Recent Advances in Blueberry Transformation

Abstract

Although Vaccinium cultivars have been generated exclusively through the traditional methods of controlled hybridization and deliberate selection, genetic transformation could provide a powerful approach to supplement conventional breeding methods for Vaccinium by introducing genes of interest. A reliable transformation system depends on efficient plant regeneration, reliable gene delivery, and effective selection. To date, in blueberry, adventitious shoot regeneration using leaf explants has been the most desirable regeneration system; Agrobacterium-mediated transformation is the major gene delivery method; and effective selection has been reported using either the nptII or the bar gene as selectable markers. In 2004, stable transformation was reported for four highbush blueberry cultivars, with a transformation frequency ranging from 5.0–15.3%. In 2006, the first field trial of transgenic blueberry with herbicide resistance was performed. In 2009, a blueberry C-repeat binding factor (CBF) gene (GenBank AF234316) was transformed into a southern highbush blueberry cultivar Legacy in order to improve cold tolerance by elucidating the CBF-regulated network in blueberry. This progress has demonstrated that blueberries can be improved through genetic engineering. In the future, as more genomic resources for Vaccinium become available, more genes of interest will be identified and isolated. Genetic transformation will allow us to evaluate these genes as to their functions. Currently, there is industry-wide concern about the application of transformation technology in blueberries, even though numerous transgenic crops have been deployed. Hopefully, new engineering strategies, such as intragenic transformation, RNAi, and marker-free technologies, will reduce public concerns about transgenic Vaccinium plants.

Keywords:

• blueberry
• genetic engineering
• plant regeneration
• transformation
• Vaccinium

INTRODUCTION

Vaccinium is a genus of terrestrial shrubs in the family Ericaceae (Syn. Heath) (Vander Kloet, 1988). It consists of approximately 450 species (Luby et al., 1991; Fang and Stevens, 2005). The most important Vaccinium crop species are found in the sections Cyanococcus, Oxycoccus, Vitis-Idaea, Myrtillus, and Vaccinium. Vaccinium fruits are health-promoting foods because of their relatively high antioxidant and anti-inflammatory capacities (Prior et al., 1998; Ehlenfeldt and Prior, 2001; Conner et al., 2002a, 2002b; Zheng and Wang, 2003; USDA-ARS, 2007).

Three Vaccinium fruit crops (blueberry, cranberry, and lingonberry) have been domesticated in the 20th century (Galletta and Ballington, 1996; Lyrene et al., 2003; Hancock et al., 2008). Of these, the highbush blueberry is by far the most important commercial crop. From 1995 to 2007, worldwide blueberry acreage grew from 23,116 ha to 58,601 ha (Lehnert, 2008). According to the latest ARS report from the USDA, cranberries and blueberries rank number one and two, respectively, in antioxidant values among 19 common fruits (USDA-ARS, 2007). Demand for berries from the various Vaccinium species (blueberries, cranberries, bilberry, and lingonberry) will likely continue to grow in the near future because of their nutritional and therapeutic properties (Prior et al., 1998; Sun et al., 2002; Ferguson et al., 2006; Neto, 2007a, 2007b; Neto et al., 2008).

Conventional breeding of Vaccinium species by germplasm selection and hybridization is usually time-consuming and labor-intensive. Traditional breeding approaches usually take over 10 years to produce finished cultivars. A major limitation is that not every desirable trait can be easily found in the natural germplasm pool. In addition, the multiple gene exchanges involved in intra- or inter-specific hybridizations lead to transfer of not only desirable but also undesirable genes to progeny; thus, the unique traits that make a variety special can be hard to regain after hybridization without the simultaneous inclusion of negative traits from the other parent.

Plant biotechnological approaches have been developed that can supplement and extend conventional breeding methods for cranberry (Serres et al., 1992, 1997a, 1997b; Polashock and Vorsa, 2002a, 2002b; Zeldin et al., 2002) and blueberry (Graham et al., 1996; Song and Sink, 2004; Song et al., 2008). For the clonally-propagated Vaccinium species, micropropagation is a powerful tool, not only for the rapid scale-up of newly introduced cultivars, but also for establishment of an in vitro genebank of Vaccinium sp. Plant regeneration is a requisite step in methodologies for stable transformation of Vaccinium. Transformation allows manipulation of individual genes of interest. In this review, we will summarize recent progress in plant regeneration and transformation of blueberry. New strategies for engineering Vaccinium species will also be discussed.

