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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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Mark Bridgen

The potential value of somaclonal variation for economically important plants is well-documented. The process of somaclonal variation can arise from a controlled or a random source of variation. Variability can be obtained by applying cellular pressures and selection. Valuable resistance to diseases and nematodes has already been accomplished with somaclonal variation; now, plant tolerance to pests has been realized. Tetranychus urticae, the two-spotted spidermite, and Trialeurodes vaporariorum, the greenhouse whitefly, were disinfected and introduced to aseptic shoot cultures of Torenia fournieri. These pests were allowed to feed until such time that their populations decreased due to the absence of food. The plant cells that remained after feeding were induced to form adventitious shoots and plantlets. These regenerated plantlets were acclimated to greenhouse conditions and evaluated for tolerance to the pest to which they were subjected in vitro. Highly significant differences were found in somaclones for both the two-spotted spidermite and greenhouse whitefly when compared to control plants. A wide range of variability was observed among the somaclonal population. There were significantly fewer mite eggs laid on plants regenerated from in vitro cultures screened with two-spotted spidermites than on seed-sown controls. Regenerants from cultures screened with whiteflies in vitro had fewer eggs, immatures and live adults than controls. The potential for somaclonal variation to be used as a method to develop insect resistant plants will be discussed.

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Mark P. Bridgen

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Elizabeth Kollman and Mark Bridgen

Alstroemeria, the Inca lily or lily-of-the-Incas, is becoming a popular garden plant in the United States. In past years, the primary interest in Alstroemeria has been for its cut flowers. However, recent cold-hardy introductions (USDA hardiness zone 5) have expanded the interest of this colorful plant as a garden perennial throughout the U.S. Previously, garden interests were restricted to warmer zones in the southern United States where Alstroemeria could over-winter. This research describes a breeding procedure which has been used with the objective to develop a cold-hardy, white flowered Alstroemeria. The interspecific hybrids were bred with the use of in ovulo embryo rescue. Reciprocal crosses were made between several white-flowered cultivars and the cold hardy Chilean species, Alstroemeria aurea during the summers of 2004 and 2005. Ovaries were collected 10–23 days after hand pollination and their ovules were aseptically excised. Ovules were placed in vitro on 25% Murashige and Skoog (MS) medium under dark conditions until germination. Three weeks after germination they were then placed on 100% MS medium, and subcultured every three to four weeks thereafter until they were large enough for rooting. After rooting and acclimation, plants were transferred to the greenhouse. Successful hybrids that were produced in 2004 were evaluated under greenhouse and field trials during 2005. Data on the flower color for each of the hybrids were recorded, as well as certain morphological characteristics that can indicate cold-hardiness. Hybrid plants are being overwintered outside in Ithaca, N.Y. (USDA zone 5), and Riverhead, N.Y. (USDA zone 7), during the next several years for a more accurate assessment of cold-hardiness. Self pollinations and reciprocal crosses with the white-flowered parent were performed on the F1 generation in the summer and fall of 2005 in order to determine segregating characteristics. Few ovules were obtained from F1 generation crosses. Successful F2generation plants are being grown in vitro and will be transferred to the greenhouse where flower color will be noted. Root squashes and pollen staining were completed to determine ploidy levels and assess male sterility of the F1 generation.

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Chunsheng Lu and Mark Bridgen

Self-pollinations of a diploid (2n = 2x = 16) interspecific hybrid from the cross of Alstroemeria aurea × A. caryophyllaea resulted in no seed set. Pollen viability studies with the hybrid demonstrated that only 5% of the pollen grains were viable. Cytological observations with the hybrid pollen mother cell (PMC) revealed abnormal chromosome behaviors, such as no pairing in Prophase I and Metaphase I, and bridges in the Anaphase I and II. Although the development of microspores appeared normal in shape until the stage of tetrad release, some chromosomes did not remain in the nucleus after completing meiosis, formed isolated groups of 1 to 4, and remained in the cytoplasm. This genetic imbalance of the microspores could be one of the causes for the abortion of the pollen grains in the late stage of development. Additional meiotic cytological studies with colchicine-induced tetraploids (2n = 4x = 32) derived from the hybrid plants showed that chromosome pairings were normal in most cases. However, self-pollination with the tetraploid plants failed to set seeds. These studies with the tetraploids further demonstrate that the sterility of the hybrid is due not only to chromosomal differences, but also to complex genic interactions.

