Julie R. Kikkert

The Biolistic® PDS-1000/He device

This paper describes the design, operation, and performance of the Biolistic® PDS-1000/He device, which is used to transform living organisms with foreign DNA. DNA is delivered to cells in association with microscopic metal particles, called microcarriers, that are propelled at high velocity towards target tissues. The microcarriers are accelerated on a plastic cylinder, called a macrocarrier, which is driven by a shock wave of helium gas. The effectiveness of the PDS-1000/He device was tested by bombarding tobacco cell suspension cultures with microcarriers that were coated with plasmid DNA containing the B-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII) genes. Two days after bombardment, there were 6835 ± 594 cell clusters per petri plate that expressed the GUS gene. Kanamycin resistant colonies were observed 6 to 8 weeks after bombardment, at a rate of 838 ± 134 colonies per bombarded plate.

Transgenic plantlets of 'Chancellor' grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions

Transgenic plantlets of ‘Chancellor’ grapevine (Vitis L. complex interspecific hybrid) were produced via biolistic transformation. Embryogenic cell suspensions were bombarded with 1 μm tungsten particles coated with pBI426 which encodes a fusion peptide between β-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for transformation of ‘Chancellor’ and should be applicable to other important grape cultivars.

Biolistic transformation of grapevine using minimal gene cassette technology

The use of minimal gene cassettes (MCs), which are linear DNA fragments (promoter+open reading frame+terminator) lacking the vector backbone sequence, was compared to the traditional use of whole circular plasmids (CPs) for transformation of grapevine. Embryogenic cell suspensions of ‘Chardonnay’ (Vitis vinifera L.) were transformed via particle co-bombardment using two nonlinked genes in either MCs or CPs. One construct contained the npt-II selectable marker and the second construct contained the MSI99 antimicrobial peptide gene. A total of five lines each from MC and CP treatments that showed positive signals by PCR for both the npt-II and MSI99 genes were selected. Southern blot analyses revealed up to five integration events in the DNA treatments. Transcription levels determined by semi-quantitative RT-PCR varied among transgenic lines. No significant differences were found in transgene transcription between lines from MC and CP transformation. The correlation between npt-II and MSI99 transcription levels was positive (P<0.05), however, no correlation between the transcription level and the number of integration events was observed. Transgenic lines presented a similar phenotype in leaf morphology and plant vigor compared to non-transgenic lines. Moreover, transgenic lines from both MC and CP DNA treatments produced fruit as did the non-transgenic lines in the third year of growth in the greenhouse. Our data confirm the effectiveness of the minimal cassette technology for genetic transformation of grapevine cultivars.

Optimization of biolistic transformation of embryogenic grape cell suspensions

Embryogenic suspensions of ‘Chancellor’ (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding ß-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 μM kanamycin. Factors that affect biolistic transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 μm (M10) resulted in more transient gene expression than 0.771 μm diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the biolistic device holds much promise as a method to achieve stable transformation of grapevines.

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (Vitis sp.) genotypes

SummaryAnthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.

Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles.

SummaryWe have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.

Grapevine Genetic Engineering

Genetic engineering is a powerful tool for plant improvement. Genes cloned from virtually any biological organism can be inserted singly or in combination into a plant. This allows for targeted improvement of elite cultivars, insertion of genes outside the usual gene pool of a species, and study of gene function. Numerous horticultural and agronomic crops have been genetically engineered worldwide. Already in some commercial markets are insect resistant maize and cotton, herbicide resistant soybeans and maize, and virus resistant papaya and summer squash, for example (Ahl Goy and Duesing, 1995; Dunwell, 1999).

BIOLOGICAL PROJECTILES (PHAGE, YEAST, BACTERIA) FOR GENETIC TRANSFORMATION OF PLANTS

SummaryBacteriophage lambda particles, yeast cells, and bacterial cells were tested as projectiles to deliver marker/reporter genes into plant cells via the biolistic process. When phage particles were complexed to tungsten or gold particles and used to bombard tobacco cells, fewer than 15 cell clusters per plate transiently expressed β-glucuronidase (GUS). Cells of wildtype Saccharomyces cerevisiae were too large to be effective projectiles, but use of a reduced-size mutant resulted in a small number of transformants. Escherichia coli cells complexed with tungsten were the most effective projectile for plant transformation. Various methods to prepare E. coli were tested to reduce particle size, improve binding of bacteria to metal particles, and/or minimize particle clumping. In maize, the number of transformants was highest when bacteria/tungsten particles were air-dried onto macrocarriers from an aqueous solution. When maize cells were bombarded with bacteria/tungsten projectiles, rates of transient gene expression (2000 per plate) and stable transformation (50 per plate) were only two- to threefold lower than when purified DNA was used. Transformation of tobacco with E. coli projectiles was improved when the bacteria were treated with a series of ethanol and ether washes, then dried into a powder. Nevertheless, tobacco transformation was still 24- (transient) and 200-fold (stable) less than when purified DNA was used. Biological projectiles can be effective for plant transformation and are advantageous because once a DNA construct is made and put into the appropriate microorganism, the need to isolate and purify DNA for the biolistic process is eliminated, which saves time and lessens DNA shear. Such projectiles may be especially well suited where high molecular weight DNA constructs are needed.

Phosphinothricin stimulates somatic embryogenesis in grape (Vitis sp. L.)

The effect of phosphinothricin concentration on embryo production from an embryogenic callus of ‘Chancellor’ (Vitis L. complex interspecific hybrid) was tested. Embryogenic callus was cultured on medium supplemented with nine phosphinothricin concentrations (0, 0.1, 0.5, 1, 1.5, 2, 3, 5, and 10 mg/l). The highest number of embryos per plate was observed at 0.5 mg/l phosphinothricin. The use of phosphinothricin to stimulate embryo production did not affect embryo germination and plantlet formation. Three germination techniques were compared. Embryo dehydration or growth on Transfergelsolidified medium gave higher germination rates than chilling treatments. Most germinated somatic embryos produced secondary embryos from the hypocotyl after a few weeks of culture. Regardless of the germination technique, the plantlet conversion rate was very low.

Genotypic Diversity and Resistance to Azoxystrobin of Cercospora beticola on Processing Table Beet in New York

Cercospora leaf spot (CLS), caused by Cercospora beticola, is one of the major diseases affecting productivity and profitability of beet production worldwide. Fungicides are critical for the control of this disease and one of the most commonly used products is the quinone outside inhibitor (QOI) azoxystrobin. In total, 150 C. beticola isolates were collected from two commercial processing table beet fields in Batavia, NY in 2014. The mating types of the entire population were determined, and genetic diversity of a subset of samples (n = 48) was assessed using five microsatellite loci. Sensitivity to azoxystrobin was tested using a spore germination assay. The cytochrome b gene was sequenced to check for the presence of point mutations known to confer QOI resistance in fungi. High allelic diversity (He = 0.50) and genotypic diversity (D* = 0.96), gametic equilibrium of the microsatellite loci, and equal ratios of mating types were suggestive of a mixed mode of reproduction for C. beticola. Resistance to azoxystrobin was prevalent because 41% of the isolates had values for effective concentrations reducing spore germination by 50% (EC50) > 0.2 μg/ml. The G143A mutation, known to cause QOI resistance in C. beticola, was found in isolates with EC50 values between 0.207 and 19.397 μg/ml. A single isolate with an EC50 of 0.272 μg/ml carried the F129L mutation, known to be associated with low levels of QOI resistance in fungi. This is the first report of the F129L mutation in C. beticola. The implications of these findings for the epidemiology and control of CLS in table beet fields in New York are discussed.