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Suzanne M.D. Rogers and Kalyani Dias

When plants are subjected to stress conditions, they are believed to be developing defensive mechanisms. Those mechanisms could be studied by analysing and comparing the proteins from stressed and nonstressed plant materials. Photomixotrophically grown soybean suspension cultures were shocked with 150 mM, 200 mM, and 250 mM salt concentrations for 1 hr. and 3 hrs. The cells were then given 2, or 4 hr. recovery period. After treatment, proteins were quantified, using Bradford Assay, and then separated on SDS PAGE gels. In Coomasie stained gells, there were different banding patterns in shock treated samples, compared to the control. But there were no differences identified between different shocking times or recovery period treatments. The results from Silver staining and growth studies will be presented.

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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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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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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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Genhua Niu, Makio Hayashi and Toyoki Kozai

Potato (Solanum tuberosum L. cv. Benimaru) plantlets were cultured under four lighting cycles (photoperiod/dark period: 16 h/8 h, 4 h/2 h, 1 h/0.5 h, and 0.25 h/0.125 h) photoautotrophically (without sugar in the medium), and photomixotrophically (with sugar in the medium) in vitro for 28 days. Simulations of time courses of CO2 concentration in the vessel (Ci) and dry weight accumulation of the plantlets cultured photoautotrophically were conducted using a previously developed model (Niu and Kozai, 1997). While underestimation and overestimation of time courses of Ci in some treatments were observed, the simulated results of Ci and dry weight accumulation of the plantlets generally agreed with the measured ones. The difference of net photosynthetic rate response to Ci throughout the culture period was examined between the plantlets cultured photoautotrophically and photomixotrophically. Quantitative relationship between daily net photosynthetic rate (daily net production) and vessel ventilation rate per plantlet was simulated under various CO2 levels outside the vessel for given sizes of potato plantlets cultured photoautotrophically in vitro to aid appropriate CO2 enrichment and vessel design in commercial micropropagation.

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Chieri Kubota, Makiko Ezawa, Toyoki Kozai and Sandra B. Wilson

The effects of initial sucrose (suc) concentrations in the medium (S0) on the carbon balance and growth of sweetpotato [Ipomoea batatas (L.) Lam. `Beniazuma'] and tomato (Lycopersicon esculentum Mill. `HanaQueen') plantlets were studied under controlled environmental conditions. Plantlets were cultured with 0, 7.5, 15, or 30 g·L-1 of S0 under high photosynthetic photon flux (160 to 200 μmol·m-2·s-1) and CO2 enriched (1400 to 2050 μmol·mol-1) conditions. Net photosynthetic rate per leaf area (Pl) decreased and dry weight per plantlet (Wd) increased with increasing S0, but did not differ significantly between S0 of 7.5 to 30 g·L-1 for sweetpotato or 15 to 30 g·L-1 for tomato. Carbon influxes and effluxes of the plantlets by metabolism of medium suc and/or photosynthesis, and respiration were estimated based on measurements of in situ and steady state CO2 exchange rates and sugar uptake during culture. At S0 from 7.5 to 30 g·L-1, photosynthesis was responsible for 82% to 92% and 60% to 67% of carbohydrate assimilation for sweetpotato and tomato, respectively. Estimated carbon balances of plantlets based on the estimated and actual increases of moles of carbon in plant tissue demonstrated that in situ estimation of carbon balance was reasonably accurate for sweetpotato at S0 of 0 to 15 g·L-1 and for tomato at S0 of 0 g·L-1 and that the actual contribution of photosynthesis for tomato at high S0 might be lower than the values estimated in the present experiment. Results showed that initial suc concentration affected the relative contribution of photosynthesis on their carbon balances and that the responses were species specific. The failure of validation at S0 in a range specific to each species suggested the need for further study on carbon metabolism of in vitro plantlets cultured with sugar in the medium.

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

several advantages over photomixotrophic micropropagation (PM). The advantages include minimal microbial contamination, increased photosynthesis, growth and rooting in vitro, and survival percentages ex vitro when the in vitro environment is controlled to

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

Broccoli (Brassica oleracea L. Botrytis group `Green Duke') seeds were cultured photoautotrophically (without sugar) or photomixotrophically (with sugar) in vitro for 3 weeks at 23 °C and150 μmol·m-2·s-1 photosynthetic photon flux (PPF). In vitro seedlings were stored for 0, 4, 8, or 12 weeks at 5 °C in darkness or under 5 μmol·m-2·s-1 of white (400–800 nm), blue (400–500 nm), or red (600–700 nm) light. Photosynthetic ability and soluble sugar contents were determined after removal from storage. Photomixotrophic seedlings contained approximately five times more soluble sugars than did photoautotrophic seedlings. Dark storage reduced soluble sugars in both photoautotrophic and photomixotrophic plants, but photosynthetic ability was maintained for up to 8 weeks in the latter whereas it decreased in the former. Illumination in storage increased leaf soluble sgars in both photoautotrophic and photomixotrophic seedlings. Soluble sugars in stems decreased during storage regardless of illumination, but remained higher in illuminated seedlings. Red light was more effective in increasing or maintaining leaf and stem soluble sugars than was white or blue light. Regardless of media composition or illumination, storage for more tan 8 weeks resulted in dramatic losses in quality and recovery, as well as photosynthetic ability. Seedlings stored for 12 weeks comletely lost their photosynthetic ability regardless of media composition or illumination. The results suggest that carbohydrate, supplied in the media or through illumination, is essential for maintenance of photosynthetic ability during low-temperature storage for up to 4 or 8 weeks.

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

Broccoli (Brassica oleracea L. Botrytis group `Green Duke') seeds were cultured in vitro photoautotrophically (without sugar in the medium) or photomixotrophically (with sugar in the medium) for 3 weeks at 23 °C and 150 μmol·m-2·s-1 photosynthetic photon flux (PPF). Vessels were then stored at 5 °C under 1.6, 4.1, or 8.6 μmol·m-2·s-1 of white (400-800 nm), red (600-700 nm), or blue (400-500 nm) light. Concentrations of CO2 inside the vessels were monitored until equilibrium was reached. Light compensation point was reached at 3.5 μmol·m-2·s-1 for photoautotrophic seedlings and at 6.5 μmol·m-2·s-1 for photomixotrophic seedlings. Therefore, in the long-term storage experiment, seedlings were stored for 4, 8, or 12 weeks at 5 °C in darkness or under 5 μmol·m-2·s-1 (average light compensation point) of white, red, or blue light. Illumination during storage was necessary to maintain dry mass, leaf area, and regrowth potentials of in vitro seedlings. All seedlings stored in darkness were of poor quality and died when transferred to the greenhouse. Red light during storage increased seedling dry mass and chlorophyll content and improved overall appearance, whereas blue light decreased chlorophyll content and increased stem elongation. The addition of 2% sucrose to media increased dry mass and leaf area and maintained overall seedling quality during illuminated storage. However, plantlets stored for more than 4 weeks did not survive poststorage greenhouse conditions, regardless of light treatment.