- Email: [email protected]
Long term, transgene expression in Lilium longiflorum ‘Nellie White’ grown outdoors and in the greenhouse
Scientia Horticulturae 167 (2014) 158–163
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Long term, transgene expression in Lilium longiflorum ‘Nellie White’ grown outdoors and in the greenhouse Kathryn Kamo ∗ Floral and Nursery Plants Research Unit, U.S. National Arboretum, U.S. Department of Agriculture, Beltsville, MD 20705-2350, United States
a r t i c l e
i n f o
Article history: Received 23 August 2013 Received in revised form 28 October 2013 Accepted 9 December 2013 Keywords: Easter lily Transformation Genetic engineering Flower bulb crops Reporter gene Selectable marker genes
a b s t r a c t Lilium longiflorum cv. Nellie White plants were transformed with either the bar-uidA fusion gene or the nptII and uidA genes and grown for two seasons in the greenhouse and outdoors in containers. All transgenes were under control of the CaMV 35S promoter. During the first year there was no difference in bar protein level between plants grown in the greenhouse and outdoors, but the second year two of the seven transgenic plant lines grown outdoors showed a significantly higher level of the bar protein than plants grown in the greenhouse. The relative level of bar protein expression between all seven transgenic plant lines remained the same for lilies grown in the greenhouse and outdoors during two growing seasons. One plant line contained the nptII gene, and nptII protein expression was comparable when plants were grown in the greenhouse or outdoors for two seasons. Four plant lines expressed GUS as determined by histochemical staining of their leaves. Three of these four plant lines showed dark blue staining along the cut edges of the leaf when plants were grown either in the greenhouse or outdoors for both seasons. The fourth plant line showed either dark blue staining along the cut edge of the leaf or small, light blue spots on parts of the leaf depending on the plant stained. Expression of the three genes was confirmed using semi-quantitative RT-PCR. Published by Elsevier B.V.
1. Introduction Lilies are one of the most important flowers marketed as a cutflower, pot plant, and for the garden. They are multiplied by bulbs, and a small bulb will produce a large flower that can be marketed after three seasons of growth in the field. The main problem in the U.S. when growing lilies in the field is nematodes. Because we are interested in genetic engineering of lilies for nematode resistance, it is important to investigate the expression of a transgene during growth under the actual environmental conditions outdoors and to verify continuous transgene expression during several seasons of growth. Several studies have shown that environmental conditions affect transgene expression. The majority of petunia plants transformed with the maize A1 gene that encodes a dihydroflavonol reductase had flowers that were a red salmon color when grown in the greenhouse while only 5% of the plants exhibited a lighter hue
Abbreviations: CaMV 35S, cauliflower mosaic virus; MS, Murashige and Skoog’s medium; nptII, neomycin phosphotransferase; PAT, phosphinothricin acetyltransferase. ∗ Corresponding author at: 10300 Baltimore Avenue, Building 010A Room 126, Floral and Nursery Plants Research Unit, USDA ARS, BARC West, Beltsville, MD 20705-2350, USA. Tel.: +1 301 504 5350. E-mail address: [email protected] 0304-4238/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.scienta.2013.12.011
(Meyer et al., 1992). In comparison, 60% of these petunias showed a lighter hue when these petunias were grown in the field indicating an environmental influence. Heat treatment (37 ◦ C) was shown to inactivate both the phosphinothricin-N-acetyltransferase (pat) gene in suspension cells of Medicago sativa and the luciferase gene under control of the CaMV 35S promoter in tobacco plants (Walter et al., 1992; Neumann et al., 1997). Levels of transgene expression were higher in field-grown potatoes expressing either VP60 (the structural capsid protein of the rabbit haemorrhagic disease) or the non-toxic B subunit of the cholera toxin combined with the nptII gene as compared to potatoes grown in the greenhouse (Mikschofsky et al., 2011). These results indicated that field cultivation of potatoes for pharmaceutical production was superior to that in the greenhouse. Others studies have found that transgene expression was not affected by environmental conditions. Water and nutrient stress and heat shock did not alter transgene expression of barley plants containing the bar and uidA genes under control of a ubi1 promoter (Meng et al., 2006). Eggplants transformed with the iaaM gene under control of the DefH9 promoter for introduction of parthenocarpy were found to be comparable when plants were grown in the greenhouse and outdoors (Acciarri et al., 2002). This study examined expression of the uidA gene that codes for GUS expression, bar, and nptII genes in Lilium longiflorum plants grown two seasons in the greenhouse and outdoors in containers.
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
2. Materials and methods All chemicals used for plant tissue culture, transformation, and biochemical analysis were obtained from Sigma–Aldrich (St. Louis, MO) unless stated otherwise. 2.1. Plant materials grown in vitro Lilium longiflorum cv. Nellie White plants were grown in vitro in Magenta jars containing Murashige and Skoog’s medium (MS; Murashige and Skoog, 1962) containing 3% sucrose and the following in mgl−1 : 1.0 glycine, 1.0 thiamine, 0.5 nicotinic acid, 1.0 pyridoxine, 100 myo-inositol, and solidified with 0.2% Phytagel. Plants were transferred monthly to fresh medium. They were grown at 25 ◦ C under a 12 h light photoperiod using cool white fluorescent lights at 40–60 mol m−2 s−1 . 2.2. Plants grown in the greenhouse and outdoors Lily plants grown in vitro were transferred to 7.6 cm diameter clay pots containing Metromix 200 (Scotts Company, Marysville, OH) and grown at 4 ◦ C for 6 weeks before they were transferred to the greenhouse or outdoors. Each 7.6 cm diameter clay pot contained a single lily plant that was placed in a 30.5 cm diameter clay pot containing Metromix to prevent contents in the smaller pot from drying out quickly outdoors. In 2010 the plants were grown from May through August in the greenhouse and outdoors. The greenhouse temperature during this time period averaged 36.7 ◦ C during the day and 22.2 ◦ C during the night. The high and low temperatures were 41.1 and 15.0 ◦ C, respectively. The average outdoor temperature was 34.4 ◦ C during the day and 22.2 ◦ C during the night. Outdoors the temperature ranged from 15.6 to 37.2 ◦ C. In 2011 lilies were grown from May through August in the greenhouse and outdoors. The average greenhouse temperature 34.0 ◦ C during the day and 22.0 ◦ C during the night with a range of 18.3–40.6 ◦ C. Outdoors the average daytime temperature was 33.9 ◦ C and the average evening temperature was 22.2 ◦ C. The range was 15.6–37.2 ◦ C.
