' km and 300 mg I_I tim. In 3-4 weeks, rootsstarted appearing and after an additional 1-3 weeks, shoots were ready for acclima-tization (Fig. le).

For acclimatization, rooted shoots were washed in sterile water to eliminate agarresidues and transferred to 3-inch-square peat pots containing Metro-Mix 5 10 pot-ting medium (SUNGRO Horticulture. Bellevue, WA USA). The potted plantletswere introduced into plastic bags which were sealed and maintained in the tissueculture growth room with a 16/8 h light/dark cycle, light intensity of 45-50 1iEn1 2 s '.and a temperature of 24±1 C (Fig. If). After -2 weeks, the plastic bags werefully opened (Fig. I g). Following 2 weeks in open plastic bags during which timeplants were irrigated with deionized water as needed, the plants were sprayed with

Genetic Inginccriiig ol Plum (1'uiwtt. donie.na L.) lot Plant Inipiovement 25!

an aqueous solution (1:20. v/v) of Anti Stress 2000 (Polymer Au, Fresno, Calif.)and cultured under ambient greenhouse conditions (Fig. I h).

The high transformation rates coupled with the rapid plant establishment method-ology, make possible the application of this technique, not only for the introduction

of agronomically useful genes into P domes/ice but also for high-throughput func-

tional genomic studies.

2.2 New Traits Introduced into Plum

Brief descriptions of successful attempts to genetically modify important traits in

plum using the Agrobacierium-mediated plum hypocotyl transformation system are

described below.

2.2.1 Honeysweet (C5)

In 1989 the USDA laboratory at Kearncysville (WV) began work oil develop-ment of resistance to plum pox virus (PPV) through genetic engineering. The firststudies utilized the papaya ringspot virus (PRV) coat protein (CP) gene, which hadbeen used to develop PRV resistant papayas (Gonsalves. 1998). This virus CP gene

had significant homology to the PPV-CP gene and virus resistance was expected to

he CF-mediated (Bcachy et al., 1990). The heterologous protection against PPV

in plum based on PRV-CP expression was effective for several years in green-

house tests, but after 32 months resistance 'broke down', symptoms were evidentand virus was detected throughout the plant (Scorza et al., 1995). The PPV-CP

gene was then isolated, sequenced, and cloned (Ravelonandro ci al., 1992) and

used for Agrobacteriwn-mcdiated transformation of pluni following the methods ofMante et al. (1991). Again, it was hypothesized that protection would be CP-hased.Transferring the gene into plum, producing the genetically engineered (GE) plants,and propagating them for testing took two years. During the following two years,greenhouse tests for resistance were conducted at the USDA-ARS BSI3-P contain-ment greenhouse at Ft. Detrick. Maryland. One transgenic plum plant that appearedhighly resistant in greenhouse tests did not express PPV-CP (Ravelonandro et al.,

1 997-, Scorza et al., 2001). This clone 'CS'. since patented as 'HoneySwcet', becamethe focus of research on the mechanism and stability of resistance to PPV. While theCS clone appeared to he highly resistant in greenhouse tests. field testing was neces-sary in order to evaluate resistance under typical orchard conditions and in differentgrowing environments. Testing of this resistant clone in areas where PPV was estab-lished was through collaboration with research partners in Europe (Poland, Romaniaand Spain). After appropriate field test permits were granted in each country. fieldtrials were initiated in 1996-1997. which was six to seven years following the initialplum transformations. At USDA-ARS. Kearneysville a field trial was planted undera USDA-Animal and Plant Health Inspection Service (APHIS) permit. This was not

282 C. Petri et al.

to test for resistance since PPV was not present in the U.S. and plants could notbe inoculated in the field, but rather to evaluate the trees for their horticultural traitsincluding growth habit and fruit quality, as well as to initiate risk assessment studies.By 2002 the field tests in Europe clearly demonstrated the resistance of C5 to PPVinfection through aphid vectors and by graft inoculation (Hily et al.. 2004). Con-tinuation of these tests through 2005 further confirmed these findings (Malinowskietal., 2006). Investigations of the mechanism of resistance in CS showed low levelsof transgene mRNA, methylation and production of siRNA specific to the silencedPPV-CP, indicating that resistance was through post-transcriptional gene silencing(PTGS) (Scorzaet al., 2001; Hily et al.. 2004, 2005).

'HoneySweet' was deregulated by API-US in 2006 (Scorza et al., 2007), andapproved it is by the U.S. Food and drug administration (FDA). It is currently beingevaluated by the Environmental Protection Agency (EPA). Additional informa -tion on 'HoneySweet' can be found at www.agbios.com (http://www.agbios.com!dbase.php).

