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Leaf temperature (C)

Figure 1. General response of plant developmental rate to temperature.

development from the expiants.

Leaf area is an important quality variable of expiants. The photoautotrophic growth of potato plantlets is affected by the leaf area of the explants; explants with larger leaves give greater initial net photosynthetic rates per plantlet, and therefore provide greater growth rates (Miyashita et al., 1996). Explant leaf removal is a common practice in conventional micropropagation, but it is noted that tomato explants retaining leaves generated almost twice as much dry mass after 3 weeks as those with leaves removed under conventional, photomixotrophic culture conditions (Kubota et al., 2001). Criteria for selecting usable explants must be more strict for photoautotrophic than for photomixotrophic micropropagation. The quality of explants in conventional micropropagation will not necessarily meet the criteria for photoautotrophic micropropagation.

sucrose conc. 0 a I'1

sucrose conc. 0 a I'1

500 1000 1500 2000 2500 CO2 concentrations in the vessel (Cin) [|jmol mol-1]

Figure 2. Net photosynthetic rates per leaf dry weight (Pn) ofpotato explants as affected by CO2 concentration in the vessel and sugar concentration in the medium The Pn were measured as soon as explants were transferred to medium with or without sugar. (after Nakayama et al, 1991).

500 1000 1500 2000 2500 CO2 concentrations in the vessel (Cin) [|jmol mol-1]

Figure 2. Net photosynthetic rates per leaf dry weight (Pn) ofpotato explants as affected by CO2 concentration in the vessel and sugar concentration in the medium The Pn were measured as soon as explants were transferred to medium with or without sugar. (after Nakayama et al, 1991).

3.3.3. Somatic embryos and photosynthetic efficiency somatic embryogenesis is a key technology for mass production of elite clones and it has been introduced commercially, producing transplants for planting in clonal forestry. one of the challenges preventing the wider application of somatic embryos is low percent germination of somatic embryos and conversion to plantlets. Photoautotrophic micropropagation may contribute favorably in this area since somatic embryos have chlorophyll in their cotyledons and/or newly emerged true leaves, in which active photosynthesis can be expected. Long (1997) also suggested the possibility of using photoautotrophic methods for improving germination of somatic embryos. The preferable photoautotrophic environmental conditions that enhance germination and conversion of somatic embryos may be different from those for explants or plantlets. In cacao somatic embryos, high percentages of conversion to plants were obtained under high CO2 concentration of 2000 ^mol mol-1 compared with that under ambient CO2 concentration (Figueira and Janick, 1993). Based on the lower number of stomata and, perhaps, high resistance to CO2 diffusion into somatic embryos, the high level of CO2 concentration reported in Figueria and Janick (1993) may be necessary for enhancing the net photosynthetic rate of somatic embryos.

Development of photosynthetic ability of somatic embryos has been intensively studied in recent years. Rival et al. (1997) compared photosynthetic parameters (photochemical activities, CO2 exchange and carboxylase enzymatic activities) among different developmental stages, from somatic embryos to plantlets. Afreen et al. (2002a and b) examined the photosynthetic ability of different developmental stages of coffee somatic embryos and found that cotyledonary and germinated embryos have photosynthetic ability. The uniqueness of their approach is the 14 days pretreatment of cotyledonary and germinated somatic embryos under increased PPF conditions which stimulated the development of photosynthetic abilities and increased the CO2 uptake rate in those somatic embryos. Therefore, when grown under photoautotrophic conditions those embryos showed growth increments (compared with initial growth) and the dry mass was almost double the initial dry mass when grown under enriched CO2 (approx. 1100 ^mol mol-1) and high PPF (100 ^mol m-2 s-1). Those findings also support the hypothesis that germination of somatic embryos will be improved significantly by controlling the environmental conditions that promote photosynthesis. Photoautotrophic growth of somatic embryos is discussed in much more detail in Chapter 7.

3.4. Chlorophyll fluorescence (photosynthetic ability)

Photosynthesis involves the conversion of light energy into chemical energy mediated by light sensitive chlorophyll molecules in the leaf. Over the last decade, the measurement of chlorophyll fluorescence kinetics has provided considerable information on the organization and function of the photosynthetic apparatus. During the process of photosynthesis, each quantum of light absorbed by a chlorophyll molecule raises an electron from the ground state to an excited state. Upon de-excitation from a chlorophyll a molecule from excited state 1 to ground state, a small proportion (3-5% in vivo) of the excitation energy is dissipated as red fluorescence. The indicator function of chlorophyll fluorescence arises from the fact that fluorescence emission is complementary to alternative pathways of de-excitation which are primarily photochemistry and heat dissipation. Generally, fluorescence yield is highest when photochemistry and heat dissipation are lowest. Therefore, changes in the fluorescence yield reflect changes in photochemical efficiency and heat dissipation.

