Anther culture has been one of the most successful techniques for generating haploid plants over a wide range of species. It is a reasonably simple procedure that can be accomplished successfully without sophisticated laboratory facilities; yet, the plants generated through anther culture can be used to demonstrate the application of many modern methods that have direct applicability to plant breeding. Anthers of diploid potato clones that have been selected for competence in anther culture can be cultured in a simple medium to yield androgenic embryos after 5 weeks. Plant regeneration requires an additional 3 to 4 weeks. Regenerated plants should be large enough 2 weeks after transfer to basal medium for ploidy determination by any of three methods depending on available facilities: chromosome counts in root tips; chloroplast counts in stomatal guard cells; or flow cytometry of nuclei released from in vitro plantlets. DNA can be extracted from anther-derived plantlets using a rapid extraction procedure to demonstrate segregation of PCR (polymerase chain reaction)-based markers such as RAPD (randomly amplified polymorphic DNA), RAMPs (randomly amplified microsatellite polymorphisms), or microsatellites. Microsatellite markers that were heterozygous in the anther donor can be used to verify haploidy in anther-derived plants. If an anther culture laboratory is scheduled early in a semester, such molecular analysis can be planned for late in the same semester.
Richard E. Veilleux
Leslie Blischak* and Richard. E. Veilleux
Gamete selection was examined as a breeding tool in developing Phalaenopsis hybrids that are more cool or warm temperature tolerant. Four pairs of hybrid cultivars of Phalaenopsis were cross-pollinated, and then exposed to two temperature extremes, 30 °C / 25 °C and 14 °C/9 °C, during pollen tube development and subsequent fertilization. One of each pollinated orchid cultivar was placed in either of two growth chambers and exposed to an 11-h photoperiod with an irradiance of 180 μmol·m-2·s-1 and a relative humidity of 70% during the day and 50% at night for 3-7 days depending on the temperature treatment. The plants were returned to the greenhouse after the initiation of fruit set and the pods were collected after 150 days. Seeds collected from these treatments were surface-sterilized, placed on Phytamax medium and evaluated for protocorm development after 73 days on a thermogradient table ranging from 10 to 30 °C. For the first family for which reciprocal crosses were available, the number of protocorms per plate ranged from 0 in the coldest treatments to 290 at 28 °C. For cold pollinated seeds, protocorm development was optimum at 22 and 28 °C (means of 290 and 250 protocorms per plate, respectively) whereas the greatest protocorm development for warm pollinated seeds occurred at 20 °C (103 protocorms per plate). Of the 1471 total protocorms scored, 1095 were from cold pollinations, whereas 376 were from the warm pollinations. Additional replication is required to confirm the greater germinability of cold-pollinated seed at higher temperatures.
Chongkhohao Singsit and Richard E. Veilleux
A technique was developed to count chloroplasts in guard cell pairs of leaflets of anther-derived potato (Solanum phureja Juz. and Buk) plants grown in vitro to determine how they compare to chloroplast counts for similar plants grown in vivo. The mean chloroplast density in guard cells of in vitro leaflets was visualized using fluorescein diacetate (FDA) stain and observed under a microscope. A consistently higher mean was observed for in vitro than for in vivo plants but monoploid could readily be distinguished from diploid regenerants whether grown in vitro or in vivo. A rapid method of ploidy estimation based on chloroplast counts on in vitro leaflets may hasten identification of monoploids to be used for in vitro applications to genetic reconstruction of potato.
Rose E. Palumbo and Richard E. Veilleux*
A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. Hemizygous primary transformants (To) were produced from three leaf discs: 17 diploid plants from one leaf disc, three and nine tetraploids from the other two leaf discs. GFP expression was observed qualitatively under fluorescence microscopes and quantitatively with a GFP meter. Segregation ratios for tetraploid T1 seedlings fit models for single duplex insertions (35 transgenic: 1 non) or double simplex insertions (15 transgenic: 1 non). Diploid T1 seedlings segregated for deleterious traits: dwarfed size and curled leaves, as well as the GFP transgene. Similar segregation patterns in diploid families implied that all diploids may have been from the same transformation event. The cumulative segregation showed the dwarfed and curled plants fit a single recessive gene ratio (3 normal: 1 mutant), and GFP fit a double-copy insertion ratio (15 transgenic: 1 non). Six T1 selections were free of deleterious traits, consistently high expressers of GFP, and produced fertile pollen.
Michael E. Compton and Richard E. Veilleux
Genetic recombination rates of hybrid plants regenerated from three tissue culture. systems were compared by backcrossing regenerated plants with mutant parents and comparing the observed crossover frequencies with those expected based on control plants raised from seed. Increased recombination rates and map distances were observed among plants from micropropagated shoot tips (4.5%-5.9%), cotyledon calli (3.7%-8.5%), and thin cell layers (2.8%-6.5%) between the sunny (sy) and baby leaf syndrome (bls) markers which flank the centromere on chromosome 3. Conversely, a decrease in map distance was observed between bls and the solanifolia (sf) locus which is more distal to the centromere on the same arm of chromosome 3 as bls. Increased map distance among plants regenerated from micropropagated shoot tips, cotyledon calli, and thin cell layers was also observed between white virescence (wv) and anthocyanin reduced (are) loci on chromosome 2.