159
The specific activity of GUS expression was determined according to the method of Jefferson et al. (1987). Leaves were ground in a Lysing Matrix tube with one ceramic ball containing 500 l of extraction buffer (50 mM NaH2 PO4 , pH 7.0, 10 mM EDTA, pH 8.0, 0.1% Triton X-100, 0.1% sarkosyl, and 10 mM -mercaptoethanol) using the FastPrep system (QBiogene, Carlsbad, CA) and then centrifuged at 16,000 × g for 5 min in a microcentrifuge. The supernatant was added to assay buffer (extraction buffer with 1 mM methyl umbelliferyl--D-glucuronide) (Molecular Probes, Eugene, OR) and incubated at 37 ◦ C for various time periods before being stopped by 0.2 M sodium carbonate. Fluorescence of the sample was measured using a BioRad Versa Fluor Fluorometer (Bio-Rad, Richmond, CA) at 365 nm excitation and 455 nm emission. The bicinchoninic protein assay reagent (Pierce Co., Rockford, IL) was added to cell extracts for measurement of the protein concentration according to the manufacturer’s instructions. Leaf extracts were used from three plants for each plant line to determine the specific activity of -glucuronidase, and this assay was repeated. 2.5. Bar and nptII protein expression Relative expression of the bar protein were determined using the LibertyLink® PAT/bar ELISA plate kit (Envirologix, Portland, ME)
2.3. Transformation Lilies had been transformed with either pCAMBIA 2301 that contains the uidA and npt genes each under control of the CaMV 35S promoter or pDM327 that contains the bar-uidA fusion gene under control of the CaMV 35S promoter (Kamo and Han, 2008). The PDS-He gene gun (Bio-Rad, Richmond, CA) was used to bombard embryogenic callus with plasmid DNA, and transformed plants were selected on MS medium containing either 2 mgl−1 dicamba or 1 mgl−1 picloram combined with 1 mgl−1 phosphinothricin (AgroEvo, Pikeville, NC) for selection. Transformed plants were maintained in vitro for seven years before being used for this study. 2.4. UidA expression The uidA gene codes for GUS expression. Leaves were stained histochemically for GUS expression by placing them for 16 h at 37 ◦ C in a phosphate buffer, pH 7.0 staining solution as described by Jefferson et al. (1987) supplemented with 0.1% Triton X-100, 20% methanol, 0.5 mM potassium ferricyanide, and 0.5 mM ferrocyanide. The staining solution was removed and samples were then incubated in 70% ethanol for one week for removal of chlorophyll. Leaves from three-four plants for each transformed plant line were stained, and non-transformed ‘Nellie White’ was used as the negative control.
Fig. 1. Leaf extracts from a non-transformed plant (NT) and plants transformed with pDM327 consisting of the bar-uidA fusion gene under control of the CaMV 35S promoter were used in an ELISA assay to determine relative levels of the bar protein in either 2010 (top graph) or 2011 (bottom graph). Bars indicate the mean±standard error. Bars with different letters have significantly different means between the two years (P ≤ 0.05).
160
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
Fig. 2. RNA levels for the bar gene as determined by semi-quantitative RT-PCR. Plant lines are shown above each lane. Plants were grown in either the greenhouse (GH) or outdoors in pots (O) during either 2010 or 2011. The PCR band for the bar gene is indicated by the arrow in the top gels in A, B, and C. Molecular size markers (M) are indicated in bp in A, and the positive control was pDM327 (+). Each lower gel in A, B, and C shows the relative RNA levels for each sample as indicated by the PCR band for the LhUBQ4 gene.
according to the manufacturer’s instructions. Leaves were collected in a 1.5 ml microcentrifuge tube on ice and then ground in the kit’s extraction buffer using a Kontes pestle. Samples were vortexed 10 s followed by centrifugation at 5000 × g for 5 min. The supernatant was transferred to a new tube and placed at −80 ◦ C. A PAT-enzyme conjugate was added to the wells of an ELISA plate immediately followed by the addition of wash/extraction buffer from the kit, and then 50 l of plant extract were added to each well. The plate was rotated by hand for 30 s and then covered with Parafilm for a 2 h incubation at 25 ◦ C. Wash/extraction buffer from the kit was used to flood the wells, and then the plate was emptied. Substrate was added and the plate incubated at 25 ◦ C for 30 min. A 1 N HCl stop solution was added to the wells, and the plate was read at OD450 nm. The NPT II ELISA Pathoscreen kit (Agdia, Elkhart, IN) was used according to the manufacturer’s instructions to determine expression of the nptII protein in leaf extracts. Leaves had been collected and stored at −80 ◦ C until used. Leaf tissue was ground in the kit’s extraction buffer using a Kontes pestle, and the supernatant (100 l) was recovered after centrifugation at 5000 × g for 5 min. Supernatant was placed in the wells of the ELISA plate along with 100 l of extraction buffer for an incubation at 25 ◦ C for 2 h before the contents of the plate were emptied. Wells were filled with phosphate buffered saline with Tween 20 (PBST) solution, emptied, and this step was repeated 6 times to wash the wells. Conjugate was
added followed by a 2 h incubation at 25 ◦ C. The plate was washed with PBST six times before adding 100 l of the kit’s substrate solution. The plate was incubated 15 min at 25 ◦ C followed by the addition of 3 M sulfuric acid to stop the reaction. The plate was read at OD450 nm. Standards included with the kit were used to construct a concentration curve for determining the nptII concentration of each sample. 2.6. Semi-quantitative RT-PCR RNA was isolated from leaves using the RNeasy Plant Mini kit followed by removal of contaminating DNA using the RNase-Free DNase set (Qiagen, Valencia, CA). Equal amounts of RNA were used from each plant sample for making cDNA with the Thermoscript RT-PCR system (InVitrogen, Carlsbad, CA). The program for cDNA synthesis was 25 ◦ C for 10 min, 62 ◦ C for 50 min, and then 85 ◦ C for 5 min. DNA was treated with RNaseH. PCR was performed using gene-specific primers. The bar PCR primers were forward 5 -GTCAACTTCCGTACCGAGCCGCAG3 and reverse 5 -CATGCCAGTTCCCGTGCTTGAAG-3 to amplify a DNA fragment of 379 bp from the bar gene. The primers forward 5 -TCGGCTATGACTGGGCACAACAGA-3 and reverse 5 -AAGAAGGCGATAGAAGGCGATGCG-3 were used to amplify a 700 bp DNA fragment from the nptII gene. A 253 bp DNA fragment was amplified from the uidA gene
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
2.7. Statistical analysis Three leaf samples were collected for each transformed plant line, and non-transformed ‘Nellie White’ served as the negative control. The leaf extract from each plant line was used to fill duplicate wells of an ELISA plate, and ELISA assays were replicated twice. The background absorbance from a well filled with extraction buffer was subtracted from the absorbance for each well filled with the leaf extract. Duplicate wells of a plate were averaged to obtain a value for each plant line. The mean was calculated from the 12 absorbance values for each plant line, and a Student’s t test used to compare either the greenhouse value with the outdoor value within a given year or the greenhouse, and outdoor values were compared between the two seasons. Means were considered significantly different at P ≤ 0.05.