REGENERATION

Success in plant regeneration is usually influenced by biotic factors, including genotype and explant type, and abiotic factors, such as culture media and environmental conditions. Previous reports on blueberry plant regeneration are summarized in Table 1. Most of the regeneration observed to date has occurred via organogenesis, although there is evidence that levels of adventitious shoot regeneration can be improved in blueberry.

TABLE 1 Literature survey on regeneration of blueberry

Species	References	Medium	Results and comments
V. angustifolium Ait.	Nickerson and Hall, 1976	Murashige and Skoog (1962) medium (MS) + 2,4-dichorophenoxyacetic acid (2,4-D)	Calluses were induced from internode sections.
	Nickerson, 1978	Anderson's medium (1975) (AN) + 75 μM 2ip + 23 μM indoleacetic acid (IAA)	Adventitious shoots up to 12 mm tall (5–7 per explant) were developed on 60% of the cotyledons and 80% of the hypocotyl sections within 6 weeks.
	Nickerson, 1980	Nitsch and Nitsch's (1969) medium + 0.47 μM kinetin	Roots were induced from calluses.
	Dweikat and Lyrene, 1988	Knop's (1865) medium + 24.6 μM 2ip	Shoot number averaged 42.6 per explant.
V. corymbosum L.—Highbush blueberry	Billings et al., 1988	Woody plant medium (Lloyd and McCown, 1981) (WPM) + 2ip at 15 μM for ‘Bluehaven’ or 10 μM for ‘Berkeley’	Shoot regeneration frequency was 70% for ‘Berkeley’ and 100% for ‘Bluehaven’. Mean number of shoots per explant was 7.0 and 10.7 for ‘Berkeley’ and ‘Bluehaven’.
	Callow et al., 1989	½ MS + 25 μM 2ip	38.2% of the leaf explants from ‘Bluecrop’ produced at least one shoot; shoots emerged mainly from the midrib and wounded areas.
	Rowland and Ogden, 1992, 1993	WPM [modified as follows (mg/L): 684 Ca(NO3)2·4H2O, 190 KNO3, 73.4 C10H13FeN2NaO8, 0.1 thiamine-HCl, and not included K2SO4, CaCl2, FeSO4, and Na2EDTA] + 20 μM zeatin riboside	Under the most optimal conditions, ‘Sunrise’ had a regeneration frequency of 88% and 21 shoots/explants. However, no regeneration occurred on ‘Bluecrop’ and ‘Duke’. Zeatin riboside was more effective than zeatin and 2ip in promoting shoot regeneration from leaf explants.
	Hruskoci and Read, 1993	Zimmerman and Broome (1980) (Z-2) + 25 μM zeatin or 25 μM thidiazuron (TDZ)	Zeatin was more effective than TDZ in promoting shoot regeneration from internode explants.
	Cao and Hammerschlag, 2000	Modified WPM (Rowland and Ogden, 1992) + 20 μM zeatin riboside or TDZ at 1 or 5 μM	100% of the leaf explants of ‘Duke’, ‘Geogiagem’, ‘Sierra’, and ‘Jersey’ produced shoots (13.0, 13.0, 12.6, and 4.6 shoots/explant). ‘Bluecrop’ regenerated poorly.
	Cao et al., 2002; orCao and Hammerschlag, 2000	Pretreatment (PT): PT1: N6 macro salts + LS micro salts + TDZ 5 μM + NAA 1.1 μM + 3% sucrose + 300 mg/L casein hydrolysate, pH 5.4; PT2: Modified WPM (Rowland and Ogden, 1992) + 20 μM zeatin riboside + NAA 2.5 μMRegeneration: Modified WPM + 5 μM TDZ	A maximum of 98% of explants regenerated shoots with a mean of 11 shoots/explant.
	Song and Sink, 2004	Regeneration medium for ‘Aurora’, ‘Brigitta’, and ‘Elliott’: Modified WPM (Rowland and Ogden, 1992) + 5 μM TDZ + 1.1 μM NAA; Regeneration medium for ‘Bluecrop’ and ‘Legacy’: Modified WPM + 24.6 μM 2ip + 9.1 μM zeatin Regeneration medium for ‘Duke’: Modified WPM + 1 μM TDZ	Adventitious shoot regeneration occurred for all six cultivars at high frequencies (86.7–100%); optimal regeneration conditions varied among the cultivars. TDZ and NAA were effective in promoting shoot regeneration. Those cultivars that easily produced shoots from the buds of stem pieces placed horizontal on stock culture medium also regenerated shoots most readily from leaf explants.
	Meiners et al., 2007	WPM + 20 μM zeatin	20 μM zeatin was effective in promoting shoot regeneration of ‘Ozarkblue’.
	Liu et al., 2010	WPM containing TDZ (4.54 or 9.08 μM), zeatin (18.2 μM), or zeatin riboside (5.7 or 11.4 μM) either separately or in combination with NAA at 2.69 μM	The optimum medium for shoot regeneration of four southern highbush cultivars was genotype-dependent; the physiology of leaf explants appeared to be important for shoot regeneration.

