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Freddi A. Hammerschlag*, Sandra Garces, Margery Koch-Dean, Stephanie Ray, Kim S. Lewers, John L. Maas and Barbara J. Smith

Diseases affecting strawberries have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose caused by the fungus Colletotrichum acutatum is one of the most serious disases of strawberry worldwide. Although chemical controls are being used to treat anthracnose, generating disease resistant plants is a more attractive solution to the problem because chemicals can pose a health hazard, have a negative impact on the environment and may only be moderately effective. Tissue culture-induced (somaclonal) variation provides us with one strategy for generating disease-resistant genotypes. An in vitro screening system was used to evaluate several commercially important cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie, and shoots regenerated from leaf explants of these cultivars for resistance to C. acutatum isolate Goff (highly virulent). Somaclones with increased levels of anthracnose resistance were identified for all the cultivars. The greatest increases in disease resistance were observed for somaclones of cultivars Chandler, Pelican and Sweet Charlie that exhibited 6.8-, 12-, and 4.2-fold increases in resistance, respectively. These studies provide evidence that: 1) in vitro screening can be used to evaluate strawberry germplasm for anthracnose resistance, 2) soma-clonal variation is influenced by stawberry genotype, and 3) generating somaclonal variants may be a feasible approach to obtaining strawberry plants with increased levels of anthracnose resistance.

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Luisa Santamaria and Sherry Kitto

Solanum quitoense, also known as naranjilla or lulo, is a native species of Ecuador and Colombia. Its value is based on the uncommon sweet-sour flavor of its fruits, which is appreciated in the national and international markets. The worst problem for this crop is the root-knot nematode Meloidogyne incognita. The main objective of our research is to develop root-knot nematode–resistant naranjilla via somaclonal variation. Seeds of Solanum quitoense `Baeza' germinated quicker than those of `Dulce'. Seeds given a 2-week dark treatment had 100% germination compared to 75% germination for seeds placed under lights (16-h photoperiod, 60 mmol·m–2s–1). Single-node explants proliferated an average of nine nodes after 1 month of culture. Microcuttings (two nodes, 3.5 cm) stuck in sand and placed under a humidity dome under mist had an average of five roots averaging 25 cm in length after 3 weeks. Stems regenerated shoots better than petioles or leaves and explant orientation/polarity had no effect on regeneration. Root cultures of Solanum quitoense inoculated in vitro with Meloidogyne incognita showed susceptibility to root-knot nematodes.

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Mark Bridgen

Alstroemeria, also known as Lily-of-the-Incas, Inca Lily, or Peruvian Lily, has been bred at the Univ. of Connecticut since 1985. In vitro procedures have been integrated with traditional breeding techniques to create new and exciting cultivars. Embryo culture has been used to generate interspecific, intraspecific, and intergeneric hybrids that would not have been possible with traditional breeding. Somaclonal variation has been used to create new plants from spontaneous and induced mutations, but, in most cases, the plants have not been acceptable commercially. Chromosome doubling with colchicine has been used for fertility restoration of sterile diploids. Somatic embryogenesis has also been studied quite extensively; somatic embryos are easily obtained from zygotic embryos of Alstroemeria. In vitro fertilization procedures are currently being studied in order to hasten embryo development after hybridization has occurred. Because Alstroemeria plants are slow to propagate by traditional rhizome division, micropropagation is used to multiply new cultivars rapidly. Because the production of pathogen-free plants is one of the goals of our breeding and new plant introduction programs, meristem culture and thermotherapy are also being studied. All of these techniques will be described during the workshop.

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David A. Evans

For many branded food products, the raw material is purchased as a commodity. Food companies provide added-value based on processing technology or marketing. The tools of plant biotechnology, which shorten the time for crop improvement and permit development of novel germplasm, offer the food industry the opportunity to modify raw materials and develop proprietary branded products. Such modifications will permit development of plant cultivars specifically selected for traits with added-value for the processor or the consumer. Biotechnology-developed cultivars offer the opportunity to develop produce that can be branded. The cell genetics tools of clonal propagation, somaclonal variation, gametoclonal variation, and protoplast fusion permit new cultivar development in an intermediate time scale, making them attractive for market introduction of proprietary products. When integrated with conventional breeding, these intermediate-term technologies will permit modification of fruits and vegetables over the course of the next several years. The longer-term technologies of plant genetic engineering will continue to have an impact on manipulation of specific traits, resulting in second-generation products specifically designed for consumers.

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Richard Meilan, Caiping Ma and Steven H. Strauss

We assessed the stability of transgene expression in 79 transgenic lines (i.e., transformation events) of hybrid poplars during several years of field trials. The transgenic lines were comprised of 40 lines of hybrid cottonwoods (P. trichocarpa × P. deltoides) that were grown at three field sites, and 39 lines of hybrid aspens (section Leuce, P. alba × P. tremula) that were grown at a single field site. All the lines were transformed with a binary construct that included two genes that confer tolerance to glyphosate (GOX and CP4), a gene encoding resistance to the antibiotic kanamycin (nptII), and a visible marker gene (GUS). Agrobacterium tumefaciens was used for transformation; callogenesis and organogenesis occurred under kanamycin selection. In addition to repeated applications of herbicide to test stability of transgene expression, for the first time, we challenged ramets of 40 lines that had not previously been tested for herbicide resistance in their fourth season of vegetative growth. We report on the stability of herbicide resistance and GUS expression and evidence for somaclonal variation in growth and leaf morphology.