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Chunsheng Lu and Mark Bridgen

An interspecific hybrid of Alstroemeria aurea × Alstroemeria caryophyllaea was rescued by immature ovule culture and was completely sterile. To restore the fertility of the hybrid, young, vigorous shoots and buds were treated aseptically with three colchicine levels (0.2, 0.4, and 0.6% in DMSO solution) at four treatment durations (6, 12, 18, and 24 hours), before being cultured onto a shoot regeneration medium for regrowth and development. The growth and development of all treated shoots were retarded by the colchicine. New shoots were regenerated from 61% of the surviving cultures after one month. The degree of recovery was not significantly different among treatments, although the highest concentration (0.6%) and the longest time treatment (24 hours) resulted in some morphological abnormalities. Cultures with newly regenerated shoots/buds were able to initiate roots and, eventually, sixty plantlets were transplanted into the greenhouse after acclimatization. Cytological examination of the root tip cells of the plantlets indicated that tetraploids (2n=4x=32) as well as aneuploids plants were generated from the colchicine treatment, whereas all plants from the control were diploids (2n=2x=16). Details explaining cytological changes and the fertility of the colchiploids will be presented.

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Chunsheng Lu and Mark Bridgen

During the winter of 1991-92. four cultivars of Alstroemeria: `F-180'. `l-5'. `Parigo Pink' and `Parigo Red' were treated with eight different overwintering covers: straw, straw with plastic covering, sawdust, sawdust with plastic covering, hoops with plastic covering, hoops with microfoam covering, microfoam and a control with no cover. All covers had significant effects on the survival of `Parigo Pink' and `Parigo Red'; mulching with straw only gave the best winter protection. There were also significant genotypic differences among the four cultivars: 73% of `Parigo Pink' and `Parigo Red' plants survived after winter, but none of `F-180' or `l-5' survived. In addition, pre-winter evaluation indicated that there were significant genotypic differences among the four cultivars with cold resistance. The cold resistance was highly correlated with winter hardiness. It was concluded that: (1) pre-winter evaluation could be an efficient indicator for winter hardiness selection on Alstroemeria and (2) application of straw provided sufficient winter protection for zone 6 Alstroemeria. Other approaches of mulching need to be further identified in order to protect all Alstroemeria for overwintering in the northeastern United States.

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Eduardo Olate and Mark Bridgen

The effect of harvesting procedures on the production of cut flowers of Alstroemeria was determined by growing six different cultivars from three different response groups with three different harvesting techniques. The orchid-type cultivars tested were `Parigo Red' and `Parigo Pink', the butterfly-type cultivars were `FreedomP', `PatriotP', and `Patricia Lynn', and the hybrid-type cultivar was `Sweet LauraP'. The different harvesting treatments were “cutting,” “pull,” and “no harvesting” (control). Cultivars and treatments were evaluated for date of harvest, number of florets per flowering stem, stem length and number of flowering stems produced per plant. The harvest period covered week 14 to week 34 of 1999. The number of florets produced per inflorescence was not affected by any harvesting treatments. The average length of the flowering stems with butterfly-type and hybrid-type cultivars of Alstroemeria decreased over the period of harvest. These two types of cultivars did not differ from each other as a result of the harvesting technique, but did differ from the control plants that were not harvested. The stems from control plants of the butterfly-type and hybrid-type cultivars were longer than those of the harvested plants. The orchid-type cultivars did not exhibit this “short stem effect” as the other two types of cultivars. In both orchid-type cultivars (`Parigo Red' and `Parigo Pink') and in the hybrid-type cultivar (`Sweet LauraP') the total number of flowering stems that were produced was greater with the “cutting” technique than with the “pulling” technique of harvest. In the case of butterfly-type cultivars, there was no difference of the number of flowering stems produced between the two harvesting systems, but they differed from the control plants.

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Mark P. Bridgen

Traditional and biotechnological breeding techniques are being united to develop exciting new plants and to improve existing cultivated plants by introducing natural variability from germplasm resources. Intervarietal, interspecific and intergeneric crosses can be accomplished by using plant embryo culture techniques, sometimes also referred to as embryo rescue. Embryo culture involves the isolation and growth of immature or mature zygotic embryos under sterile conditions on an aseptic nutrient medium with the goal of obtaining a viable plant. The technique depends on isolating the embryo without injury, formulating a suitable nutrient medium, and inducing continued embryogenic growth and seedling formation. The culture of immature embryos is used to rescue embryos from hybrid crosses that were once thought to be incompatible because they would normally abort or not undergo the progressive sequence of ontogeny. The culture of mature embryos from ripened seeds is used to eliminate seed germination inhibitors, to overcome dormancy restrictions, or to shorten the breeding cycle. New and exciting cultivars of Alstroemeria, also known as Lily-of-the-Incas, Inca Lily, or Peruvian Lily, have been bred by using zygotic embryo culture; these techniques and applications will be discussed.

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Mark P. Bridgen