2.2.2 Intron Hairpin PPV-CP

Constructs with self-complementary sequences separated by an intron produce'hairpin' RNA [intronhairpin-RNA (ihpRNA)] structures that efficiently elicitPTGS. In the case of 'Honeysweet' resistance was not produced by an ihpRNAvector, but rather PTGS developed as a result of peculiarities of the insertion event(Scorza et al., 2001)

Hily et al. (2007) designed different intron-hairpin RNA PPV-Cp constructs(Fig. 2c). Transgenic plum lines transformed with the ihpRNA-B14 and ihpRNA-E2 constitutively produced a short (21 nt) and a long (25-26 nt) class of siRNA(Fig. 2a, b). The siRNA accumulation levels in the transgenic clones were found tobe similar to P domesticci CS (Hily et al., 2005), suggesting that the clones wouldbe highly resistant to PPV. Currently, these clones and additional PPV-CP hairpintransformed lines (Petri et al.. 2008) are under evaluation in the greenhouse (Scorza,personal communication).

2.2.3 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase Antisense Plums

Fruit are harvested at a time determined by the handling properties that will yield thehighest quality fruit that can withstand storage and transport. Genetic engineeringefforts have been conducted to improve fruit quality in the market by manipulatinggenes that can affect the rate of softening. To this end, a project to reduce or delaythe amount of ethylene produced by the fruit was undertaken to determine whetherlower ethylene levels resulted in a firmer fruit that could remain in the tree longerto develop more tree-ripened flavors, yet resist damage incurred during harvesting.processing and shipping (Callahan and Scorza. 2007).

Plum hypocotyls were transformed with an antisense construct of a peach ACCoxidase ACCO) gene (the enzyme responsible for the last step in ethylene synthe-sis) under the control of the aMV35S promoter (Gonzalez-Padilla et al., 2003).

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lated Prunus doniestica containing the intron-hairpin-RNA (ihpRNA)-B 14 construct (b) and the

ihpRNA-E2 construct (h). C5 or C3, a resistant and a sensitive transgenic plum, were used aspositive and negative controls respectively for siRNA production. Nucleic acid preparations wereseparated on a 20 denaturing polyacrylamide gel and hybridized with radioactive DNA probescorresponding to both strands of the full-length plum pox virus—coat protein (PPV-CP) sequence(Scorz.a et al.. 1994). Positions of the 22 and 27 nucleotides (nt) markers are indicated at the left.Relative quantification was performed on a 1% nondenaturing agarose gel using ethidium bro-mide (EtBr) staining. (c) ihp-RNA-B14 construct designed by Hily et al. (2007) based on thepHELLSGATE8 vector. (OCS, octopine synthase; Pdk, pyruvate orthophosphate dikinase intron).This construct carries the full length (I kb) of the PPV-CP sequence (black rectangle) driven by

the cauliflower mosaic virus (CaMV) (35S) promoter (Figure from Hily et al. (2007))

Eighteen lines were derived from hypocotyls from 'Bluebyrd'. DNA blots indi-cated that the majority of these lines had single insertions of the peach antisense

ACCO gene (Callahan and Scorza, 2007). Analyses of the data suggested that insome transgenic lines ethylene production and softening was delayed. Sugar lev-els in most of these lines were, on the average, lower than in the non-transformed

'Bluebyrd' (Callahan and Scorza, 2007).

2.2.4 Gastrodianin Antifungal Protein

The Gastrodia anti-fungal protein (GAFP) is a monocot mannose-binding lectin

isolated from the Asiatic orchid Gastrodia elata (Hu et al., 1988). This lectin has

284 C. Pail et ii!.

provided increased resistance in transgenic tobacco against root diseases from dif -ferent phylogenetic lineages (Cox et al., 2006). Agrobacteriwn tuinefticiens medi-ated transformation yielded three GAFP-I expressing plum lines (Prunus domestica'Stanley') designated 4 J, 41, and 5D (Nagel et al., 2008). These lines possessedone, two, and four copies of the GAFP-J gene, respectively, as demonstrated byDNA blotting. Lines 4 J and 41 were not phenotypically different from the non-transformed control line, but line 5D showed significant divergence in leaf mor-phology and growth habit. Compared to the inoculated control line, lines 4 J and41 exhibited increased tolerance to Phvtophthora root rot (PRR), caused by P cm-namomi. When inoculated with the root-knot nematode, Meloidogvne incognita,the 4 J and 41 lines showed a significantly lesser degree of root galling than theinoculated control line, although nematode reproduction in these lines was not sig-nificantly different from the control line. The results of this study suggest that theexpression of GA PP-i in the roots of a woody plant may confer some level of resis-tance to PRR and root-knot nematode. Long-term field trials will be necessary toconfirm these findings in an applied context.

