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José M. Iriondo, Carmen Moreno, and César Pérez

Micropropagation methods for six rockrose species (Cistus albidus L., C. clusii Dunal, C. ladanifer L., C. laurifolius L., C. psilosepalus L., and C. salvifolius L.) were established. Cultures, initiated from nodal segments of seedlings, were grown on MS medium, alone, or supplemented with 0.88 μm BAP or 0.93 μm Kin. Multiple shoot formation was obtained after the first subculture (30 days) from which new nodal segments were taken and grown on the same culture medium to maintain proliferation. Shoots obtained at the third subculture were rooted alone or supplemented with different concentrations of IBA. The plantlets of the six species, thereby obtained, were successfully acclimatized to ex vitro conditions. Chemical names used: 6-benzylaminopurine (BAP), indole-3-butyric acid (IBA), 6-furfurylaminopurine (Kin).

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Charlotte R. Chan and Robert D. Marquard

Traditional seed propagation (warm/cold stratification) was compared to embryo culture of Chionanthus virginicus L. to determine if germination could be promoted and time necessary to produce a sizable plant could be reduced. Embryos of C. virginicus were extracted from immature fruit collected 9, 16, and 23 Aug. 1995 and grown in vitro on Anderson's rhododendron medium. They germinated in 4 weeks and were transferred ex vitro to flats. Mature fruit from the same source were grown simultaneously using warm/cold stratification. The two groups were evaluated periodically over a 2-year period for percent germination, plant size, and seedling success. The embryo-cultured plants had a lower survival rate (16% vs. 44%) and were more labor intensive. After 2 years, embryo-cultured plants were 13.4-fold the mass and 4.7-fold taller than traditionally grown plants. Ten-month-old cultured plants were comparable in size to 2-year-old plants grown traditionally.

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Rida A. Shibli, M. Ajlouni, A. Jaradat, and M. Shatnawi

Some factors that affect the in vitro conservation of wild pear (Pyrus syrica) microshoot cultures were studied. Sorbitol and mannitol at 0.2 to 4.0 M reduced growth significantly and extended the subculture intervals to 5 months when cultures where kept at 15°C. Increasing sucrose to 12% in the medium was not highly effective and the subculture intervals did not exceed 3.0 months. After 2 years of maintaining cultures on slow-growth medium, cultures grew slowly when transferred to fresh control medium. Shoots started to proliferate after three subcultures (6.0 weeks apart) on medium containing 1.0 mg/L BA and 0.1 mg/L NAA. New microshoots were rooted on medium containing 2.0 mg/L IBA and rooted microshoots gave 90% survival when acclimatized ex vitro under intermittent mist.

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Karim H. Al-Juboory

Shoots of greenhouse-grown Pothos were surface disinfested and explanted on modified Murashige and Skoog (MS) medium. Later they were treated with pulsed XeCl excimer laser radiation for 30 sec. Cultures treated with 12 or 25 pulses of excimer laser radiation showed only 23% and 10% contamination, respectively, versus 75% control. Inaddition, we demonstrated that pulsed XeCl excimer laser radiation affected the subsequent growth and regenerability of in vitro plants. The reason for this increased growth needs further investigation. Both BA and TDZ were important for increasing the number of shoots generated from a microshoot as well as inducing shoot organogenesis from Pothos callus. Of the 50 rooted ex vitro plants from this experiment only 30% were variegated like parental clone. The others were either pure green or albino, suggesting chimeral segregation.

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Laura A. Dellevigne, Jeffrey W. Adelberg, and Peter Vergano

Three-dimensional polypropylene enclosures have been fabricated for the in vitro culture and ex vitro growth of Cattleya orchid propagules. The enclosures consist of: 1) microporous polypropylene membrane for nutrient transfer between liquid media and the growing tissue. 2) molded polypropylene side wall sized for growth of Cattleya orchid plants and flanged to allow heat seals with membranes, and 3) polypropylene membrane(s) top member for light and gaseous transmission. Three commercial clones of Cattleya have been sealed into these enclosures and grown for eight months on unmended MS medium. Contaminated liquid media was effectively isolated from the propagules within the sealed enclosures, and following a bleach treatment with sterile rinses, propagules were returned to aseptic culture. Greenhouse growth of plant tissues in these enclosures will be discussed. Optimization for growth of Cattleya has begun with studies of gas, light and temperature regimes within the sealed enclosures and a comparison of growth on two different nutrient formulations.