1.0 0.8
OD450
using forward primer 5 -TAACCTTCACCCGGTTGCCAGAGG3 and reverse primer 5 -CTTTAACTATGCCGGAATCCATCG-3 . Primers forward 5 -GGTATCCCTCCGGACCAG-3 and reverse 5 ATGGTGTCCGAACTCTCCAC-3 amplified a 194 bp fragment from the LhUBQ4 gene of lily as an internal control (Yamagishi, 2011). PCR reactions were run on a MJ Research PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA) programmed at 94 ◦ C for one min followed by 30 cycles of 94 ◦ C for 20 s, 67 ◦ C for 30 s, 72 ◦ C for 2 min, and then one cycle of 72 ◦ C for 10 min. PCR products were separated by electrophoresis on a 1% agarose gel using TBE buffer (0.045 M Tris-borate, 0.001 M EDTA, pH 8.0) and visualized using ethidium bromide.
161
0.6
Greenhouse Outdoor
0.4 0.2 0.0
2011
2010
Plant line 2A Fig. 3. Leaf extracts from a non-transformed plant (NT), and the lily plant line 2A transformed with pCAMBIA2301 consisting of the npt II and uidA gene under control of the CaMV 35S promoter were used in an ELISA assay to determine relative levels of the nptII protein in 2010 and 2011. Bars indicate standard error.
2010 and 2011 following an ELISA assay of the plants were not seen at the RNA level (Fig. 2). There were very weak bands not readily visible on the gel photographs for the bar gene for lines 1B grown both years in either the greenhouse or outdoors, the two lines of 2B grown in 2011, the one line of 6A grown outdoors in 2011, and all four lines of 40(1) grown both years in either the greenhouse or outdoors. The highest to lowest RNA levels were observed to be line 4B, line 4(1), line 6A, line 4C, line 2B, and a bar band was barely visible for lines 1B and 40(1). This correlated with the highest
3. Results and discussion Plant lines 1B, 2B, 4B, 4C, 4(1), 6A, 40(1) were transformed by gene gun bombardment in 2002 with pDM327, and all lines contained the bar gene. Lines 1B, 4B, 4C, and 40(1) have the uidA gene but not lines 2B, 4(1), and 6A. Line 2A was recovered following bombardment with pCAMBIA2301 and contains both the nptII and uidA genes. Transgenic lily plants were previously confirmed to contain the transgene by PCR and Southern hybridization (Kamo and Han, 2008). Inheritance of the bar gene occurred in 15% of the T1 progeny of plant lines 4B, 4(1), and 6A but not lines 4C and 40(1) indicating the possibility that lines 4C and 40(1) may be chimeric (Kamo, 2011). PCR analysis did not provide supporting evidence that any of these five plant lines were chimeric as all leaf samples from three different plants of each plant line were found to contain the transgene even though the plants had been transferred for six years on non-selective media. 3.1. Bar protein and RNA levels Seven lily plant lines transformed with the bar gene produced a protein product that reacted with an antibody to the bar protein as determined by an ELISA assay (Fig. 1). There were no significant differences in bar protein level when transgenic lilies were grown in either the greenhouse or outdoors in 2010. In 2011 two of the seven transgenic lily lines (2B, 4B) grown outdoors showed a statistically higher level of the bar protein than lilies grown in the greenhouse. In comparison, transgenic potatoes showed either similar or higher levels of two transgenes coding for antigens when grown in the field instead of the greenhouse (Mikschofsky et al., 2011). The relative level of bar protein between the lily lines remained the same for lilies grown in the greenhouse and outdoors in 2010 and 2011 (Fig. 1). In both 2010 and 2011, bar expression was highest in line 4B followed by similar levels of expression for both 4(1) and 6A which had higher levels than 4C, and 4C was higher than both 40(1) and 2B that showed similar levels and were higher than line 1B. These relative differences in bar protein levels observed during
Fig. 4. RNA levels for the (A) nptII and (B, C) uidA genes as shown by semiquantitative RT-PCR. Plant lines are shown above each lane. Plants were grown in either the greenhouse (GH) or outdoors in pots (O) during either 2010 or 2011. The PCR band for the nptII gene is indicated by the arrow in the top gels in A and the uidA gene in B and C. The molecular size markers (M) are indicated in bp, and the positive control (+) was pCAMBIA2301 in A and pDM327 in B. Each lower gel in A, B, and C shows the relative RNA levels for each sample as indicated by the PCR band for the LhUBQ4 gene.