Explants

While the Vaccinium leaves harvested from in vitro plants are generally small, they are easily obtained and are readily regenerated. Nickerson (1978) first reported on lowbush blueberry regeneration from hypocotyl or cotyledon sections; however, shoot regeneration from seedling explants is less preferable to that from leaf explants because Vaccinium crops are clonally propagated. Most attempts now focus on regenerating adventitious shoots from leaf explants.

Media and Plant Growth Regulators

As summarized in Table 1, WPM is the most common basal medium used for regeneration of Vaccinium species. The cytokinin 6-(γ,γ–dimethylallylamino)-purine (2ip) was the first hormone evaluated to promote shoot regeneration from leaf explants since it was already effective for blueberry micropropagation. On WPM supplemented with 15 μM 2ip, 70–100% of leaf explants from ‘Berkeley’ and ‘Bluehaven’ regenerated shoots (Billings et al., 1988). On half strength MS medium plus 5 to 25 μM 2ip, adventitious shoots were produced on leaf explants of ‘Bluecrop’, ‘Bluejay’, and ‘Jersey’. Rowland and Ogden (1992) found that zeatin riboside was more effective than either of the cytokinins, 2ip and zeatin, in promoting shoot regeneration for leaf explants of ‘Sunrise’; whereas, ‘Bluecrop’ and ‘Duke’ produced no shoots on any of the media tested. Thidiazuron (TDZ) at 1 or 5 μM, or zeatin riboside at 20 μM improved adventitious shoot organogenesis from leaf explants of five highbush cultivars (Cao and Hammerschlag, 2000; Cao et al., 2002).

When shoot regeneration of ten highbush cultivars was evaluated in medium containing different plant growth regulators, the optimum treatment(s) varied among highbush cultivars (Song and Sink, 2004; Liu et al., 2010). TDZ and NAA were most effective in promoting adventitious shoot formation for eight cultivars, while either 11.4 μM of zeatin riboside or 18.2 μM of zeatin was optimum for shoot regeneration of the other two cultivars.

TRANSFORMATION

Transgenic breeding relies primarily on the existence of suitable target genes and effective gene transformation approaches. While transgenic breeding can play an important role in introducing desirable horticultural traits not possible by conventional breeding, it is obvious that reliable transformation protocols must first be developed to deploy this strategy (Polashock and Vorsa, 2002a; Song and Sink, 2005). Successful transformation and regeneration of blueberry and cranberry plants have been reported (Table 2), but there has been no report on the transformation of other Vaccinium crops.