Phillip A. Wadl* and Richard E. Veilleux
In order to facilitate the high throughput transformation required to use Fragaria vesca (wild strawberry) as a tool in genomic research, functional genomics, and gene discovery not only for strawberry but for fruit crops in general, we need to increase its regeneration frequency and transformation efficiency using Agrobacterium. Ten accessions of F. vesca representing a range of germplasm with worldwide distribution were obtained from the USDA National Germplasm Repository, Corvallis, Ore. for use in shoot regeneration experiments. Seed germination with or without vernalization ranged from 0% to 90%. In vitro growth varied for the accessions with five accessions eliminated from further experiments due to poor growth. In preliminary experiments with 125 leaf explants and 40 petiole explants combined representing PI 551573, PI 602923, and F. vesca `Alpine'; 100% of the uncontaminated explants regenerated at least one shoot after 8 weeks on medium supplemented with 1 mg·L-1 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron or TDZ) and 0.2 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D). In a replicated study of `Alpine' comparing regeneration on the above TDZ/2,4-D medium with control medium [0.25 mg·L-1 indole-3-butyric acid (IBA)/3 mg·L-1 benzyladenine (BA)], regeneration frequency at 6 weeks for leaf or petiole explants on control medium was 8% (n = 180) compared to 27% (n = 210) on the TDZ/2,4-D medium. This optimized shoot regeneration protocol for F. vesca `Alpine' is currently under investigation in transformation experiments with several other accessions and Agrobacterium constructs.
Leslie A. Blischak and Richard E. Veilleux
Gamete selection was examined as a breeding tool in developing Phalaenopsis hybrids that are more extreme temperature tolerant. Four pairs of hybrid cultivars were cross-pollinated, and then exposed to two temperature extremes, 30 °C/25 °C and 14 °C/9 °C, during initial pollen tube growth. One of each pollinated orchid cultivar was placed in either of two growth chambers and exposed to an 11-hour photoperiod with an irradiance of 180 mmol·m-2·s-1 and a relative humidity of 70% for 3–7 days. The capsules were collected after 150 days. Protocorm development was evaluated after 73 days on a thermogradient table ranging from 10 to 30 °C. For cold-pollinated seeds, protocorm development was optimum at 22 and 28 °C (means of 290 and 250 protocorms per plate, respectively), whereas the greatest protocorm development for warm-pollinated seeds occurred at 20 °C (103 protocorms/plate). Protocorms were evaluated for leaf and root formation 125 days after initial plating. Transfer to warm or cold incubators occurred as seedlings matured. One year after the initial plating, seedlings were evaluated on the following criteria: weight, number of leaves, leaf width, leaf length, leaf area, number of roots, and root length. The pollination treatment significantly affected the number of roots per seedling, whereas table position during germination significantly affected weight. The weight, number of leaves, and average root length were significantly affected by the pollination treatment and incubator interaction. The interaction between pollination treatment and table position significantly affected weight, number of roots, and average root length. Additional replication is required to confirm the greater germinability of cold-pollinated seed at higher temperatures.
Richard E. Veilleux and A. Raymond Miller
F1 hybrids between high leptine-producing clones (8380-1, PI 458310 and 55-1) of Solanum chacoense Bitt. and anther culture competent or anther-derived clones of S. phureja Juz. & Buk. that did not produce leptines were generally weak plants that grew slowly and died before flowering. Exceptional hybrids could be found that were capable of completing a life cycle, especially during the hot summer months in the greenhouse. All F1 hybrids produced leptines in the leaves but not the tubers, albeit at lower levels than in the S. chacoense parent. Anther-derived monoploids from the F1 hybrids exhibited a range of leptine production from none to levels approaching the S. chacoense parent. Backcross populations of an F1 hybrid to the S. chacoense and S. phureja parents were examined for leptine production. Backcross hybrids were generally much more vigorous than the F1 hybrids. All of the S. chacoense backcrosses produced leptines ranging from intermediate to high levels; four of the twelve S. phureja backcrosses exhibited low leptine levels. A general dominance of leptine synthesis was therefore exhibited, although the nonleptine-producing parent affected the expression of leptines in the hybrids.
Yeun-Kyung Chang, Richard E. Veilleux and Muhammad Javed Iqbal
Phalaenopsis is the second most valuable potted plant in the United States. Information on the genetic diversity and relationships among species and hybrids is important for breeding purposes and species conservation. In this study, genetic variability of 16 Phalaenopsis species and hybrids was analyzed using amplified fragment length polymorphism (AFLP) markers. Ten AFLP primer combinations amplified 1353 DNA fragments ranging in size from 100 to 350 bp and 1285 (95%) of them were polymorphic. The genetic similarity among Phalaenopsis species and hybrids ranged from 0.298 to 0.774 based on Dice coefficient. The dendrogram derived by the unweighted pair group method with arithmetic mean analysis clustered the germplasm into two main groups. Bootstrap values for the groups supported 70% of the clustering. A significant linear relationship (r = 0.724, P < 0.0001) was observed between known pedigrees and AFLP-derived genetic similarity for 136 pairwise comparisons of Phalaenopsis species and hybrids. The results of this study demonstrate the usefulness of AFLP analysis in Phalaenopsis and its potential application in breeding and species conservation.