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C. Kubota, N. Abe, T. Kozai, K. Kasahara and J. Nemoto

`HanaQueen' tomato plantlets were cultured under conditions with different levels of sugar, photosynthetic photon flux, CO2 concentration, and number of air exchanges of the vessel. Effects of medium substrates (Gelrite or vermiculite) and explant preparation (with or without leaves) on growth of the plantlets were also examined. After 20 days in culture, photoautotrophically cultured plantlets with leafy explants, under increased PPF, CO2, and ventilation rate of the vessel had twice as much dry weight as those cultured conventionally with non-leafy explants under low PPF, CO2, and ventilation rate of the vessel. Dry weight of the plantlets was significantly greater when cultured with leafy than non-leafy explants. Net photosynthetic rate of the plantlets increased linearly as culture period when cultured without sugar, and remained almost zero when cultured with sugar, regardless of other culture conditions. Results obtained in this experiment have shown that tomato plantlets can be grown photoautotrophically, and the net photosynthetic rate was greater under photoautotrophic than under conventional photomixotrophic conditions.

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Chieri Kubota, Chalermpol Kirdmanee and Toyoki Kozai

Cymbidium (cv. Lisa rose) PLB (protocorm-like bodies) were cultured in liquid 1/2 MS medium with/without 20 mg g-1 sucrose under continuous lighting conditions. The vessels were shaken at 100 rpm under PPF (photosynthetic photon flux) of 20 and 140 μmol m-2 s-1 and CO2 concentrations outside the vessel (Cout) of 450 and 2000 μmol mol-1 conditions. Photoautotrophic growth was obtained at high PPF and high Cout. The chlorophyll content of the PLB in the medium without sucrose at high PPF and high Cout was almost 3 times that with sucrose at low PPF and low Cout. The number of newly developed PLB with sucrose at low PPF and low Cout was 1.6 times that without sucrose at high PPF and high Cout; the dry weight per unit PLB with sucrose at low PPF and low Cout was almost 3 times that without sucrose at high PPF and high Cout. Photoautotrophic growth of the PLB might be further promoted at higher CO2 concentration (> 1%).

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Meijun Zhang, Duanduan Zhao, Zengqiang Ma, Xuedong Li and Yulan Xiao

production. The losses during acclimatization accounted for 20% to 50% of the plantlets ex vitro. Photoautotrophic micropropagation (PA), using a sugar-free medium and leafy explant, in which plantlets use CO 2 in the air as the sole carbon source, has

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How-Chiun Wu and Chun-Chih Lin

vitro conditions. The use of photoautotrophic propagation to improve overall explant growth has been successful in numerous plant species. As described by Kozai and Kubota (2005) , the main advantages of photoautotrophic propagation are: 1) stimulation

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Jeffrey Adelberg, Kazuhiro Fujiwara, Chalermpol Kirdmanee and Toyoki Kozai

Two triploid clones of melon from the same tetraploid parent were grown in vitro with and without sugar, rooted without sugar in media both in a laboratory controlled environment chamber (in vitro) and a greenhouse acclimatization unit (ex vitro), and compared for subsequent nursery growth in the greenhouse unit. The clone `(L-14 c B) × L-14' produced more shoots in both photomixotrophic (with sucrose) or photoautotrophic (sugar-free) conditions. Both genotypes were equally likely to root in sugar-free media, and `(L-14 × B) × L-14' rooted as well from either photoautotrophic and photomixotrophic shoots but `(L-14 × B) × Mainstream' rooted less frequently from photoautotrophic shoots. Seventy-six percent (76%) of the shoots were able to root photoautotrophically in vitro, whereas 47% of the ex vitro shoots were rooted. About 85% of plantlets from all treatments survived after transfer to the nursery. After growth in the greenhouse nursery, the sizes of plants (fresh and dry weight, leaf area) were the same for either clone, from either photoautotrophic or photomixotrophic shoots. Also, after growth in the nursery, plantlets that had been rooted in vitro were larger than those rooted ex vitro. Photoautotrophic rooting demonstrates a concept for integrating micropropagation and plug-type vegetable transplant production.

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Chieri Kubota, Natsuko Kakizaki, Toyoki Kozai, Koichi Kasahara and Jun Nemoto

Nodal explants of tomato (Lycopersicon esculentum Mill.) were cultured in vitro to evaluate the effects of sugar concentration, photosynthetic photon flux (PPF), CO2 concentration, ventilation rate of the vessel, and leaf removal on growth and photosynthesis. After 20 days of culture, the dry weights of plantlets derived from explants with leaves and cultured photoautotrophically (without sugar in the medium) under high PPF, high CO2 concentration, and high ventilation rate were more than twice as great as those of plantlets derived conventionally from explants without leaves and cultured photomixotrophically (with sugar in the medium) under low PPF, low CO2 concentration, and low ventilation rate (107 and 45 mg per plantlet, respectively). Under photomixotrophic micropropagation conditions, the dry weights of plantlets from explants with leaves increased more than did those of plantlets from explants without leaves. High PPF, high CO2 concentration, and high ventilation rate increased net photosynthetic rate and promoted growth of the plantlets under photomixotrophic micropropagation conditions. Photomixotrophic conditions produced the greatest dry weight and the longest shoots, but photoautotrophic conditions produced the highest net photosynthetic rate. The number of leaves did not differ significantly between photoautotrophically and photomixotrophically cultured plantlets. Thus, photoautotrophic micropropagation is applicable to the production of high quality tomato transplants.