TABLE 2 Literature survey on genetic transformation of Vaccinium species

Species	References	Delivery system	Gene	Selection system	Results and comments
V. corymbosum L.—Highbush blueberry	Rowland, 1990	Agrobacterium strains T37, C58, A281, A518, or B6 Co-cultivation time: 10 weeks	Natural transfer of DNA from non-disarmed strains	None	Highbush blueberry was susceptible to infection by at least three Agrobacterium strains—A281, T37, and C58.
	Rowland and Ogden, 1993	Agrobacterium strain C58C1 Co-cultivation time: 2 days	NPT II	10 mg/L Km + 500 mg/L Cef	No stable transgenics were obtained.
	Graham et al., 1996	Agrobacterium strain LBA4404 Co-cultivation time: 2 days	NPT II + an intron-interrupted gusA directed by the cauliflower mosaic virus 35S promoter	No antibiotics were included in regeneration medium	GUS-positive transformants were obtained, but Southern analysis was not conducted to confirm stable transformation.
	Cao et al., 1998	Agrobacterium strains LBA4404 and EHA105 Co-cultivation time: 2, 3, 4, or 5 days	NPT II + an intron-interrupted gusA directed by the 35S promoter	No selection or a two-week selection using50 mg/L Km + 100 mg/L Cef	Four days co-cultivation resulted in better transient gusA expression. EHA105 was more effective than LBA4404 in delivery of transgenes.
	Song and Sink, 2004, 2006	Transient transformation studies: Agrobacterium strains GV3101, EHA105, and LBA4404	NPT II + an intron-interrupted gusA directed by the chimeric super promoter (Aocs)3AmasPmas	None	EHA105 was more effective than LBA4404, or GV3101. Use of AS at 100 μM for 6 days was optimum for the co-cultivation step.
		Co-cultivation: 2, 4, 6, 8, or 10 daysStable transformation: Agrobacterium strains EHA105 Co-cultivation: 6 daysRegeneration medium: Modified WPM + 5 μM TDZ + 1.1 μM NAA		10 mg/L Km + 250 mg/L Cef	Stable transformation with frequencies of 15.3% for ‘Aurora’, 5.0 % for ‘Bluecrop’, 10.0 % for ‘Brigitta’ and 5.6 % for ‘Legacy’ was confirmed by Southern analysis.
	Song et al., 2007, 2008	Agrobacterium strain EHA105 Vectors: Four chimeric bialaphos resistance genes (bar) driven by different promoters. Co-cultivation time: 6 daysRegeneration medium: Modified WPM + 5 μM TDZ + 1.1 μM NAA	Bar	0.1 mg/L glufosinate ammonium + 250 mg/L Cef	The bar served both as a selectable marker and produced herbicide resistance.
V. macrocarpon Ait.—Large Cranberry	Serres et al., 1992	Particle bombardment Vector: pTVBTGUS	NPT II + gusA + Bacillus thuringiensis subsp. kurstaki crystal protein (Bt)	Km at 300 mg/L	1. Stable transgenic plants were recovered from bombarded stem sections.
	Zeldin et al., 2002	Vector: PUC-bar	Bar driven by the 35S promoter		2. Stable transgenic plants were obtained with herbicide resistance

Currently, Agrobacterium-mediated transformation and particle bombardment gene delivery are the two most widely used methods for generating transgenic plants. Transgenic cranberries have been produced by particle bombardment, while A. tumefaciens-mediated transformation has been used on blueberry (Table 2).

The highbush blueberry has been shown to be highly susceptible to infection by A. tumefaciens strains (Rowland, 1990). To date, two groups have obtained transgenic blueberry plants using A. tumefaciens-mediated transformation (Graham et al., 1996; Song and Sink, 2004). Graham et al. (1996) was the first to transform blueberries using the half-high cultivar ‘North Country’ (V. corymbosum L. x V. angustifolium Ait.). These transformants were obtained without antibiotic selection due to the extreme sensitivity of the leaf explants to kanamycin (Km); however, they were not confirmed by Southern analysis. In 2004, Southern-blot confirmed transgenic plants of four commercial varieties, ‘Aurora’, ‘Bluecrop’, ‘Brigitta’, and ‘Legacy’, of highbush blueberry (V. corymbosum L.) were obtained by Song and Sink (2004). A detailed step-by-step description of the successful transformation procedure has been published (Song and Sink, 2006).

A reliable transformation system depends on efficient gene delivery, effective selection, and efficient regeneration systems. Transient gene expression systems have been used to optimize these conditions for inoculation and co-cultivation in A. tumefaciens–mediated transformation. Dr. Hammerschlag's group at the USDA-ARS, Beltsville, Maryland, studied several factors influencing gusA expression mediated by A. tumefaciens strains LBA4404 and EHA105, including time of co-cultivation, explant age, genotype, and sucrose concentration in the medium. They found that 4 days of co-cultivation with EHA105 yielded efficient transient GUS expression, and that genotype and explant age were important factors (Cao et al., 1998).

Transient expression studies have also been performed at Michigan State University to select suitable conditions for inoculation and co-cultivation (Song and Sink, 2004). Blueberry leaf explants were found to be susceptible to three strains of Agrobacterium, EHA105, LBA4404, and GV3101 (Koncz and Schell, 1986). However, EHA105 yielded higher frequencies of transient GUS expression than the other two. The differences in the transient GUS expression of these three Ti-plasmids in blueberry paralleled that reported in rice (Li et al., 1992), chrysanthemum (Boase et al., 1998), and conifers (Humara et al., 1999). Co-cultivation time and acetosyringone both influenced transient GUS expression. Six days of co-cultivation on filter paper overlaid over medium yielded high levels of transient GUS expression, without any necrosis of the leaf explants. The efficiency of transient GUS expression was also improved by using acetosyringone (100 μM) and suitable medium. The blue stained cells, which were apparently susceptible to A. tumefaciens, were also the ones most active at the tissue regeneration sites. These results indicate that there is a high potential for regeneration of transformed blueberry cells.

