2.1. Temperature, light intensity andphotoperiod
Traditionally, harvested fresh horticultural produce are stored under low temperature, since lowering temperature can slow the metabolic processes (e.g., respiration) and thereby prevent undesirable loss of dry mass and associated deteriorations of quality. Post harvest temperature is generally selected to be the lowest possible temperature that does not cause chilling injury. For storage of plantlets, placing vessels under low temperature and dark environment has been practiced in commercial laboratories to slow down the growth to intentionally delay the subculture timings especially for limited plant species that are relatively tolerate to low temperatures. In conventional micropropagation, the medium contains and therefore provides energy during the storage. However, a drawback of sugar-containing medium in a prolong storage period is that it often causes an increased chance of contamination during storage.
According to Reed (1993), storage conditions for in vitro genetic conservation of temperate genera are typically 4 or 5 C in darkness. However, positive effects of illumination during storage were also reported. Dorion et al. (1991) successfully stored rose plantlets at 2 to 4 C under 9 to 18 |imol m-2 s-1 PPF at 8 h photoperiod for 6 months. Baubault et al. (1991) reported that in vitro Rhododendron plantlets were successfully stored at 4 C under 30 |imol m-2 s-1 at 14 h photoperiod for up to 12 months. Reed (1999) also reported the positive effect of illumination on mint plantlets during storage, indicating the recommendation as the combination of 4 C with 12 h photoperiod.
Under photoautotrophic culture conditions, the medium does not provide an energy source to the plantlets during storage, and therefore, maintaining carbon balance during storage by sustaining a minimum amount of photosynthesis is important for success in maintaining photosynthetic and regrowth ability of photoautotrophic plantlets during storage. Among a number of environmental factors affecting plantlet growth and quality deterioration during storage, temperature and light environments are the most important factors to manipulate. The same principle can apply to storage of transplants.
Illumination during storage has been shown in many horticultural species to extend storability of transplants (Heins et al., 1992, 1994; Kubota and Kozai, 1995). Heins et al. (1992) also found that transplants could be stored under higher air temperature when light was provided during storage than in the dark. Kubota and Kozai (1995) found that the optimum light intensity for storage was the light compensation point of photosynthesis at the storage temperature, when the light was provided continuously (24 h per day), showing that the CO2 exchange rates of plantlets exhibiting the best storability were maintained at null during storage.
4 6 Weeks
Figure 1. Dry mass of broccoli plantlets grown in vitro for 3 weeks and stored for 6 weeks under various combinations of air temperature and photosynthetic photon flux (PPF) (after Kubota and Kozai, 1994). Numerical values after letter T and P denote the storage air temperature (C) and PPF (Jmol m'2 s'1), respectively. Seedlings were regrown in the same growth conditions (23 C, 160 ¡Jmol m'2 s'1 PPF and 16 h photoperiod) for 2 weeks subsequent to storage. The dry mass increase of non-stored seedlings was shown for comparison. PPF of 2 ¡Jmol m'2 s'1 was the light compensation point at 5 and 10 C of the seedlings examined in the experiment.
under continuous lighting conditions, the light compensation point maintains the carbon balance of the plant at null so that the dry mass per plant does not change. For example, dry mass of photoautotrophic broccoli plantlets in vitro were maintained unchanged through the 6 week storage under a light compensation point (2 ¡mol m-2 s-1 PPF) at 5 and 10 C air temperature and these conditions also maintained regrowth ability of the plantlets (Kubota and Kozai, 1994) (Figure 1). Dry mass of eggplant (Solanum melongena L.) seedlings increased linearly with increasing PPF, from 0 to 16 ¡mol m-2 s-1 (Kozai et al., 1996), indicating that 5 ¡mol m-2 s-1 was the light compensation point that maintained dry mass at the storage temperature (9 C). Kubota et al. (1995) stored photoautotrophically cultured broccoli plantlets at 5, 10 or 15 C under 2 or 5 ¡mol m-2 s-1 PPF and showed that lowering air temperature in conjunction with a PPF either close to or higher than the light compensation point preserved regrowth ability of the plantlets. Higher PPF than the light compensation points, however, caused undesirable shoot elongation and dry mass increase of the plantlets. After 6 weeks of storage, a small difference of 2 and 5 |imol m-2 s-1 PPF caused considerable differences in plantlet dry mass and quality after storage, suggesting that light environment during storage should be carefully selected for each crop.
Photoperiod is also a factor affecting carbon balance and thereby dry mass of plantlets during storage. Under lighting cycles consisting of photo- and dark periods, the light intensity needs to be increased and carbon gain during the photoperiod needs to compensate the loss of carbon during the dark period. The light intensity providing plants the null daily carbon balance and thus keeping dry mass unchanged is called the "daily light compensation point" where the daily integrated gross photosynthetic rate is balanced with the daily integrated respiration rate. Kubota et al. (2002) stored eggplant plug seedlings at various combinations of light intensity and photoperiod and showed that, as long as the daily PPF is equal to the daily light compensation point (shown to be 430 mmol m-2 d-1 at 9 C), seedling growth and quality after 4 weeks of storage were not affected by the combinations of PPF and photoperiod. This will allow flexibility in designing the light environment for storage and may make light installation in storage more practical and more realistic in transplant production.
