Effects of Quantum Flux Density on Photosynthesis and Chloroplast Ultrastructure in Tissue-Cultured Plantlets and Seedlings of Liquidambar styraciflua L. towards Improved Acclimatization and Field Survivall

Liquidambar styraciflua L. seedlings and tissue-cultured plantlets were grown under high, medium, or low (315, 155, or 50 microeinsteins per square meter per second photosynthetically active radiation) quantum flux densities. Net photosynthesis, chlorophyll content, and chloroplast ultrastructure of leaves differentiated from these conditions were investigated. Seedling photosynthetic rates at light saturation were positively related to light pretreatments, being 6.44, 4.73, and 2.75 milligrams CO2 per square decimeter per hour for high, medium, and low light, respectively. Cultured plantlets under all light conditions had appreciably higher photosynthetic rates than noncultured seedlings; corresponding rates were 12.14, 13.55, and 11.36 milligrams CO2 per square decimeter per hour. Chlorophyll in seedlings and plantlets was significantly higher in low light-treated plants. Seedling leaves had chloroplasts with abundant starch regardless of light pretreatment. In high light, starch granules were predominant and associated with disrupted granal structure. Low light seedling chloroplasts had smaller starch grains and well-formed grana. In contrast, tissue culture-differentiated leaves were devoid of starch; grana were well organized in higher quantum flux density treatments, but disorganized at low flux densities. Tissue culture-derived plants require an acclimatization period during the transition from culture to field or greenhouse conditions. The acclimatization process usually consists of placement in high humidity conditions (under mist or humidifiers), with a gradual decrease of humidity and increase in light intensity over time. Although some plants have no problem during hardeningoff, significant losses are often incurred in some species (27). High mortality and increased crop time needed for acclimatization economically limit the use of in vitro techniques for propagation purposes in some species, particularly woody plants. Causes for plantlet mortality are partially due to water stress affects. Tissue culture plantlets have a divergent anatomy compared to noncultured plants which includes reduced quantities of epicuticular wax (10, 12), reduced cuticular development (26), extensive intercellular spaces (3, 13, 26), and stomata which are raised, larger, of higher density (27), and with reduced stomatal closure (2). In addition to water stress affects, photosynthetic factors may ' Supported in part, by Department of Energy contract 7860-X02, and by State and Hatch funds allocated to the Georgia Experiment Station. play a role in plantlet acclimatization and survival. Cultured plantlets of Brassica oleraceae had lower Chl levels, Hill activity, and CO2 fixation than noncultured plants (13). This poor development of the photosynthetic system in culture was cited as a major factor in transplant vulnerability. Red raspberry plantlets exhibited significantly lower net CO2 uptake rates than field control plants, i.e. 2.5 versus 10 to 15 mg CO2 dm 2 h-' at saturating light intensities (7). Laetsch and Stetler (17) compared the ability of cultured pith, induced buds, and mature tobacco leaves to fix carbon using '4C02 incorporation. Carbon fixation patterns were qualitatively the same, but showed quantitative differences. The light to dark incorporation ratio for "4C02 was at least three times as great in the leaf tissue as in growing cultured tissue. In ultrastructural evaluations of Liquidambar, we reported that in vitro plantlet leaves have cells with large vacuoles, limited cytoplasmic content, and flattened chloroplast with an irregularly arranged internal membrane system compared to acclimatized or field grown leaves (26). The lack of internal chloroplast membrane development and lack of differentiation of a palisade parenchyma suggests that the photosynthetic capacity of these plantlets may be lower than noncultured plants. In addition, plantlets are generally grown with media supplemented with sucrose. Of question is whether cultured plantlets are totally heterotrophic, photoautotrophic, or photomixotrophic. Also of question is whether the low light intensities prevalent in most culture systems is limiting in terms of chloroplast differentiation and photosynthetic capacity. It is well known that photosynthetic characteristics of plants are influenced by the light climate in which they are grown (1, 19, 28). Information concerning photosynthesis in culture and the effects of modifying in vitro environmental conditions can be utilized towards developing plantlets in culture which are better adapted to field survival. The objectives of this study are (a) to evaluate the photosynthetic capacity of tissue cultured and noncultured plants in terms of CO2 fixation, pigment content, and chloroplast ultrastructure and (b) to assess the effects of quantum flux density on leaf chloroplast differentiation in culture. MATERIALS AND METHODS Liquidambar styraciflua L., sweetgum, was aseptically cultured from adventitious shoots initiated from hypocotyl segments of 1-month-old seedlings, using methods previously described (23, 24). Cultures were maintained in a growth room at 25 ± 2°C, under cool-white fluorescent lamps at 30 lsE m-2 s-' at culture level, under a 14-h photoperiod. After 85% of the explants had rooted, plantlets were selected for uniformity and vigor. Plantlets 637 www.plantphysiol.org on July 14, 2017 Published by Downloaded from Copyright © 1985 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 78, 1985 were moved into a growth room maintained at 26 ± 20C with a 16-h photoperiod and placed under one of three quantum flux densities: 50 ± 5 (low light), 155 ± 10 (medium light), 315 ± 15 (high light) E m-2 s-'. PAR was measured using a LI-190S quantum sensor. Illumination was provided by Sylvania metal halide lamps suspended 1.2 m above table height. Light intensity was adjusted by the use of cheesecloth. One-month-old seedlings germinated in coarse perlite were grown in the same growth room, under the same light treatments for comparison, and fed weekly with the same inorganic salts found in the culture medium. Light treatments were continued for 4 weeks. After this time, there were at least two new, fully developed leaves on each plantlet or seedling. Pn2 of both seedlings and plantlets was determined by the amount of CO2 fixed using a Beckman 21 5B differential CO2 analyzer. Intact seedlings and plantlets were placed in 125-ml flasks and 25 x 150 mm glass tubes, respectively, with roots placed in distilled H20. Light, temperature, and gas flow rate were controlled and continuously monitored. The light source was the same as that used in the growth room. Quantum flux density was adjusted by addition of layers of cheesecloth. Temperature around the intact plant was maintained at 25 ± 2°C with the aid of a circulating water bath. The incoming gas was premixed at concentrations of 21% 02 containing 290 to 310 ,ul/L CO2. The gas flow rates were 200 ml min-' or 100 ml min-' through the closed measuring chamber depending on the culture size. Leaf areas were measured with a Lambda area meter 3000. Chl was determined as described by Moran (21). Absorbance was measured with a Beckman ACTACV spectrophotometer. For anatomical evaluations, the second newly developed leaves of plantlets and seedlings under the three light treatments were sampled. Samples were taken at 0900 h and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, postfixed with 2% buffered osmium tetroxide, and dehydrated through an ethanol series. Samples were infiltrated and embedded in Spurr's (25) low viscosity medium. Ultrathin sections were cut with a diamond knife on a Sorval MT-2 ultramicrotome and poststained with 2% uranyl acetate and Reynolds' lead citrate. Tissues were examined using a Phillips 200 TEM.