162
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
Fig. 5. Leaves from a non-transformed lily (NT) and four transgenic lily lines containing the uidA gene were stained for GUS expression. Plants were grown in the greenhouse (GH) or outdoors in containers (Out) during the 2010 and 2011 growing seasons.
level of bar protein for line 4B followed by 4(1), 6A, and 4C. Levels of the bar protein and bar RNA were low for the remaining lines 2B, 40(1), and 1B. The reason for the significantly higher level of bar protein observed in 2011 compared to 2010 in six of the seven lines is unclear. All plant samples collected in 2010 and 2011 were processed and analyzed the same day using one ELISA kit, but it appears that there was some factor that affected the ELISA but not the RNA results. Expression of the bar gene was stable in lilies because it was not silenced even though the plants were exposed to the high temperature that caused silencing of the pat gene in cell suspensions of Medicago sativa (Walter et al., 1992). Silencing of the bar and pat genes did not occur in Gladiolus plants when plants were subjected to high temperatures (Kamo, 2008) indicating differences in plant species. 3.2. NptII protein and RNA levels Only one plant line was recovered that was transformed with the nptII gene. The relative levels of nptII protein (0.84–0.86 ng/L) were similar when lilies of line 2A were grown in the greenhouse and outdoors during 2010 and 2011 (Fig. 3). RNA levels for the nptII gene were also similar when line 2A was grown in the greenhouse or outdoors for two years (Fig. 4A). 3.3. GUS protein and RNA levels GUS expression was readily detected by histochemical staining in the four lines that contained the uidA gene (Fig. 5). Leaf tissue turned dark blue at the cut regions for lines 4B, 4C, and 40(1) grown in both the greenhouse and outdoors during 2010 and 2011. Only line 1B showed a variable response between plants of this line. Some 1B leaves taken from plants grown in the greenhouse
or outdoors showed faint blue spots and other leaves showed the dark blue staining at the cut regions. Variation between transformants of a single clone has been reported previously. Potato plants transformed with the snowdrop lectin (Galanthus nivalis agglutinin) and bean chitinase under control of the CaMV 35S promoter were found to have variability in the levels of transgene protein produced within each line of clonal replicates (Down et al., 2001). GUS expression was silenced in the lily line 2A transformed with pCAMBIA2301 as there was no leaf tissue that showed a blue reaction following histochemical staining (data not shown). Levels of GUS expression could not be compared for lilies grown in 2010 and 2011 because they were too low for determining the specific activity of -glucuronidase that codes for GUS expression (data not shown). Comparative RNA levels for the uidA gene showed that levels of RNA for the various plant lines were from high to low: 4B, 2A, 4C, and very low as shown by faint bands on the gel for lines 1B and 40(1) (Fig. 4B,C). It appeared that the RNA level of the uidA gene was higher for line 4C when plants were grown outdoors rather than in the greenhouse both years. RNA for the uidA gene was not seen on the gel for line 2A that was transformed with pCAMBIA2301 (data not shown). Levels of GUS expression were significantly different for four of the six Gladiolus plant lines expressing GUS when grown two seasons outdoors and for one of the four plant lines grown two seasons in the greenhouse (Kamo, 2008). GUS and bar expression were observed in poplars growing three seasons in a field, and expression was similar throughout the years for both transgenes (Li et al., 2008). In conclusion, this study showed that the expression of the bar, nptII, and uidA genes was usually similar when transgenic lilies were grown in the greenhouse or outdoors. The relative level of bar protein between the lily lines remained the same for lilies grown in the greenhouse and outdoors in 2010 and 2011. These lilies were transformed nine years ago demonstrating long term,
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
stable expression of the uidA, bar, and nptII genes in lilies when grown under different environmental conditions. Acknowledgements Thanks to Anne O’Connor for excellent technical assistance throughout this study. Funds to support this project were received from the Gloeckner Foundation. pDM327 was received from David McElroy (Verdia, Redwood City, CA). Mention of a trademark, proprietary product or vendor does not imply its approval to the exclusion of other products or vendors that may also be suitable. References Acciarri, N., Restaino, F., Vitelli, G., Perrone, D., Zottini, M., Pandolfini, T., Spena, A., Rotino, G.L., 2002. Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation. BMC Biotechnology 2, 4–8. Down, R.E., Ford, L., Bedford, S.J., Gatehouse, L.N., Newell, C., Gatehouse, J.A., Gatehouse, A.M.R., 2001. Influence of plant development and environment on transgene expression in potato and consequences for insect resistance. Trans. Res. 10, 223–236. Kamo, K., 2008. Transgene expression for Gladiolus plants grown outdoors and in the greenhouse. Sci. Hort. 117, 275–280.