In addition to efficient plant regeneration systems and gene delivery approaches using A. tumefaciens, effective selection is also necessary for the successful production of transgenic plants. Two selectable marker genes, nptII and bar, were tested for blueberry transformation and both yielded transgenic plants (Song and Sink, 2004; Song et al., 2007).

The sensitivity of plant tissues to antibiotics, such as Km, hygromycin, or glufosinate-ammonium (GS), is highly dependent upon species, genotype, and type and concentration of antibiotic. In the only report on the levels of sensitivity of blueberry to antibiotics, Km and ticaricillin, the half-high blueberry ‘North Country’ was found to be hypersensitive to both, as reviewed above (Graham et al., 1996). However, both cefotaxime and timentin have been used by other researchers in the successful production of transgenic blueberry plants. The optimized transformation and selection protocols yielded efficient production of transgenic plants at frequencies of 15.3% for ‘Aurora’, 5.0% for ‘Bluecrop’, 10.0% for ‘Brigitta’, and 5.6% for ‘Legacy’ (Song and Sink, 2004).

The effect of GS on regeneration of blueberry leaf explants has also been investigated. Blueberry explants are very sensitive to GS; however, the combination of 0.1 mg l⁻¹ GS with 250 mg l⁻¹ timentin yielded effective shoot inhibition without immediate death of explants of ‘Legacy’. This GS-selection protocol has led to successful production of herbicide-resistant transgenic plants of ‘Legacy’ (Song et al., 2007, 2008).

The screenable reporter gusA driven by either the cauliflower mosaic virus (CaMV) 35S or a chimeric super promoter (Aocs)₃AmasPmas, each terminated by T-nos, has been transformed into blueberry cultivars using the nptII as a selectable marker (Ni et al., 1995; Graham et al., 1996; Song and Sink, 2004). After selection with the herbicide GS, three chimeric bar genes with the promoter nopaline synthase (nos), CaMV 35S, or CaMV 34S, yielded transgenic plants; whereas, the synthetic (Aocs)₃AmasPmas super promoter did not lead to successful regeneration of transgenic plants. The herbicide Rely (Bayer CropScience, Research Triangle Park, NC, USA) was applied at five levels using a track sprayer (GS in mg·L⁻¹: 0, 750, 1,500, 3,000, and 6,000) on 3-month-old plants in the laboratory, representing three separate transgenic events each for the 35S and nos promoter. Evaluations of leaf damage 2 weeks after spraying indicated that all transgenic plants exhibited much higher herbicide resistance than non-transgenic plants. After application of eight times the standard level of GS (6,000 mg·L⁻¹) in the field, over 90% of the leaves on transgenic plants with the 35S-bar showed no symptoms of herbicide damage, whereas 95% of the leaves on non-transgenic plants were abscised. The transgenic plants with the 35S-bar showed higher herbicide resistance than those with the nos-bar, in which 19.5–51.5% of the leaves had no damage (Song et al., 2008).

Most recently, a CBF/DREB transcription factor gene isolated from V. corymbosum (Polashock et al., 2010) and posted in GenBank (FJ222601) has been transformed into the relatively cold-sensitive highbush blueberry ‘Legacy’. Over 60 independent transgenic events have been produced (Song et al., unpublished). The preliminary indications are that over-expression of this CBF will improve the cold-tolerance of leaves and perhaps flower buds in this southern blueberry cultivar (Song et al., unpublished).

POTENTIAL IMPACTS OF TRANSGENIC BLUEBERRY

Establishment of transformation systems has opened the window for transgenic breeding of blueberry and might allow production of cultivars with unique characteristics that can not be obtained through conventional breeding. The challenges are to further improve transformation frequency, to find target genes for unique improvement of Vaccinium species, and to make transgenic products more acceptable to the general public.

Transgenic breeding on blueberry species could be used to introduce various traits, including: (1) resistance/tolerance to insects, diseases, and herbicides; (2) stress resistance/tolerance to drought and cold; and (3) fruit qualities, such as control of ripening, fruit softening, shelf life, nutrition, and antioxidants (Giovannoni, 2004). In the future, as more genomic resources for Vaccinium become available, more genes of interest will be identified and isolated. Genetic transformation will allow us to evaluate these genes as to their functions. The blueberry mapping and gene discovery work currently being undertaken by Rowland and her associates and the sequencing of the diploid blueberry genome by generating long read structural scaffolds using paired end 454 libraries, likely will provide important insights into the blueberry genome that can be used in blueberry improvement (Brown et al., 2010).