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Hope Jones and Chieri Kubota

In vitro culture of orchid plantlets within conventional photomixotrophic micropropagation (PMM) systems (sucrose containing media in a non-enriched CO2 environment) often induces vigorous growth and multiplication. However, transition to ex vitro conditions frequently results in significant plantlet loss during the acclimatization process. Recent studies investigating micropropagation within photoautotrophic (PAM) systems (sucrose-free media in enriched CO2 conditions) have demonstrated improved plantlet survival during the acclimatization period due to greater root growth and stomata adaptation. Laelia purpurata var. alba, an orchid with many endangered relatives, was chosen as a model orchid species to investigate if plantlet culture within PAM in vitro systems has the potential to improve propagation success and ex vitro survival of endangered Laelia species. Protocorm-like bodies with developed two fully extended leaves were transferred into PMM (photosynthetic photon flux 50 μmol·m-2·s-1 under non-enriched CO2 conditions) and PAM (photosynthetic photon flux 150 μmol·m-2·s-1, CO2 level enriched to 1500 μmol·mol-1) systems. After 6 weeks, plantlet rooting within the PMM system was variable and inconsistent, while all PAM plantlets produced healthy robust root systems. Average fresh weights and percent shoot development were not significantly different between treatments. Induction of improved root growth by PAM systems may improve orchid plantlet survival rates during acclimatization and advance our ability to increase endangered orchid populations.

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Sandra B. Wilson, Nihal C. Rajapakse and Roy E. Young

Hosta (Hosta tokudama Makeawa `Newberry Gold') plantlets were micropropagated photoautotrophically (without sucrose in medium) or photomixotrophically (with 2% sucrose in medium) for 3 weeks at 23 °C under 80 μmol·m-2·s-1 photosynthetic photon flux (PPF) prior to long-term storage. Plantlets were stored for 4, 8, or 12 weeks at 5, 10, or 22 °C in darkness or under white (400-800 nm), blue (400-500 nm), or red (600-700 nm) light at or near light compensation points. Illumination during storage was necessary to maintain dry weight and regrowth potentials of plantlets in vitro, but light quality had no effect on these parameters. All photoautotrophic plantlets stored in darkness were of poor quality at the time of removal from storage and died when transferred to the greenhouse. Dark-stored photomixotrophic plantlets survived storage for 12 weeks at 5 °C, but declined in appearance (visual quality) as the storage duration increased. Decline in visual quality was greater when plantlets were stored at 10 and 22 °C. Leaf dry weight of illuminated plantlets increased and percentage of leaf yellowing decreased as storage temperature increased. Recovery of illuminated plantlets from photomixotrophic storage was best when plantlets were stored at 22 °C. These plantlets were characterized by increased visual quality (color and form) and increased dry weight compared with those in other treatments. After 60 days in the greenhouse, the dry weight of these plantlets was similar for 4-, 8-, and 12-week storage durations, indicating flexibility in storage time if specific light and temperature provisions are met.

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Yulan Xiao and Toyoki Kozai

A photoautotrophic or sugar-free medium micropropagation system (PAM) using five large culture vessels (volume = 120 L each) with a forced ventilation unit for supplying CO2-enriched air was developed and applied to commercial production of calla lily (Zantedeschia elliottiana) and china fir (Cunninghamia lanceolata) plantlets. The culture period of calla lily plantlets in the PAM was reduced by 50%, compared with that in a conventional, photomixotrophic micropropagation system (PMM) using small vessels each containing a sugar-containing medium. Percent survival ex vitro of calla lily plantlets from the PAM was 95%, while that from the PMM was 60%. The production cost of calla lily in the PAM was reduced by about 40%, compared with that in the PMM, and the initial investment per plantlet for the PAM was ≈10% lower than that for the PMM. The sales price of ex vitro acclimatized calla lily plantlet was increased by 25% due to its higher quality, compared with plantlets produced in the PMM.

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Jeongwook Heo, Sandra B. Wilson and Toyoki Kozai

An improved forced ventilation micropropagation system was designed with air distribution pipes for uniform spatial distributions of carbon dioxide (CO2) concentration and other environmental factors to enhance photoautotrophic growth and uniformity of plug plantlets. Single-node stem cuttings of sweetpotato [Ipomoea batatas (L.) Lam. `Beniazuma'] were photoautotrophically (no sugar in the culture medium) cultured on a mixture of vermiculite and cellulose fibers with half-strength Murashige and Skoog basal salts in a scaled-up culture vessel with an inside volume of 11 L (2.9 gal). CO2 concentration of the supplied air and photosynthetic photon flux on the culture shelf were maintained at 1500 μmol·mol-1 and 150 μmol·m-2·s-1, respectively. Plantlets grown in forced ventilation systems were compared to plantlets grown in standard (natural ventilation rate) tissue culture vessels. The forced (F) ventilation treatments were designated high (FH), medium (FM), and low (FL), and corresponded to ventilation rates of 23 mL·s-1 (1.40 inch3/s), 17 mL·s-1 (1.04 inch3/s), and 10 mL·s-1 (0.61 inch3/s), respectively, on day 12. The natural (N) ventilation treatment was extremely low (NE) at 0.4 mL·s-1 (0.02 inch3/s), relative to the forced ventilation treatments. On day 12, the photoautotrophic growth of plantlets was nearly two times greater with the forced ventilation system than with the natural ventilation system. Plantlet growth did not significantly differ among the forced ventilation rates tested. The uniformity of the plantlet growth in the scaled-up culture vessel was enhanced by use of air distribution pipes that decreased the difference in CO2 concentration between the air inlets and the air outlet.