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Richard K. Kiyomoto and Mark H. Brand

Experiments were conducted on tissue proliferation (TP) development and in vitro and ex vitro growth of tissues from plants with (TP+) and without TP (TP-). In 1993 the increase in TP in one-, two-, and three-yr-old `Holden' and `Besse Howells' was 3%, 52%. and 32% and 10%, 26% and 21%, respectively. No differential mortality was observed. Shoot tip cultures initated from TP+ and TP- `Montego' showed 10-12 mo were required for miniaturiziation and multiplication in TP- shoot tips and 4 mo in TP+ shoot tips. TP- cultures require 10 uM 2-iP for normal shoot proliferation; whereas TP+ cultures had to be transferred to hormone-free medium after 6 mo to maintain normal shoot morphology. Cutting propagation from TP- and TP+ plants older than 5 yr, showed persistence of morphological aberrations associated with TP+ plants.

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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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S.M. Eichorst, R.B. Rogers, and M.A.L. Smith

Use of a liquid media during micropropagation has promoted improved proliferation and rooting response in several species. In this experiment, a double phase system (a combination of liquid and agar solidified medium) was applied to three cultivars of miniature roses (Rosa chinensis var. minima) to determine the effects on shoot quality and subsequent ex-vitro rooting. Applications of liquid media to the surface of agar solidified media were made at 0, 2, and 4 weeks. Evaluation via computerized image analysis after eight weeks of proliferation revealed equal or greater values for shoot length, area and weighted density (equivalent to fresh weight) for cultures receiving overlay, regardless of timing, compared to the solid media control. Additionally, application of a liquid overlay improved rooting response by up to 20% over the control and resulted in a tendency for a greater number of roots of greater length and area than the treatment without liquid media overlay.

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Les Frey, Yehoshua Saranga, and Jules Janick

Somatic embryogenesis was induced from internodal callus of `Scania', `Improved White Sim', and `Sandra' carnation (Dianthus caryophyllus L.). The optimum protocol for the induction of somatic embryogenesis included initiation of callus in liquid basal Murashige and Skoog medium supplemented with 3.0 μm 2,4-D followed by transfer to liquid basal medium lacking 2,4-D for embryo development. Somatic embryos originated from single cells and early embryonic development proceeded conventionally (i.e., via globular, heart-shaped, and torpedo stages), but clearly developed apical or root meristems were not always formed. A few embryos developed into seedlings and were acclimatized to ex vitro conditions. Chemical name used: 2,4-dichlorophenoxyacetic acid (2,4-D).

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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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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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Yves Desjardins and André Gosselin

Strawberry plantlets (Fragaria X ananassa Duch. cv. Kent) were submitted to a factorial arrangement of 2 photosynthetic photon fluxes (PPF) (80 and 150 μmol·m-2·s-1, PAR) and 2 CO2 concentrations (330 and 3000 ppm) during the in vitro rooting stage. Leaves were tagged and placed in a growth chamber tor acclimatization. Photosynthetic capability of leaves from different origins was determined by measuring the initial and total activity of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco), but the contribution of Phosphoenolpyruvate carboxylase (PEPCase) to fixation was also examined High CO2 concentration and PPF significantly increased fresh weight and surface area in vitro and after 4 weeks ex vitro. Improved growth was not the result of increased autotrophy in vitro since initial rubisco activity was 10 times lower than that of de novo formed leaves and declined under high CO2 and PPF. Carbon dioxide concentration and PPF had no effect on total activity of rubisco. Low activation state and total activity of rubisco in in vitro leaves is the cause of poor photosynthetic activity in vitro Persistent in vitro leaves after 4 weeks of acclimatization did not have higher total activity of rubisco, but the activation state was 4 times larger than the corresponding activity in vitro which might thus provide for non-negligible contribution to photosynthetic carbon assimilation. The possible inhibition of photosynthesis by the presence of sugar in the medium is discussed.

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Michael E. Kane, Edward F. Gilman, Matthew A. Jenks, and Thomas J. Sheehan

Procedures for in vitro establishment, rapid shoot proliferation, and ex vitro plantlet acclimatization of Cryptocoryne lucens de Witt were determined. Shoot cultures were established from surface-sterilized shoot tips cultured on Linsmaier and Skoog salts and vitamins medium (LS) solidified with 0.8% (w/v) agar and supplemented with 2.0 μm BA and 0.5 μm NAA. The effect of BA (0 to 20 μm) and 0.5 μm NAA on shoot multiplication from single-node and clustered triple-node shoot explants was determined after 35 days. The most efficient shoot proliferation (7.7 shoots/explant) occurred from single-node shoot explants cultured on LS + 20 μm BA and 0.5 μm NAA. Maximum plantlet establishment was achieved by direct sticking of triple-node (cluster) microcuttings in either soilless planting medium or polyurethane foam cubes. Production of highly branched salable plants from microcuttings was possible within 18 weeks. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).

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Michael Marcotrigiano and Susan P. McGlew

In an effort to accelerate breeding programs and to study somaclonal variation, a micropropagation system was devised for cranberries (Vaccinium macrocarpon). Using a factorial design, explants taken from greenhouse grown plants were placed on Anderson's medium containing different concentrations of 2ip' GA3, and IBA, with 4 cultivars tested over 3 subcultures. In other experiments, explant source, macro and micro salt formulations, and rooting treatments, were studied. Optimal multiplication and shoot quality occurred when single node explants taken from greenhouse grown plants were placed on Anderson's media containing 150 uM 2iP, 1.0 uM IBA and no GA3. Histological examinations indicate that initial response is axillary bud proliferation but upon subculture adventitious shoot formation may be possible. Proliferated shoots could be rooted ex vitro in plug trays under plastic tents and without hormone treatments. Optimal rooting occurred under high light conditions in a 1:1 (v:v) peat:sand mix. Plants were easily transplanted into the field in spring and will be evaluated by comparison to conventionally propagated material.