163
Kamo, K., 2011. Inherited transgene expression of the uidA and bar genes in Lilium longiflorum cv. ‘Nellie White’. Floriculture Ornamental Biotech. 5, 35–39. Kamo, K., Han, B.H., 2008. Biolistic-mediated transformation of Lilium longiflorum cv. Nellie White. HortScience 43, 1864–1869. Li., J., Brunner, A.M., Meilan, R., Strauss, S.H., 2008. Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons. Tree Physiol. 29, 299–312. Meng, L., Ziv, M., Lemaux, P.G., 2006. Nature of stress and transgene locus influences transgene expression stability in barley. Plant Mol. Biol. 62, 15–28. Meyer, P., Linn, F., Heidmann, I., Meyer, H., Niedenhof, I., Saedler, H., 1992. Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype. Mol. Gen. Genet. 231, 345–352. Mikschofsky, H., Heilmann, E., Schmidtke, J., Schmidt, K., Meyer, U., Leinweber, P., Broer, I., 2011. Greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression. Plant Mol. Biol. 76, 131–144. Murashige, T., Skoog, F., 1962. A revised medium for rapid assays with tobacco tissue cultures. Physiol. Plant 15, 473–497. Neumann, K., Dröge-Laser, W., Köhne, S., Broer, I., 1997. Heat treatment results in a loss of transgene-encoded activities in several tobacco lines. Plant Physiol. 115, 929–947. Walter, C., Broer, I., Hillemann, D., Pühler, A., 1992. High frequency, heat treatmentinduced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa. Mol. Gen. Genet. 235, 189–196. Yamagishi, M., 2011. Oriental hybrid lily Sorbonne homologue of LhMYB12 regulates anthocyanin biosyntheses in flower tepals and tepal spots. Mol. Breed. 28, 381–389.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Long term, transgene expression in Lilium longiflorum ‘Nellie White’ grown outdoors and in the greenhouse Kathryn Kamo ∗ Floral and Nursery Plants Research Unit, U.S. National Arboretum, U.S. Department of Agriculture, Beltsville, MD 20705-2350, United States
a r t i c l e
i n f o
Article history: Received 23 August 2013 Received in revised form 28 October 2013 Accepted 9 December 2013 Keywords: Easter lily Transformation Genetic engineering Flower bulb crops Reporter gene Selectable marker genes
a b s t r a c t Lilium longiflorum cv. Nellie White plants were transformed with either the bar-uidA fusion gene or the nptII and uidA genes and grown for two seasons in the greenhouse and outdoors in containers. All transgenes were under control of the CaMV 35S promoter. During the first year there was no difference in bar protein level between plants grown in the greenhouse and outdoors, but the second year two of the seven transgenic plant lines grown outdoors showed a significantly higher level of the bar protein than plants grown in the greenhouse. The relative level of bar protein expression between all seven transgenic plant lines remained the same for lilies grown in the greenhouse and outdoors during two growing seasons. One plant line contained the nptII gene, and nptII protein expression was comparable when plants were grown in the greenhouse or outdoors for two seasons. Four plant lines expressed GUS as determined by histochemical staining of their leaves. Three of these four plant lines showed dark blue staining along the cut edges of the leaf when plants were grown either in the greenhouse or outdoors for both seasons. The fourth plant line showed either dark blue staining along the cut edge of the leaf or small, light blue spots on parts of the leaf depending on the plant stained. Expression of the three genes was confirmed using semi-quantitative RT-PCR. Published by Elsevier B.V.
1. Introduction Lilies are one of the most important flowers marketed as a cutflower, pot plant, and for the garden. They are multiplied by bulbs, and a small bulb will produce a large flower that can be marketed after three seasons of growth in the field. The main problem in the U.S. when growing lilies in the field is nematodes. Because we are interested in genetic engineering of lilies for nematode resistance, it is important to investigate the expression of a transgene during growth under the actual environmental conditions outdoors and to verify continuous transgene expression during several seasons of growth. Several studies have shown that environmental conditions affect transgene expression. The majority of petunia plants transformed with the maize A1 gene that encodes a dihydroflavonol reductase had flowers that were a red salmon color when grown in the greenhouse while only 5% of the plants exhibited a lighter hue
Abbreviations: CaMV 35S, cauliflower mosaic virus; MS, Murashige and Skoog’s medium; nptII, neomycin phosphotransferase; PAT, phosphinothricin acetyltransferase. ∗ Corresponding author at: 10300 Baltimore Avenue, Building 010A Room 126, Floral and Nursery Plants Research Unit, USDA ARS, BARC West, Beltsville, MD 20705-2350, USA. Tel.: +1 301 504 5350. E-mail address: [email protected] 0304-4238/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.scienta.2013.12.011
(Meyer et al., 1992). In comparison, 60% of these petunias showed a lighter hue when these petunias were grown in the field indicating an environmental influence. Heat treatment (37 ◦ C) was shown to inactivate both the phosphinothricin-N-acetyltransferase (pat) gene in suspension cells of Medicago sativa and the luciferase gene under control of the CaMV 35S promoter in tobacco plants (Walter et al., 1992; Neumann et al., 1997). Levels of transgene expression were higher in field-grown potatoes expressing either VP60 (the structural capsid protein of the rabbit haemorrhagic disease) or the non-toxic B subunit of the cholera toxin combined with the nptII gene as compared to potatoes grown in the greenhouse (Mikschofsky et al., 2011). These results indicated that field cultivation of potatoes for pharmaceutical production was superior to that in the greenhouse. Others studies have found that transgene expression was not affected by environmental conditions. Water and nutrient stress and heat shock did not alter transgene expression of barley plants containing the bar and uidA genes under control of a ubi1 promoter (Meng et al., 2006). Eggplants transformed with the iaaM gene under control of the DefH9 promoter for introduction of parthenocarpy were found to be comparable when plants were grown in the greenhouse and outdoors (Acciarri et al., 2002). This study examined expression of the uidA gene that codes for GUS expression, bar, and nptII genes in Lilium longiflorum plants grown two seasons in the greenhouse and outdoors in containers.
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
2. Materials and methods All chemicals used for plant tissue culture, transformation, and biochemical analysis were obtained from Sigma–Aldrich (St. Louis, MO) unless stated otherwise. 2.1. Plant materials grown in vitro Lilium longiflorum cv. Nellie White plants were grown in vitro in Magenta jars containing Murashige and Skoog’s medium (MS; Murashige and Skoog, 1962) containing 3% sucrose and the following in mgl−1 : 1.0 glycine, 1.0 thiamine, 0.5 nicotinic acid, 1.0 pyridoxine, 100 myo-inositol, and solidified with 0.2% Phytagel. Plants were transferred monthly to fresh medium. They were grown at 25 ◦ C under a 12 h light photoperiod using cool white fluorescent lights at 40–60 mol m−2 s−1 . 2.2. Plants grown in the greenhouse and outdoors Lily plants grown in vitro were transferred to 7.6 cm diameter clay pots containing Metromix 200 (Scotts Company, Marysville, OH) and grown at 4 ◦ C for 6 weeks before they were transferred to the greenhouse or outdoors. Each 7.6 cm diameter clay pot contained a single lily plant that was placed in a 30.5 cm diameter clay pot containing Metromix to prevent contents in the smaller pot from drying out quickly outdoors. In 2010 the plants were grown from May through August in the greenhouse and outdoors. The greenhouse temperature during this time period averaged 36.7 ◦ C during the day and 22.2 ◦ C during the night. The high and low temperatures were 41.1 and 15.0 ◦ C, respectively. The average outdoor temperature was 34.4 ◦ C during the day and 22.2 ◦ C during the night. Outdoors the temperature ranged from 15.6 to 37.2 ◦ C. In 2011 lilies were grown from May through August in the greenhouse and outdoors. The average greenhouse temperature 34.0 ◦ C during the day and 22.0 ◦ C during the night with a range of 18.3–40.6 ◦ C. Outdoors the average daytime temperature was 33.9 ◦ C and the average evening temperature was 22.2 ◦ C. The range was 15.6–37.2 ◦ C.