Light compensation points of plant photosynthesis are affected by other environmental conditions that affect photosynthetic and respiration rates (temperature and CO2 concentration). Kozai et al. (unpublished) showed, using unrooted chrysanthemum cuttings, the relationship between light intensity (PPF), temperature, and CO2 concentration that give null carbon balance (null net photosynthetic rate) of plants. The relationship can be depicted in a 3-axied graph as a surface, namely "compensation surface", where given values of two environmental variables of the three (air temperature, CO2 concentration and PPF) determine value of the third environmental variable. This concept was also demonstrated by Fujiwara et al. (2001) in the low temperature storage of tomato (Lycopersicon esculentum Mill.) grafted seedlings showing various combinations of PPF and CO2 concentration that provide null carbon balance for the seedlings at 10 C air temperature. Based on these findings, transplants/plantlets can be theoretically stored at lower PPF when air temperature is lower and/or CO2 concentration is higher. However, as shown by Fujiwara et al. (1999c and 2001), a lowest threshold of PPF that can maintain normal metabolic activities during storage is likely to exist. For example, tomato grafted seedlings were stored under various combinations of CO2 concentration and PPF that were determined to be light compensation points at the corresponding CO2 concentration (2.5, 1.9, 1.3, 0.9, and 0.5 |imol m-2 s-1 PPF at 0.05, 0.25, 0.50, 0.75, and 1.00% CO2, respectively and the best storability of the seedlings after 28 days were at 1.9 and 1.3 |imol m-2 s-1 PPF under 0.25 and 0.50% CO2, respectively, while significant quality degradation was observed at 0.9 and 0.5 |imol m-2 s-1 PPF (Fujiwara et al., 2001). Perhaps a certain minimal level of light is necessary to provide gross photosynthesis (energy) and/or to induce regulatory effects (signals) sufficient for maintaining chlorophyll synthesis and other important metabolic functions (Fujiwara, personal communication).
When plantlets were cultured photomixotrophically (with sugar in the medium), light compensation points were generally higher than those plantlets culture photoautotrophically, primarily due to a higher dark respiration rates as affected by higher sugar concentrations in the medium and therefore in the plantlet tissue. Kubota and Kozai (1995) reported that 20 - 50% greater dark respiration rates were observed for photomixotrophic broccoli plantlets compared with those for the photoautotrophic plantlets over air temperatures ranging from 3 to 25 C.
Sugar in the medium seems to be able to maintain dry mass of plantlets under a wider range of environmental conditions during low temperature storage. For example, Kubota and Kozai (1995) compared photoautotrophic and photomixotrophic broccoli plantlets stored under the same conditions (5, 10, or 15 C) under darkness or 2 |imol m-2 s-1 PPF. Photoautotrophic plantlets maintained the dry mass unchanged under illumination at 5 or 10 C, while photomixotrophic plantlets did so at all conditions except for 15 C under darkness. Apparently sugar in the medium compensated for the respiratory loss of carbon and contributed to maintaining the dry mass during the storage. However, chlorophyll concentrations of leaves were maintained at higher levels with illumination during storage and chlorophyll fluorescence parameters indicated the high photosynthetic activities of chlorophyll when stored under light. In fact, the broccoli plantlets stored in darkness either lost their regrowth ability or showed significant leaf damage presumably due to photoinhibition during the subsequent culture period, indicating the necessity of illumination to maintain photosynthetic and regrowth ability of the photomixotrophic plantlets for assuring normal growth after removing them from the storage.
Light quality is one of the environmental factors affecting plant growth and development. However, physiology of plants under different light qualities at low temperature has not been well investigated. Along with findings on optimum environmental conditions for storing transplants, effects of light quality on transplants stored at low temperature have been examined (Fujiwara et al., 1999a; Kubota et al., 1996 and 1997; Wilson et al., 1998a and b). Broccoli plantlets grown in tissue culture vessels exhibited greater stem elongation and decrease in chlorophyll concentration under red and blue light than those under white light after 6 weeks in storage at 5 C (Kubota et al., 1996). However, Wilson et al. (1998a, 1998b) showed that quality of broccoli seedlings was best maintained under red light compared with white or blue light. Fujiwara et al. (1999b) also reported that the quality of harvested culinary herbs was better maintained under red light than white light. The red light source employed in Wilson et al. (1998a and 1998b) and
Fujiwara et al. (1999b) was light emitting diodes, while that in Kubota et al. (1996 and 1997) was fluorescent lamps covered with a spectral filter. Therefore conflicts of the findings with regard to the plant responses to red light during storage may include potential effects of different spectra employed in these experiments.
Light emitting diodes have been considered as a light source for plant production (Bula et al., 1991; Brown et al., 1995). Application of LEDs to low temperature storage may be more feasible than for biomass production, since the light fixture size is smaller and reduction in irradiance at low temperatures is relatively less than for fluorescent lamps. If such lighting systems become available at a reasonable price, introduction of low temperature storage under dim lighting conditions will be facilitated for various horticultural operations including tissue culture propagation facilities.
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