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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.

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Surawit Wannakrairoj

Patumma, a native of Thailand, is a new ornamental crop. Its increased demand for export make the species vulnerable to extinction. Cloning methodology is thus needed for the production of both existing and newly developed clone. Young inflorescent segment and lateral bud from rhizome can be used as explants. For decontamination of the lateral bud, the success was depended both on pre-treatment and disintestation procedure. The bud from dried rhizome was better than one from fresh rhizome. Prior to disinfestation with sodium hypochlorite, pre-treatment of rhizome in 52°C water for 5 minutes could replaced the use of antibiotic. Plantlet were placed on modified MS media with 0, 6.67, 13.32, 19.98 and 26.64 μmol/l benzyladenine (BA) or 0.19, 0.56, 1.67 and 5 μmol/l kinetin. The best multiplication rate of 4.83 fold was obtained when longitudinally-divided rhizome was on the medium with 13.32 μmol/l BA. The result also showed that wild-collected and selected clones responded to the media similarly. When the MS media modified with 13.32, 15.54, 17.76 and 19.98 μmol/l BA in combination with 15, 30 and 45 g/l sucrose were tested, the multiplication rate of non-divided plantlets were all the same. Acclimatization by open the culture vessel for 3 day improved plantlet survival rate ex vitro.

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Jung Eek Son, Yil Jang, and Jung Hyuk Seo

Supporting materials for rooting have a considerable influence on the growth and quality of in vitro plantlets. Various supporting materials (rockwool, perlite, vermiculite, and polyurethane) and nutrient supply cycles (12, 24, 36, and 48 hours) were examined to find the optimum conditions for photoautotrophic micropropagation of potato plantlets in the nutrient-circulated micropropagation (NCM) system. In the NCM system, nutrient solution was circulated between the culture vessel and the nutrient reservoir. A plug cell tray with 70 plantlets was placed inside. The number of air exchanges was 10 hours under forced ventilation. Nodal leafy cuttings of plantlets were cultured at CO2 concentrations (mol·mol-1)/PPF s (mol·m-2·s-1) of 350/80, 700/120, and 1500/250 on day 5-11, 12-18, and 19-28, respectively, for all treatments. All growth factors of in vitro plantlets grown for 28 days using rockwool, perlite and vermiculite were greater than those grown using polyurethane. Dry weight of plantlets grown using rockwool was eight times greater than those grown using polyurethane. The same results were obtained in the growth and survival percentages 14 days after transplanting to ex vitro conditions. Optimum nutrient supply cycles were 12, 24, and 48 hours when perlite, rockwool, and vermiculite were used as supporting materials, respectively. It was considered that the range of optimum nutrient supply cycle was affected by water retention characteristics of supporting materials. This study proved that the supporting material and the nutrient supply cycle were very important environmental factors in photoautotrophic mass propagation.

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Nancy Phiman and Michael E. Kane

Beach stabilization by replanting dune species such as Uniola paniculata L. (Sea Oats), is an accepted practice to control erosion in the southeastern United States. Increased restrictions on collection of sea oat seed and plant material for propagation is of increasing concern. Development of micropropagation protocols for establishment and production of sea oats from donor plants of known phenotype would be useful for selecting and producing plants with commercially valuable characteristics. Terminal and lateral shoot tips (3 mm wide and 4 mm high) from containerized plants were surface sterilized and established on Linsmaier & Skoog mineral salts and organics supplemented with 87.6 mM sucrose, 2.2 μM benzyladenine solidified with 0.8% TC® Agar. Terminal tiller shoot tips were more responsive than lateral shoot tips. Four monthly subcultures were. required for stabilized shoot multiplication from culture lines established from terminal tiller shoot tips. Shoot organogenesis frequently occurred from the cut leaf surfaces of subcultured shoot clusters. Microcuttings were established ex vitro in plug cells containing sand or vermiculite.