159
The specific activity of GUS expression was determined according to the method of Jefferson et al. (1987). Leaves were ground in a Lysing Matrix tube with one ceramic ball containing 500 l of extraction buffer (50 mM NaH2 PO4 , pH 7.0, 10 mM EDTA, pH 8.0, 0.1% Triton X-100, 0.1% sarkosyl, and 10 mM -mercaptoethanol) using the FastPrep system (QBiogene, Carlsbad, CA) and then centrifuged at 16,000 × g for 5 min in a microcentrifuge. The supernatant was added to assay buffer (extraction buffer with 1 mM methyl umbelliferyl--D-glucuronide) (Molecular Probes, Eugene, OR) and incubated at 37 ◦ C for various time periods before being stopped by 0.2 M sodium carbonate. Fluorescence of the sample was measured using a BioRad Versa Fluor Fluorometer (Bio-Rad, Richmond, CA) at 365 nm excitation and 455 nm emission. The bicinchoninic protein assay reagent (Pierce Co., Rockford, IL) was added to cell extracts for measurement of the protein concentration according to the manufacturer’s instructions. Leaf extracts were used from three plants for each plant line to determine the specific activity of -glucuronidase, and this assay was repeated. 2.5. Bar and nptII protein expression Relative expression of the bar protein were determined using the LibertyLink® PAT/bar ELISA plate kit (Envirologix, Portland, ME)
2.3. Transformation Lilies had been transformed with either pCAMBIA 2301 that contains the uidA and npt genes each under control of the CaMV 35S promoter or pDM327 that contains the bar-uidA fusion gene under control of the CaMV 35S promoter (Kamo and Han, 2008). The PDS-He gene gun (Bio-Rad, Richmond, CA) was used to bombard embryogenic callus with plasmid DNA, and transformed plants were selected on MS medium containing either 2 mgl−1 dicamba or 1 mgl−1 picloram combined with 1 mgl−1 phosphinothricin (AgroEvo, Pikeville, NC) for selection. Transformed plants were maintained in vitro for seven years before being used for this study. 2.4. UidA expression The uidA gene codes for GUS expression. Leaves were stained histochemically for GUS expression by placing them for 16 h at 37 ◦ C in a phosphate buffer, pH 7.0 staining solution as described by Jefferson et al. (1987) supplemented with 0.1% Triton X-100, 20% methanol, 0.5 mM potassium ferricyanide, and 0.5 mM ferrocyanide. The staining solution was removed and samples were then incubated in 70% ethanol for one week for removal of chlorophyll. Leaves from three-four plants for each transformed plant line were stained, and non-transformed ‘Nellie White’ was used as the negative control.
Fig. 1. Leaf extracts from a non-transformed plant (NT) and plants transformed with pDM327 consisting of the bar-uidA fusion gene under control of the CaMV 35S promoter were used in an ELISA assay to determine relative levels of the bar protein in either 2010 (top graph) or 2011 (bottom graph). Bars indicate the mean±standard error. Bars with different letters have significantly different means between the two years (P ≤ 0.05).
160
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
Fig. 2. RNA levels for the bar gene as determined by semi-quantitative RT-PCR. Plant lines are shown above each lane. Plants were grown in either the greenhouse (GH) or outdoors in pots (O) during either 2010 or 2011. The PCR band for the bar gene is indicated by the arrow in the top gels in A, B, and C. Molecular size markers (M) are indicated in bp in A, and the positive control was pDM327 (+). Each lower gel in A, B, and C shows the relative RNA levels for each sample as indicated by the PCR band for the LhUBQ4 gene.
according to the manufacturer’s instructions. Leaves were collected in a 1.5 ml microcentrifuge tube on ice and then ground in the kit’s extraction buffer using a Kontes pestle. Samples were vortexed 10 s followed by centrifugation at 5000 × g for 5 min. The supernatant was transferred to a new tube and placed at −80 ◦ C. A PAT-enzyme conjugate was added to the wells of an ELISA plate immediately followed by the addition of wash/extraction buffer from the kit, and then 50 l of plant extract were added to each well. The plate was rotated by hand for 30 s and then covered with Parafilm for a 2 h incubation at 25 ◦ C. Wash/extraction buffer from the kit was used to flood the wells, and then the plate was emptied. Substrate was added and the plate incubated at 25 ◦ C for 30 min. A 1 N HCl stop solution was added to the wells, and the plate was read at OD450 nm. The NPT II ELISA Pathoscreen kit (Agdia, Elkhart, IN) was used according to the manufacturer’s instructions to determine expression of the nptII protein in leaf extracts. Leaves had been collected and stored at −80 ◦ C until used. Leaf tissue was ground in the kit’s extraction buffer using a Kontes pestle, and the supernatant (100 l) was recovered after centrifugation at 5000 × g for 5 min. Supernatant was placed in the wells of the ELISA plate along with 100 l of extraction buffer for an incubation at 25 ◦ C for 2 h before the contents of the plate were emptied. Wells were filled with phosphate buffered saline with Tween 20 (PBST) solution, emptied, and this step was repeated 6 times to wash the wells. Conjugate was
added followed by a 2 h incubation at 25 ◦ C. The plate was washed with PBST six times before adding 100 l of the kit’s substrate solution. The plate was incubated 15 min at 25 ◦ C followed by the addition of 3 M sulfuric acid to stop the reaction. The plate was read at OD450 nm. Standards included with the kit were used to construct a concentration curve for determining the nptII concentration of each sample. 2.6. Semi-quantitative RT-PCR RNA was isolated from leaves using the RNeasy Plant Mini kit followed by removal of contaminating DNA using the RNase-Free DNase set (Qiagen, Valencia, CA). Equal amounts of RNA were used from each plant sample for making cDNA with the Thermoscript RT-PCR system (InVitrogen, Carlsbad, CA). The program for cDNA synthesis was 25 ◦ C for 10 min, 62 ◦ C for 50 min, and then 85 ◦ C for 5 min. DNA was treated with RNaseH. PCR was performed using gene-specific primers. The bar PCR primers were forward 5 -GTCAACTTCCGTACCGAGCCGCAG3 and reverse 5 -CATGCCAGTTCCCGTGCTTGAAG-3 to amplify a DNA fragment of 379 bp from the bar gene. The primers forward 5 -TCGGCTATGACTGGGCACAACAGA-3 and reverse 5 -AAGAAGGCGATAGAAGGCGATGCG-3 were used to amplify a 700 bp DNA fragment from the nptII gene. A 253 bp DNA fragment was amplified from the uidA gene
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
2.7. Statistical analysis Three leaf samples were collected for each transformed plant line, and non-transformed ‘Nellie White’ served as the negative control. The leaf extract from each plant line was used to fill duplicate wells of an ELISA plate, and ELISA assays were replicated twice. The background absorbance from a well filled with extraction buffer was subtracted from the absorbance for each well filled with the leaf extract. Duplicate wells of a plate were averaged to obtain a value for each plant line. The mean was calculated from the 12 absorbance values for each plant line, and a Student’s t test used to compare either the greenhouse value with the outdoor value within a given year or the greenhouse, and outdoor values were compared between the two seasons. Means were considered significantly different at P ≤ 0.05.