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R. Lowe and D. Donnelly

Minituber production was investigated using ex vitro `Norland' plantlets in a rockwool-based hydroponic system. Productivity was evaluated for 12- and 16-h photoperiod pre-treatment, planting density (two, four, and six plantlets/slab), vertical or horizontal orientation, pinching, and hilling. Total yield differences did not result from photoperiod pre-treatments, but 12-h pre-treatment increased the number of minitubers in the desirable 10- to 40-g size range. Increased planting density reduced yield per plant but caused small increases in yield per slab. Planting orientation, pinching, or hilling had no effect on overall fresh weight yield, number, or size distribution. Short photoperiod pre-treatment, and planting densities of four to six plantlets/slab, oriented vertically, are recommended.

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Michio Kanechi, Masakatsu Ochi, Michiko Abe, Noboru Inagaki, and Susumu Maekawa

The effects of natural ventilation and CO2 enrichment during the rooting stage on the growth and the rates of photosynthesis and transpiration of in vitro cauliflower (Brassica oleracea L.) plantlets were investigated. In vitro plantlets were established in airtight or ventilated vessels with or without CO2 supplied (≈1200 μg·L-1) through gas permeable films attached to the vessel's cap for 15 days before transplanting ex vitro. Leaves generated in vitro in ventilated vessels had a higher photosynthetic rate than those produced in airtight vessels, which lead to greater leaf expansion and shoot and root dry matter accumulation during in vitro culture and acclimatization. Enhanced photosynthesis in leaves of ventilated plantlets was positively correlated with chlorophyll content. Increasing photosynthetically active radiation from 70 to 200 μmol·m-2·s-1 enhanced the growth of in vitro plantlets under ventilated conditions but it depressed photosynthesis of the leaves grown photomixotrophically with sugar and CO2 enrichment which might be due to the feedback inhibition caused by marked accumulations of sucrose and starch. Higher CO2 levels during in vitro culture enhanced photosynthesis under photoautotrophic conditions, but inhibited it under photomixotrophic conditions. Fifteen days after transplanting ex vitro, high photosynthetic ability and stomatal resistance to transpiratory water loss of ventilated plantlets in vitro had important contributions to rooting and acclimatization. Our findings show that the ventilated culture is effective for accelerating photoautotrophic growth of plantlets by increasing photosynthesis, suggesting that, especially for plantlets growing in vitro without sugar, CO2 enrichment may be necessary to enhance photosynthetic ability.

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Kris Pruski, Tina Lewis, and Mohyuddin Mirza

Chokecherries and pincherries are commonly used in landscaping. Some of the selections such as `Garrington', `Mary Liss' and `Jumping Pound' have large fruits of good quality suitable for food processing. The species are also very well adapted to severe winter conditions of the Prairie Provinces. In our studies, in vitro propagation of the selections was undertaken. The best results with initiation of cultures were observed when dormant buds were used as explants on MS medium with 30 g/L sucrose, 0.1 mg/L NAA and 1.0 mg/L BAP (4 wks, 24/22°C day/night, 16 hrs photoperiod 3000 lux). Optimal proliferation in both species was on MS medium with 1-2 mg/L BAP, 80 mg/L AdSO4 and 170 mg/L NaH2PO4. Rosettes produced were placed on medium without hormones prior to rooting. Rooting was performed ex vitro in root-trainers (soilless mix) on the greenhouse bench under mist. Basal dip in commercial rooting powder Stimroot 1 (0.1% IBA) was equally effective to spray application (2 mg/L IAA with 0.5 mg/L NAA). Average of 77% rooting with `Garrington, 72% and 81% rooting with `Jumping Pound' and `Mary Liss' was observed respectively.

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Paula M. Pijut

Butternut (Juglans cinerea L.), a native hardwood to the northeastern United States, is a valuable species for its wood and edible nuts. Butternut is becoming endangered in its native range as a result of a virulent fungal (perennial canker) pathogen, Sirococcus clavigignenti - juglandacearum. Micropropagation techniques are being developed to clone disease-resistant specimens. Axillary buds, obtained from 2-3-month old seedlings, were induced to break buds in vitro and form a single shoot when cultured on Murashige and Skoog (MS) medium supplemented with 200 mg/l casein hydrolysate, 3% sucrose, and 2 mg/l 6-benzylaminopurine. Roots were initiated on microshoots when cultured on half-strength MS medium containing 100 mg/l casein hydrolysate, 1.5% sucrose, and 0.5 mg/l indole-3-butyric acid for seven days in the dark. Adventitious roots elongated when shoots were placed in the light on the same medium, but with 2% sucrose, and no growth regulators. Rooted plantlets were successfully acclimated ex vitro. These results provide a basis for the development of techniques to micropropagate selected, mature, disease-resistant butternut germ plasm.

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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.