Weed infestation in fields is one of the major problems in blueberry production. To control weeds, non-selective and broad-spectrum herbicides, such as glyphosate and phosphinothricin, are usually used, although they can only be applied as a directed spray under the bushes and avoiding any contact with the green stem tissues of blueberry plants. Herbicide-resistant plants, especially for lowbush genotypes, can be expected to broaden the application of non-selective herbicides and to provide a simple, inexpensive, potent, and environmentally friendly management for weed control. Currently, two transgenic herbicide resistant cropping systems are common for soybean, maize, rapeseed, and cotton: RoundupReady (active agent: glyphosate) and Liberty Link (active agent: glufosinate). The bar gene from Streptomyces hygroscopicus encodes phosphinothricin acetyl transferase that confers tolerance to glufosinate—the active ingredient of non-selective herbicide Basta (De Block et al., 1987; Thompson et al., 1987; De Block et al., 1989, 1995).It has been successfully engineered into many plant species when used as a selectable marker but also as a target trait for production of herbicide-resistant plants (Toki et al., 1992; Ritter and Menbere, 2001). Transformed cranberry and blueberry with the bar gene showed excellent resistance to glufosinate-ammonium (Zeldin et al., 2002; Song et al., 2007).

Although the Bt-expressing cranberry did not show promising resistance to insects (Polashock and Vorsa, 2002a), it was shown that transgenic breeding is a potential tool to obtain insect-tolerant plants. Similarly, transgenic breeding could enable the production of transgenic Vaccinium plants with resistance to viruses and/or diseases. The major viruses in Vaccinium species include red ringspot virus (RRSV) (Ramsdell, 1995a), blueberry shoestring virus (BBSSV) (Ramsdell, 1995b), blueberry stunt (Ramsdell, 1995c), and newly appeared blueberry scorch virus (BlScV) and blueberry shock virus (BlShV) (Martin et al., 2006); the important fungal diseases are mummy berry (Monilinia vacinii-corymbosi), phomopsis twig blight (Phomopsis vaccinii), and anthracnose fruit-rot (Colletotrichum acutatum). In addition to the expression of coat protein for protection against virus diseases, application of RNA interference (RNAi) technology could generate virus-resistant Vaccinium plants (Tenllado et al., 2004; Ravelonandro and Scorza, 2009).

There are many more potential types of genes that could be used in the improvement of Vaccinium species. Transgenic approaches are being explored to modify plant secondary metabolism for improvement of nutritional quality and flavor of fruits. Successful modification of the nutritional value of tomatoes through metabolic engineering and transformation has provided a novel example of the use of organ-specific gene silencing to enhance the nutritional value of fruits (Davuluri et al., 2004).

There is no work currently being done to release transgenic Vaccinium plants for commercial use. Several obstacles are working against the acceptance of transgenic blueberries and cranberries. The economic value of these fruit crops is limited compared to many of the agronomic crops and as a result there is only modest private stimulus to develop new biotechnological products. A second issue is that they are all outcrossed crops with widespread, native relatives in close proximity to cultivated fields. Most transgenic crop releases to date have been with species that do not have nearby congeners, eliminating the risk of the movement of the transgene into wild species populations. The release of transgenic small fruit crops will require more scrutiny and in-depth ecological studies than has been necessary with most of the other transcrops previously released (Hancock, 2003). The third issue associated with the commercial release of transgenic blueberries and cranberries is a strong reluctance by the fruit industry to introduce transgenic products, for fear that there will be a negative backlash from people leery of consuming genetically modified crops. In 2001, the North American Blueberry Council (NABC) even stated that the NABC, along with blueberry sellers around the globe, opposed any development of transgenic blueberry clones (North American Blueberry Council, 2001). This attitude persists, even though most of the commercial corn, cotton, and soybean in the USA are genetically engineered, and transgenic papaya has been grown in Hawaii now for more than a decade (Manshardt, 2007; Tecson et al., 2008).

A strong influx of federal and state funds is needed to stimulate biotechnology research for Vaccinium species, along with a careful analysis of what people's real perceptions are concerning transgenic fruit. Until this happens, transgenic small fruit crops will remain an important research tool, but not a commercial entity. We are very much encouraged by the recent USDA-Specialty Crop Research Initiative funding of two major grants to study the genomics of blueberries. Utilizing marker-free transformation systems and targeted expression of transgenes will help minimize public concern, but the fear of the technology in general must be reduced before transgenic small fruit products will find their way into homes.