1.0 0.8
OD450
using forward primer 5 -TAACCTTCACCCGGTTGCCAGAGG3 and reverse primer 5 -CTTTAACTATGCCGGAATCCATCG-3 . Primers forward 5 -GGTATCCCTCCGGACCAG-3 and reverse 5 ATGGTGTCCGAACTCTCCAC-3 amplified a 194 bp fragment from the LhUBQ4 gene of lily as an internal control (Yamagishi, 2011). PCR reactions were run on a MJ Research PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA) programmed at 94 ◦ C for one min followed by 30 cycles of 94 ◦ C for 20 s, 67 ◦ C for 30 s, 72 ◦ C for 2 min, and then one cycle of 72 ◦ C for 10 min. PCR products were separated by electrophoresis on a 1% agarose gel using TBE buffer (0.045 M Tris-borate, 0.001 M EDTA, pH 8.0) and visualized using ethidium bromide.
161
0.6
Greenhouse Outdoor
0.4 0.2 0.0
2011
2010
Plant line 2A Fig. 3. Leaf extracts from a non-transformed plant (NT), and the lily plant line 2A transformed with pCAMBIA2301 consisting of the npt II and uidA gene under control of the CaMV 35S promoter were used in an ELISA assay to determine relative levels of the nptII protein in 2010 and 2011. Bars indicate standard error.
2010 and 2011 following an ELISA assay of the plants were not seen at the RNA level (Fig. 2). There were very weak bands not readily visible on the gel photographs for the bar gene for lines 1B grown both years in either the greenhouse or outdoors, the two lines of 2B grown in 2011, the one line of 6A grown outdoors in 2011, and all four lines of 40(1) grown both years in either the greenhouse or outdoors. The highest to lowest RNA levels were observed to be line 4B, line 4(1), line 6A, line 4C, line 2B, and a bar band was barely visible for lines 1B and 40(1). This correlated with the highest
3. Results and discussion Plant lines 1B, 2B, 4B, 4C, 4(1), 6A, 40(1) were transformed by gene gun bombardment in 2002 with pDM327, and all lines contained the bar gene. Lines 1B, 4B, 4C, and 40(1) have the uidA gene but not lines 2B, 4(1), and 6A. Line 2A was recovered following bombardment with pCAMBIA2301 and contains both the nptII and uidA genes. Transgenic lily plants were previously confirmed to contain the transgene by PCR and Southern hybridization (Kamo and Han, 2008). Inheritance of the bar gene occurred in 15% of the T1 progeny of plant lines 4B, 4(1), and 6A but not lines 4C and 40(1) indicating the possibility that lines 4C and 40(1) may be chimeric (Kamo, 2011). PCR analysis did not provide supporting evidence that any of these five plant lines were chimeric as all leaf samples from three different plants of each plant line were found to contain the transgene even though the plants had been transferred for six years on non-selective media. 3.1. Bar protein and RNA levels Seven lily plant lines transformed with the bar gene produced a protein product that reacted with an antibody to the bar protein as determined by an ELISA assay (Fig. 1). There were no significant differences in bar protein level when transgenic lilies were grown in either the greenhouse or outdoors in 2010. In 2011 two of the seven transgenic lily lines (2B, 4B) grown outdoors showed a statistically higher level of the bar protein than lilies grown in the greenhouse. In comparison, transgenic potatoes showed either similar or higher levels of two transgenes coding for antigens when grown in the field instead of the greenhouse (Mikschofsky et al., 2011). The relative level of bar protein between the lily lines remained the same for lilies grown in the greenhouse and outdoors in 2010 and 2011 (Fig. 1). In both 2010 and 2011, bar expression was highest in line 4B followed by similar levels of expression for both 4(1) and 6A which had higher levels than 4C, and 4C was higher than both 40(1) and 2B that showed similar levels and were higher than line 1B. These relative differences in bar protein levels observed during
Fig. 4. RNA levels for the (A) nptII and (B, C) uidA genes as shown by semiquantitative RT-PCR. Plant lines are shown above each lane. Plants were grown in either the greenhouse (GH) or outdoors in pots (O) during either 2010 or 2011. The PCR band for the nptII gene is indicated by the arrow in the top gels in A and the uidA gene in B and C. The molecular size markers (M) are indicated in bp, and the positive control (+) was pCAMBIA2301 in A and pDM327 in B. Each lower gel in A, B, and C shows the relative RNA levels for each sample as indicated by the PCR band for the LhUBQ4 gene.