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Darren Touchell, Zenaida Viloria, and Thomas Ranney

Weigela Thunb. consists of 12 species distributed throughout Northeast Asia. Diervilla Mill. is a closely related genus containing three species endemic to North America. Taxa from both of these genera are important nursery crops. Hybrids between these genera could potentially combine the excellent cold hardiness and adaptability of Diervilla with diverse forms, foliage colors, and flower colors found in Weigela. Prior attempts to create intergeneric hybrids between these genera were unsuccessful and resulted in embryo abortion before seeds matured. To overcome this barrier, ovule culture and micropropagation procedures were used to develop intergeneric hybrids. Cleaved amplified polymorphic sequences (CAPS) analysis was used to verify hybrids. Intergeneric crosses, D. lonicera × W. middendorfiana, D. sessilifolia × W. florida (two clones), and D. lonicera × W. florida were attempted. Crosses of D. lonicera × W. middendorfiana did not produce viable hybrids. From the remaining three crosses, a total of 544 plants were obtained from 1278 ovules. About 85% of the 544 plants appeared very chlorotic or had low vigor, and senesced when transferred to multiplication medium. Only 80 of the 544 plants were successfully maintained in tissue culture, of which 10 have been successfully transferred ex vitro. CAPS analysis indicated that a majority of these plants were hybrids. Further studies are focused on improving tissue culture procedures and other methods to develop tetraploids to increase plantlet vigor and fertility.

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Karim H. Al-Juboory, J. Al-Naimi, L.K. Al-Amiry, R. Shibli, and R.M. Skirvin

Callus was initiated from leaves of Gladiolus cv. `Balady' on MS medium containing 1.0 mg/L NAA, 0.1 mg/L 2,4-D, and 0.5 mg/L kinetin. Organogenesis from callus was induced on medium containing 0.5, 1.0, 1.5, or 2.0 mg/L of either BA, kinetin, or TDZ. TDZ was more effective and resulted in a higher percentage regeneration and regenerant number. The microshoots produced were then propagated in vitro and cormel production was studied. Maximum shoot number (25.1) was obtained on medium containing 1.0 mg/L TDZ without auxin supplements in liquid shaking culture. In vitro cormel formation was significantly enhanced by B-9 and paclobutrazol. Increased sucrose concentration (4% to 5%) proved the most effective for cormel formation. Optimal dormancy break was obtained by storing cormels at 5°C for 1 month or by soaking them for 5 sec with 50 mg/L GA3. In-vitro rooting was achieved on solid medium containing NAA, IAA, or IBA, with higher root number recorded on NAA-treated cultures. Rooted microshoots were successfully acclimatized for ex vitro conditions and grown in the greenhouse. Plants produced from in-vitro propagation showed similar morphological characteristics of plants propagated by direct corm planting in the greenhouse.

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Wenhao Dai, Cielo Castillo, and Victoria Magnusson

In vitro shoot cultures for two birch species, Asian white birch (Betula platyphylla) and paper birch (Betula papyrifera), were initiated from shoot tips of mature trees and maintained in MS (Murashige and Skoog) medium containing 3% sucrose and 5–10 μM (micromolar) benzyladenine (BA). The effect of such factors as genotype, basal medium, and plant growth regulator (PGR) on proliferation was investigated. Shoots were proliferated in both MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ), BA, and kinetin (Kin). Two birch species responded differently to these factors. In general, more shoots were proliferated in WPM than in MS medium. The maximum proliferation rate of Asian white birch was achieved by being cultured in WPM containing 4–8 μM TDZ, while paper birch gave rise to the maximum proliferation rate in WPM supplemented with 20 μM BA. Interactions between genotype and medium or cytokinin were found. Shoots produced on media with TDZ had thick stems and small, dark green leaves. Microshoots can be rooted both in vitro and ex vitro with or without IBA treatment. Plants were regenerated from leaf tissues of Asian white birch. Adventitious shoots regenerated when in vitro leaves were cultured on WPM supplemented with 10–20 μM BA with 2-week dark treatment. The effect of genotype, PGR, and culture condition on in vitro regeneration of birch species is being tested.