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Erika Szendrák and Paul E. Read

The temperate native terrestrial orchids are endangered species. Their propagation from seeds poses specific problems. It is well known that orchid seeds are devoid of endosperm and in nature they need microscopic fungi in a symbiotic relationship for germination. We developed a successful asymbiotic in vitro culture method for germinating seeds of several temperate orchid species and for maintaining the cultures of young plantlets. The medium used for both germination and seedling culture was a modified FAST medium. Seeds were surface-disinfested for 10 minutes in a 10% calcium hypochlorite solution. After sowing, the cultures were kept under dark condition at 10–12°C for 4 weeks. After that the cultures remained in the dark, but the temperature was raised to 25–26°C until germination occurred. Thereafter cultures required alternating seasonal temperatures: 25–26°C from the beginning of April to the end of September and 17–19°C from October to March. For the development of the young plantlets natural dispersed light and prevailing day-length was favorable. After 2 years of aseptic culture they were suitable for transfer ex vitro. Different stages of seed germination and plant development were observed using a scanning electron microscope and will be included in this presentation. Further observation of the effects of different environmental factors is currently under investigation.

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Erika Szendrak and Paul E. Read

The effects of organic compounds most commonly used for orchid micropropagation and the physical condition of the medium were investigated for the development of young temperate orchid protocorms. Separate experiments were conducted with five different temperate orchid species: Dactylorhiza fuchsii, Dactylorhiza maculata, Dactylorhiza majalis, Orchis morio, and Ophrys lutea. Small 2- to 4-mm-wide protocorms were placed in baby food jars (three per jar) containing 50 ml modified FAST medium (Szendrak and R. Eszki, 1993) supplemented with one of eight treatments in a split-plot design with five replications. Both the liquid medium (gyrotary shaker, 125 rpm) and the gelled medium (8 g agar/L) were supplemented with one of the following compounds: 2 g peptone/L; 100 ml coconut water/L; 1 g casein+1 g lactalbumin/L; and 10 g glucose/L as a treatment with a defined compound. All treatments were kept in the dark at 25°C. The number of protocorms/jar were counted weekly over a 6-week-long period and the size and fresh weight of protocorms were measured at the end of the 6th week. In most cases, the liquid medium increased proliferation and the size of the protocorms. However, generally after the 4th week on liquid medium, the development of the protocorms often stopped, but it continued on the gelled medium till the end of the experimental period. The media supplemented with the undefined organic compounds showed a much better effect than the medium supplemented with glucose. Generally peptone and coconut water led to the best development of protocorms, but this varied with species. The development of protocorms into plantlets was normal in all cases.

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Virginia Miller-Roether, Paul E. Read, and Erika Szendrak

The American Chestnut Foundation (ACF) has conducted a breeding program aimed at developing blight-resistant chestnut trees exhibiting the phenotype of American Chestnut (Castanea dentata). We developed a protocol for in vitro micropropagation and multiplication of candidate blight-resistant plants from the ACF breeding program. The protocol included forcing dormant shoots to budbreak, culture establishment, shoot multiplication, inducing a functional root system on the microcuttings produced by this system and establishment of autotrophic plants. Because Castanea spp. is recalcitrant to rooting, a unique bilayer method of rooting was developed. The unique bilayer consisted of a clear basal medium of 50% DKW and 50% WPM (Long and Preece), with a continuous level of 0.01 mg IBA/L and 0.2 mg BA/L. The clear basal medium was over-laid with an opaque layer. Rooting response occurred for 27 of the 31 genotypes at various frequencies. Rooted plantlets were planted in 50% peat: 50% perlite in order to become autotrophic and acclimated. Acclimated trees were planted in 10″ × 2″ Deepots® and placed in the greenhouse. These trees exhibited a very vigorous functional root system. Acclimated trees were hardened off, placed in cold storage (≈4-5 °C) for 5 months. All trees placed in cold storage broke dormancy for spring growth and ≈100 trees were sent to ACF for planting into field trials.

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Virginia I. Miller, Paul E. Read, and Erika Szendrák

The American Chestnut Foundation (ACF) has conducted a breeding program aimed at developing blight-resistant chestnut trees exhibiting the phenotype of American Chestnut [Castanea dentata (Marsh) Borkh]. Because such plants are difficult to propagate, we developed a protocol for in vitro multiplication of candidate blight-resistant plants resulting from the ACF breeding programs. Dormant shoots were taken from 5- to 8-year-old trees and forced, producing softwood growth for use as a source of explants for shoot multiplication. Best shoot proliferation took place on WPM containing 0.2 mg BA/L. Explant material for the rooting experiments was taken from 6- to 12-month-old proliferating cultures. The basal rooting medium consisted of WPM containing 0.01 mg IBA/L and was overlaid with a thin opaque layer. Rooting was enhanced overall with this bilayer approach. A “D/W” medium (DKW and WPM) was also used as a rooting medium containing 0.01 mg IBA/L and 0.2 mg BA/L, which further enhanced leaf quality and rooting for some genotypes. After several transfers on the bilayer system, explant growth appeared to become less juvenile in stem and leaf development and more analogous to mature later-season growth. The rooting responses and the time for rooting to be induced were highly variable among the different genotypes.

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Erika Szendrák, Paul E. Read, Eszter R. Eszéki, Elizabeth Jámbor-Benczúi, and Aniko Csillag

Cultures of several orchid species [Barlia robertiana (Loisel.), Dactylorhiza fúchsii Soó, D. incarnata (L.) Soó, D. maculata (L.) Soó, D. majalis (Rchb.), D. saccifera (Brong) Soó, D. sambucina (L.) Soó, Gymnadenia conopsea (L.) R.Br., Himantoglossurn hircinum (L.) Spreng., Ophris sphegodes Mill., Orchis coriophora ssp. fragrans L., Orchis laxiflora ssp. palustris Lam., Orchis mascula L., Orchis morio L., Platanthera bifolia (L.) Rich., Spiranthes aestivalis (Poir.) Rich.] were initiated with fresh ripe seeds from desiccated fruit and 4-month-old in vitro seedlings. The medium used for both germination and seedling culture was a modified FAST medium. Samples for the scanning electron microscope (SEM) surveys were taken from the in vitro cultures and some plant materials were collected from their native habit. Samples were observed with a Tesla BS 300 SEM. Seeds ranged from 300 to 450 μm in length and were flask-shaped. The first germination step is opening of the seedcoat, when the first few white cells will be visible. After a few weeks, the apical meristem appears. The young protocorm is covered with numerous translucent rhizoids. In the last stage of germination, the first root and the first true leaf start to develop. After 2 years, they are suitable for transfer ex vitro. Structure of the mature organs and tissues can be examined at this stage.