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Jinggui Fang, Chih Cheng Chao, Richard J. Henny and Jianjun Chen

Plant tissue culture can induce a variety of genetic and epigenetic changes in regenerated plantlets, a phenomenon known as somaclonal variation. Such variation has been widely used in the ornamental foliage plant industry as a source for selection of new cultivars. In ornamental aroids alone, at least 63 somaclonal-derived cultivars have been released. In addition to morphological differences, many somaclonal aroid cultivars can be distinguished by amplified fragment length polymorphism (AFLP) analysis. However, a few cultivars have no detectable polymorphisms with their parents or close relatives by AFLP fingerprints. It is postulated that DNA methylation may be involved in the morphological changes of these cultivars. In this study, methylation-sensitive amplification polymorphism (MSAP) technique was used to study DNA methylation in selected somaclonal cultivars of Alocasia, Aglaonema, Anthurium, Dieffenbachia, Philodendron, and Syngonium. Results showed that polymorphisms were detected in the somaclonal cultivars, suggesting that DNA methylation polymorphisms may associate with tissue culture-induced mutation in ornamental aroids. This is the first study of methylation variation in somaclonal variants of ornamental foliage plants. The results clearly demonstrate that the MSAP technique is highly efficient in detecting DNA methylation events in somaclonal-derived cultivars.

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Li Xu, Suzhen Huang, Yulin Han and Haiyan Yuan

An improved three-stage protocol for plant regeneration via somatic embryogenesis of the horticulturally important plant Iris germanica L. was developed using shoot apex segments as explants. At the first stage of the experiment, 60% of callus was obtained from shoot apex segments of I. germanica on Murashige and Skoog’s (MS) medium supplemented with 4.52 μm 2,4-dichloropheoxyacetic acid (2,4-D) and 0.44 μm 6-benzyladenine (6-BA). When nonembryogenic calli were subcultured on MS medium with 11.31 μm 2,4-D and 0.44 μm 6-BA, maximum frequency of embryogenic callus (66.0%) was obtained. At the second stage, the treatment of 9% (w/v) sucrose resulted in the optimum somatic embryo (SE) formation (70.0%). More than 90.0% of SEs germinated with bipolar structure and regenerated into plantlets on plant growth regulator-(PGR)free MS medium during the third stage. Regenerated plantlets were successfully acclimatized in greenhouse environment with little somaclonal variation. Histological study showed that somatic embryogenesis stages were asynchronous and SEs developed from the surface and inner tissue of embryogenic calli.

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Reynato P. Umali, Nanako Kameya and Ikuo Nakamura

The banana (Musa sp., AAA) genome is continuously expanding due to the high frequency of somaclonal variation. Because of this increasing diversity, numerical and morphological methods of taxonomic and phylogenetic identification of banana cultivars became laborious, difficult, and often the subject of disagreements. The aim of this study, therefore, is to develop molecular tools for DNA fingerprinting that can discriminate Musa, AAA Cavendish subgroup cultivars. In this paper, we showed that the plastid-subtype identity (PS-ID) sequence of the noncoding region between rpl16 and rpl14 genes of plastid DNA was highly conserved except for single-base substitution and deletion. These differences separated the clones into three groups (G1, G2, and G3) and suggested that clones within groups are closely related maternally. Using arbitrary primer A13, we later identified negative RAPD markers A133.0 and A131.3 specifically for S4 (selection from Giant Cavendish subgroup, AAA) and S11 (`Morado' from `Red' and `Green Red' subgroup, AAA), respectively. Fragments corresponding to the missing bands were sequenced and used as templates to design new primers with overlapping sequences. Two of these primers, Ba3.0A and Ba1.3A, successfully generated positive markers consistently amplified as Ba3.0A0.8 and Ba1.3A0.6 for S4 and S11, respectively. It is proposed that the method just described can be a better alternative over screening more arbitrary primers in generating positive markers in cases when negative ones were already identified. Results of PS-ID subtype analysis likewise suggested potential use in identifying wild maternal progenitor in polyploid bananas.

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Mohammad Sadat-Hosseini, Kourosh Vahdati and Charles A. Leslie

regenerated from somatic embryos could exhibit somaclonal variation ( Bradaï et al., 2016 ). Heritable adverse changes due to somaclonal variation can be a problematic in tree-breeding programs, which typically encounter long juvenile phases before phenotypic

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Takahiro Tezuka, Masashi Harada, Masahumi Johkan, Satoshi Yamasaki, Hideyuki Tanaka and Masayuki Oda

( Johkan et al., 2008a , 2008b , 2008c , 2011 ). In addition to these studies, evaluation of the genetic stability of regenerated plants is required. Plants regenerated from tissue culture often show somaclonal variation ( Larkin and Scowcroft, 1981