Cypripedium is a genus of temperate terrestrial ladyslipper orchids of excellent garden size, forming clumps or 12 - 18 inches in diameter or more, with very showy flowers. Many species are easy to grow in northern gardens (USDA zones 2-7) and have excellent horticultural potential. Today, several nurseries grow a few species and hybrids from seed, although a number of garden-worthy species have not yielded to propagation by seed germination. Several exceptionally garden-worthy hybrids have been developed, but as with the hybrids of many orchids and other flowering plants, vigor and flower appearance varies from specimen to specimen. At present no method of vegetative mass production of superior cultivars is available for Cypripedium. The goals of this research are 1) further optimization of methodology to rapidly induce plantlet formation from callus derived from a wide range of horticulturally superior cypripedium hybrids, and 2) development of grow-out methodology that results in the survival of a large percentage of plantlets into flowering plants within four years from deflasking. Success in this program will allow large scale production of cypripedium cultivars for provision of superior cultivars to the floricultural market. Orchid production is valued at $140 million wholesale in the U.S. Unlike tropical orchids, which are optimally produced in southeast Asia, cypripedium are optimally produced in the northern U.S. While many floriculturally important orchids including the Cattleya alliance, Cymbidium, and Phalaenopsis are mass produced using in vitro micropropagation techniques, ladyslipper orchids have thus far not yielded to micropropagation using commercially relevant methodology. Unlike the seed of many epiphytic orchids, seed of terrestrial orchids is difficult to germinate due to the introduction during seed maturation of growth inhibitory factors that enable seed to survive the harsh weather conditions of winter. Only beginning in the 1980s has germination from seed of some species of cypripedium become routinely practiceable, and the first generally successful protocols for seed germination and maturation of seedlings appeared in the mid-1990s. Indeed, while hybrids of many orchid genera have been registered since the mid-1800s, the cypripedium hybrid was registered in 1991. The availability of uniform, vegetatively mass-produced offspring of commercially important orchids such as Cattleya, Cymbidium and Phalaenopsis dramatically transformed the worldwide markets for hybrids of those species. The market for orchids in the U.S. has been rapidly growing, from $47 million at the wholesale level in 1995 to $144 million in 2005. Virtually all of these orchids are produced using vegetative micropropagation. Today, most mass production of these tropical species occurs outside the U.S., particularly in southeast Asia where the climate dramatically reduces production costs. In contrast, Cypripedium grow in temperate climates and require prolonged freezing conditions during the winter. This makes many regions of the U.S. particularly attractive regions in which to establish commercial production. OBJECTIVES: While many floriculturally important orchids are mass produced using in vitro micropropagation techniques, Cypripedium have thus far not yielded to micropropagation using commercially relevant methodology. Orchids of various genera have been successfully micropropagated from shoot buds, node buds, root tips and leaf explants. In previous attempts at development of methods for micropropagation of Cypripedium, bud explants, root tip explants, nodal explants and leaf explants have been used. In most protocols young Cypripedium seedlings still in flask were used as sources of explant materials. Flasked Cypripedium seedlings do not produce shoot buds typical of older plants. The goals of this research are 1) further optimization of methodology to rapidly induce plantlet formation from callus derived from a wide range of horticulturally superior cypripedium hybrids, and 2) development of grow-out methodology that results in the survival of a large percentage of plantlets into flowering plants within four years of deflasking. Success in this program will allow large scale production of cypripedium cultivars for provision of superior cultivars to the floricultural market. Specific Aim 1: Evaluate and optimize the the micropropagation method developed for Cypripedium reginae (Section Obtusipetala) root tip explants in species from other commercially important subgenera and hybrids. Anticipated Results. As a result of our success with Cypripedium reginae (Phase I grant), we anticipate that conditions may be optimized for species and hybrids from all of the sections of the genus Cypripedium. Specific Aim 2: Optimize a method to induce callus formation and differentiation into plantlets using Cypripedium dormant lateral bud explants. As a backup method of micropropagation, we will also attempt to induce plantlet formation from lateral buds. While lateral buds are generally a less attractive option for micropropagation than root tips owing to the damage rendered to the donor plant, there are circumstances in which it is desirable to have such a method available. Anticipated Results. Cytokinin treatment of dormant lateral buds has been used to induce callus growth. Specific Aim 3: Develop a method that reliably supports maximum growth rate of micropropagated Cypripedium plantlets into flowering size plants. Wild Cypripedium require up to 12 years to produce flowering plants from seed. Laboratory-propagated Cypripedium plants generally require about five-six years to grow to flowering size, although a few plants grown from seed from a given pod may bloom in their fourth year. If plants can be grown to reliably flower after four years, considerable labor resources may be saved, making it possible to dramatically reduce prices that must be charged for these horticulturally important plants. Anticipated Results. Cypripedium require constant moisture around their roots but at the same time require a highly aerated medium or roots and rhizome will rapidly rot with loss of the plant. Hydroponics conditions are expected to allow optimization of automatic watering and fertilization cycles with the goal of growing seedlings rapidly to maturity. APPROACH: Specific Aim 1. We will evaluate our methods optimized to induce callus formation and differentiation of Cypripedium reginae root tip meristem into plantlets in species from Section Cypripedium and Section Macrantha as well as in several important hybrids. Harvais medium with potato homogenate will form the baseline medium. Callus formation will be observed for 16 weeks under conditions of differing ratios of auxin to cytokinin. When callus formation is observed, it will be removed and explanted to new flasks containing either: 1) the same growth factors as the donor flask, 2) only auxin at the same concentration as the donor flask, 3) only cytokinin at the same concentration as the donor flask, or 4) no growth factors. Specific Aim 2. As a backup method of micropropagation, we will also attempt to replicate and extend preliminary observations of plantlet formation from lateral buds. High cytokinin concentration alone has been shown to induce plantlet formation from lateral buds, a standard technique to reduce apical dominance of shoot meristem. All flasks will be incubated at 25 C in the dark and examined for growth once per week. There will be 3 general growth conditions: a) no complex additive will be included in the media, b) potato homogenate in the media, and c) coconut water in the media. In each series of groups, varying ratios of auxin to cytokinin will be used. Specific Aim 3. Wild Cypripedium require up to 12 years to produce flowering plants from seed. Laboratory-propagated Cypripedium plants generally require five-six years to grow to flowering size, although a few plants grown from seed from a given pod may bloom in their fourth year. If plants can be grown to reliably flower after four years, labor resources may be saved. Plantlets will be deflasked in the fall and vernalized over the winter for May plant-out. All plantlets will be grown in the same shade house under 70% shade cloth. Plantlets will be maintained in plastic flats. Plantlets will be grown in the following groups: a) under a regimen of daily watering from overhead at noon with continuous fertilization using complete hydroponics medium diluted to maintain concentration at 100, 300, or 600 ppm; b) under a regimen of daily watering from overhead at noon with fertilization at concentrations of 100, 300, or 600 ppm one time each week; c) under ebb-and-flow hydroponics conditions with twice daily flooding; d) under ebb-and-flow hydroponics conditions with six times daily flooding at ambient (measured) temperature; and e) under ebb-and-flow hydroponics conditions as in group 4, but with water maintained at 80 F. Temperature 1 cm below the surface of the media will be monitored and compared to air temperature 1 meter above the flat. Temperatures of flats will be assessed prior to and after watering at noon (overhead watering) and 4 PM (ebb-and-flow). Plantlets typically go dormant beginning in late September. Thus, during year 1, on August 15 when growth for the season is complete, plantlet height, leaf number and length, wet weight and root weight will be determined. Identical measurements will be made on August 15 of year 2.