162
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
Fig. 5. Leaves from a non-transformed lily (NT) and four transgenic lily lines containing the uidA gene were stained for GUS expression. Plants were grown in the greenhouse (GH) or outdoors in containers (Out) during the 2010 and 2011 growing seasons.
level of bar protein for line 4B followed by 4(1), 6A, and 4C. Levels of the bar protein and bar RNA were low for the remaining lines 2B, 40(1), and 1B. The reason for the significantly higher level of bar protein observed in 2011 compared to 2010 in six of the seven lines is unclear. All plant samples collected in 2010 and 2011 were processed and analyzed the same day using one ELISA kit, but it appears that there was some factor that affected the ELISA but not the RNA results. Expression of the bar gene was stable in lilies because it was not silenced even though the plants were exposed to the high temperature that caused silencing of the pat gene in cell suspensions of Medicago sativa (Walter et al., 1992). Silencing of the bar and pat genes did not occur in Gladiolus plants when plants were subjected to high temperatures (Kamo, 2008) indicating differences in plant species. 3.2. NptII protein and RNA levels Only one plant line was recovered that was transformed with the nptII gene. The relative levels of nptII protein (0.84–0.86 ng/L) were similar when lilies of line 2A were grown in the greenhouse and outdoors during 2010 and 2011 (Fig. 3). RNA levels for the nptII gene were also similar when line 2A was grown in the greenhouse or outdoors for two years (Fig. 4A). 3.3. GUS protein and RNA levels GUS expression was readily detected by histochemical staining in the four lines that contained the uidA gene (Fig. 5). Leaf tissue turned dark blue at the cut regions for lines 4B, 4C, and 40(1) grown in both the greenhouse and outdoors during 2010 and 2011. Only line 1B showed a variable response between plants of this line. Some 1B leaves taken from plants grown in the greenhouse
or outdoors showed faint blue spots and other leaves showed the dark blue staining at the cut regions. Variation between transformants of a single clone has been reported previously. Potato plants transformed with the snowdrop lectin (Galanthus nivalis agglutinin) and bean chitinase under control of the CaMV 35S promoter were found to have variability in the levels of transgene protein produced within each line of clonal replicates (Down et al., 2001). GUS expression was silenced in the lily line 2A transformed with pCAMBIA2301 as there was no leaf tissue that showed a blue reaction following histochemical staining (data not shown). Levels of GUS expression could not be compared for lilies grown in 2010 and 2011 because they were too low for determining the specific activity of -glucuronidase that codes for GUS expression (data not shown). Comparative RNA levels for the uidA gene showed that levels of RNA for the various plant lines were from high to low: 4B, 2A, 4C, and very low as shown by faint bands on the gel for lines 1B and 40(1) (Fig. 4B,C). It appeared that the RNA level of the uidA gene was higher for line 4C when plants were grown outdoors rather than in the greenhouse both years. RNA for the uidA gene was not seen on the gel for line 2A that was transformed with pCAMBIA2301 (data not shown). Levels of GUS expression were significantly different for four of the six Gladiolus plant lines expressing GUS when grown two seasons outdoors and for one of the four plant lines grown two seasons in the greenhouse (Kamo, 2008). GUS and bar expression were observed in poplars growing three seasons in a field, and expression was similar throughout the years for both transgenes (Li et al., 2008). In conclusion, this study showed that the expression of the bar, nptII, and uidA genes was usually similar when transgenic lilies were grown in the greenhouse or outdoors. The relative level of bar protein between the lily lines remained the same for lilies grown in the greenhouse and outdoors in 2010 and 2011. These lilies were transformed nine years ago demonstrating long term,
K. Kamo / Scientia Horticulturae 167 (2014) 158–163
stable expression of the uidA, bar, and nptII genes in lilies when grown under different environmental conditions. Acknowledgements Thanks to Anne O’Connor for excellent technical assistance throughout this study. Funds to support this project were received from the Gloeckner Foundation. pDM327 was received from David McElroy (Verdia, Redwood City, CA). Mention of a trademark, proprietary product or vendor does not imply its approval to the exclusion of other products or vendors that may also be suitable. References Acciarri, N., Restaino, F., Vitelli, G., Perrone, D., Zottini, M., Pandolfini, T., Spena, A., Rotino, G.L., 2002. Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation. BMC Biotechnology 2, 4–8. Down, R.E., Ford, L., Bedford, S.J., Gatehouse, L.N., Newell, C., Gatehouse, J.A., Gatehouse, A.M.R., 2001. Influence of plant development and environment on transgene expression in potato and consequences for insect resistance. Trans. Res. 10, 223–236. Kamo, K., 2008. Transgene expression for Gladiolus plants grown outdoors and in the greenhouse. Sci. Hort. 117, 275–280.
163
Kamo, K., 2011. Inherited transgene expression of the uidA and bar genes in Lilium longiflorum cv. ‘Nellie White’. Floriculture Ornamental Biotech. 5, 35–39. Kamo, K., Han, B.H., 2008. Biolistic-mediated transformation of Lilium longiflorum cv. Nellie White. HortScience 43, 1864–1869. Li., J., Brunner, A.M., Meilan, R., Strauss, S.H., 2008. Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons. Tree Physiol. 29, 299–312. Meng, L., Ziv, M., Lemaux, P.G., 2006. Nature of stress and transgene locus influences transgene expression stability in barley. Plant Mol. Biol. 62, 15–28. Meyer, P., Linn, F., Heidmann, I., Meyer, H., Niedenhof, I., Saedler, H., 1992. Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype. Mol. Gen. Genet. 231, 345–352. Mikschofsky, H., Heilmann, E., Schmidtke, J., Schmidt, K., Meyer, U., Leinweber, P., Broer, I., 2011. Greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression. Plant Mol. Biol. 76, 131–144. Murashige, T., Skoog, F., 1962. A revised medium for rapid assays with tobacco tissue cultures. Physiol. Plant 15, 473–497. Neumann, K., Dröge-Laser, W., Köhne, S., Broer, I., 1997. Heat treatment results in a loss of transgene-encoded activities in several tobacco lines. Plant Physiol. 115, 929–947. Walter, C., Broer, I., Hillemann, D., Pühler, A., 1992. High frequency, heat treatmentinduced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa. Mol. Gen. Genet. 235, 189–196. Yamagishi, M., 2011. Oriental hybrid lily Sorbonne homologue of LhMYB12 regulates anthocyanin biosyntheses in flower tepals and tepal spots. Mol. Breed. 28, 381–389.