A new construct, where GAFP is under a potato uhiquitin promoter stronglyexpressed in roots, has been engineered. This construct has been introduced, via A.tumefaciens, into tobacco and plum, and currently, plants are under evaluation in thegreenhouse (Schnabel, personal communication).

3 Future Applications

3.1 Functional Genomics

Many aspects of tree biology are common to all plants, and therefore gene func-tion studies for certain genes may be carried out in herbaceous model species suchas A rabidopsis (http://www.nsf.gov/pubs/2002/bioO2O2/model.htm) . Yet, there aremany genes that are unique in their function or expression pattern that are of neces-sity, or are most appropriately, studied in the species of interest or a closely alliedspecies. This is especially true of woody perennial fruit tree species, includingPrunus spp., in which gene function and regulation in tree growth and fruit develop-ment are poorly understood. Research in Rosaceae genomics is generating a largebody of gene sequence data and the analysis of gene function is critical for theapplication of this information to the genetic improvement of rosaceous species.

We developed two ihp constructs using a peach phytoene desaturase (PDS) gene(Fig. 3a, b), to show that the plum system provides a rapid, high throughput systemfor analyzing the function of endogenous Prunus genes (Petri et al.. 2008). Silencingof the function of the PDS gene produces a readily visible photo-bleaching pheno-type (albinism) due to the inhibition of chloroplast development. (Qin et al.. 2007).

After Agmbacteriuin-mediated plum transformation, fifteen clusters displayedthe albino phenotype (Fig. 4a). Gene-silencing efficiencies were 40 and 64C/e forPpPDSI.l and PpPDS2.1 constructs, respectively. PDS mRNA level was highlyreduced (Fig. 4b) in the albino lines and siRNA specific to the PDS sequence wasdetected (Fig. 4c).

Getictic Eneincering of Plum (Piu,iiu /'/ua Li br Plant Inipros cinent 285

PpPDS1 PpPDS2

1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1722

293 792 974 1463

a) Promoter Sense Intron Antisense Terminator35S PDS Pdk PDS OCS

attRl attR2 attR2 attRl

Fig. 3 (a) Schematic of the peach (Prunus peI:vIca) (Pp) phytoenc desaturase (PDS) sequenceillustrating the positions of the two fragments - PpPDS 1 and PpPDS2 that were used to produceintron hairpin (ihp) constructs. (b) Schematic of a PDS ihp contruct that was used to transformtobacco (N. iabacu,n) and plum (P doniestica) (Figure from Petri et al. (2008))

By demonstrating the functionality of peach PDS gene ihp constructs in plum,

we have established that the plum system is amenable for the study of gene function

in Prunus and could be used for other rosaceous species since a high level of syn-teny has been reported (Arfis et al., 2006 Jung et al., 2006). The transformation

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Fig. 4 Silencing of plum phytoene desaturase (PDS) gene expression by peach-based PDS ihpconstructs PpPDS 1.1 and PpPDS2. 1. (a) Plant I control: plants 3.4. 6 7, 8.9. 10 independents PDSsilenced transgenic plum plants showing variable albino phenotype. (b) PDS mRNA accumulation.All transgenic lines showed lower PDS mRNA level than the control. (c) Detection of PDS siRNAin lines 7. 8. 9 and 10. Lines 3. 4 and 6 were not analyzed for PDS siRNA. NT = non tested (Figurefrom Petri et al. (2008))

296 C. Petri Ct ji,

efficiency and time are superior to those br many other tree species (Petri andBurgos, 2005) and comparable to those published for other model woody plants.such as poplar (Popu/us) (Cseke et al., 2007 Song et al.. 2006). Moreover, transfor-mation with genes that promote early flowering in other tree species, such as LEAFY,

APETALA I (Pcfla et at.. 2001), FTJ (Bdhlenius et al.. 2006) or FT2 (Hsu et at.,2006), could reduce the time to flowering in the plum system, enabling rapid evalu-ation of flower and fruit specific gene functions.

Recently, a transformation protocol in Japanese plum (Prunus so/irma) has beenpublished (Urtuhia et al.. 2008). Transformation of P. sa/icina would allow forimprovement of this species through the development of GE varieties and addi-tionally, transformation of this diploid species would provide a platform for func-tional genomics studies that would be useful for all Prunus and Rosaceous species(Urtuhia et al.. 2008).

3.2 Regeneration of Marker-Free Transgenic Plums

In a transformation procedure only a few cells stably incorporate the transgene(s).Marker genes allow for the recognition and selection of transformed cells (Mikiand McHugh, 2004). These marker genes are introduced with the gene of interestand are used for identifying the rare individuals that have taken up foreign DNA.Most plant transformation systems have relied on dominant selectable markers,such as antibiotic and herbicide resistance genes, to enable the recovery of trans-genie plants. Unfortunately, their presence is also often problematic for commercialbiotechnology products because of consumer concerns and regulatory requirementsover the presence of 'excess' exogenous DNA. In Europe, release of plants carry-ing certain antibiotic resistance genes has been particularly problematic (Directive2001/18/EEC of the European Parliament and the Council of the European Union).Therefore, development of procedures to avoid the use of antibiotic selection or toallow elimination of marker genes from the transformed plant should he a researchpriority in the coming years (Petri and Burgos, 2005).

Over the past decade, researchers have developed several approaches for excis-ing marker DNA from transgenie plants and crops (See review Darbani et al..2007). Only multm-autonomou s-trail sformation (MAT)-vector system (Ebi ii u maet al., 1997) have been applied to fruit tree transformation procedures and someresults have been published in apricot (López-No( , ueraetal., 2006. 2007) and citrus(Ballester et al., 2007. 2008).

The high-throughput plum transformation system that has been developed in ourlaboratory (Petri et al.. 2008), represents an alternative system to obtain marker-freetransgenic plum plants under non-selective conditions. Highly efficient transforma-tion systems have been used to develop markerless transformation platforms in cit-rus species (Ballester et at., 2008: Dominguez et al., 2002: Ghorhel et al.. 1999),with transformation efficiencies between 3 and 7% for the different species.

Genetic Engineering of Plum (Primus £/onles!uu L.) for Plant I mprovenicnt 257

3.3 Cultivar Transformation

There are just few reports of regeneration of transformed plum shoots from clonal

explants of P doinestica. Moreover, transformation efficiencies are low and the vari-eties used are generally of limited or local importance (Table 2).

In our laboratory a transformation/regeneration protocol is currently being devel-oped from leaves of P domestica 'Improved French'. 'Improved French', alsoknown as 'French Prune', is perhaps the most economically Important Prunus

doinesrica variety in the world. It is particularly suited to drying and accounts formost of the worlds commercial production of dried plums (or 'prunes'). 'ImprovedFrench' is particularly important in California where it accounts for 99% of the driedplum production, which reached 180,000 tons in 2006 with a production value ofUS $255,684,000 (USDA, 2006), consequently the potential impact of improvingthis cultivar by genetic engineering is significant.

Several factors affecting adventitious regeneration (including shoot proliferationmedium, light, gelling agent. growth regulators, and ethylene inhibitors) were stud-ied. Regeneration rate from 'Improved French' leaves reached tip to 50% (Petri

and Scorza, submitted).Some preliminary transformation experiments have been carried out with three

different disarmed Agrobacterium ruineJiiciens strains (GV3 101 EHA 101 andLBA4404) harboring the pGA482GGi plasmid. This plasniid is a modification ofthe plasmid pGA482GG (An et al., 1985) that includes changes in the multiplecloning site and the addition of an intron in the gu.c gene for plant-specific expres-sion. Gus assays were performed 4 weeks after A. 1uniet iciens infection in order to

determinate stable transformation. Gus positive spots/zones were found in 90.5% of

leaf explants, with an average of 6.8 gus spots/zones per transformed explant. open-ing the possibility for genetic engineering of the most important dried plum cultivarin the USA.

4 Conclusions

Substantialprogress has been made in Primus functional genomics particularlyin peach where physical maps. sequenced EST libraries, and mieroarray plat-forms have been developed (reviewed by Shulaev et al., 2008). The peach genomesequence is also now underway and will lead to the development of even moresophisticated functional genomics tools. However, in the absence of an efficienttransformation system, progress in determining gene function will remain slow. Aslong as peach transformation remains difficult and inefficient. the transgenic plumsystem offers a solution to bottlenecks in Prunus research. Whether the focus of

study is peach, almond, apricot, or cherry. the plum system should serve as a usefulmodel for assessing the functions of individual genes. By combinin g transformationwith short generation cycles through the development of early flowering plum lines,it will he possible to further improve plum as a functional genomics model system

288 C. Petri et al.

r trees. As the plum s ystem becomes further refined, the pace of functional dis-covery for ke y agronomic genes in Primus will rapidly accelerate.

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