In the photoautotrophic growth of plants, biomass accumulation is totally relative to the contribution of photosynthesis. Chlorophyll fluorescence parameters such as Fv / Fm, Fv / Fo etc. are generally used to study the organization and function of photosynthetic apparatus (Gently et al., 1989). These parameters are the ratios of variable chlorophyll fluorescence (Fv = Fm - Fo) to either maximal (Fm) or ground (Fo) chlorophyll fluorescence. Fv / Fm ratio is used to evaluate the photosystem II (PS II) in the dark-adapted state with fully open PS II reaction centers (Serret et al., 2001). This value is highly correlated with the quantum yield of net photosynthesis in intact leaves (Bjorkman and Demmig, 1987). On the other hand, the ratio Fv / Fo is a reliable indicator of the potential photosynthetic capacity of leaves (Serret et al., 2001).

Serret et al. (2001) studied the association between the degree of photoautotrophy and the photosynthetic capacity of micropropagated gardenia plantlets by using chlorophyll fluorescence parameters. At each micropropagation stage, varying degrees of photoautotrophy were achieved by changing the types of closure, sucrose content in the growing medium and the PPF. Afreen et al. (2002a) assessed the photosynthetic ability of different stage coffee somatic embryos grown in the photomixotrophic and photoautotrophic conditions by using the chlorophyll fluorescence parameters. A fibre-optic based chlorophyll fluorimeter (Hansatech, King's Lynn, Norfolk, UK) was used to analyse the photochemical activity of the somatic embryos. In dark-adapted samples (2 h), the maximal quantum yield of photochemistry through PSII ((pmax) was calculated from the ratio (Fm - Fo)/Fm (Kitajima and Butler, 1975). The actual quantum yield ((p) of PSII photochemistry in light-adapted leaves was calculated from the steady-state level of chlorophyll fluorescence (Fs) and maximal fluorescence level: (p = (Fm - Fs)/Fm (Havaux et al., 1991). Values of both (pmax and (p of different stage embryos measured after 60 days of culture were not significantly different between the photoautotrophic and photomixotrophic treatments. In general, among the different stages values were greater in cotyledonary and germinated embryos in both treatments.

3.5. Photosynthetic pigments

Pigments are chemical compounds which reflect only certain wavelengths of visible light. Chlorophylls, the most important pigment in nature, are greenish in color, and contain a stable porphyrin ring around which electrons are free to migrate. Because the electrons move freely, the ring has the potential to gain or lose electrons easily, and thus has the potential to provide energized electrons to other molecules. This is the fundamental process by which chlorophyll "captures" the energy of sunlight. It is capable of channeling the energy of sunlight into chemical energy, that is, during the process of photosynthesis, green plants use light energy to produce chemical energy and chlorophyll is essential for this process. There are several kinds of chlorophyll, the most important being chlorophyll a. This is the molecule which makes photosynthesis possible, by passing its energized electrons on to molecules which will manufacture sugars. All plants, algae, and cyanobacteria which photosynthesize contain chlorophyll a. A second kind of chlorophyll is chlorophyll b, which occurs only in green algae and in the plants. Chlorophyll a is generally 3-times higher than chlorophyll b in normal in vivo plants.

In the photoautotrophic micropropagation, photosynthesis is the sole source for the carbohydrate accumulation and thus to grow under photoautotrophic conditions explants or plantlets must be chlorophyllous. Compare to photomixotrophic or sugar-containing culture medium, growing plantlets under photoautotrophic conditions do not alter the leaf chlorophyll content significantly, if the other environmental parameters especially PPF and the number of air exchanges of the vessel remain same. For example, while growing Rehmannia glutinosa plantlets in culture medium containing different concentrations of sugar (10, 15, and 30 gL-1), Cui et al (2000), found no significant difference in chlorophyll contents. In their study, the PPF (70 ^mol m-2 s-1), temperature (25C), ambient CO2 concentration (1000 ^mol mol-1) and the number of air exchanges of the vessel (4.4 h-1) remained same in all the treatments. However, in the same study, chlorophyll contents increased significantly when the number of air exchanges in the vessel was increased. Previously, forced ventilation has been found to increase the chlorophyll concentration compared to that of the natural ventilation or airtight system (Zobayed et al., 1999a). In airtight system or vessel having low number of air exchanges, generally ethylene accumulation take place which is known to decrease the leaf chlorophyll content. While culturing Prunus avium in closed vessels, Righetti (1996) found shoots were irregularly shaped, hyperhydrated with curled leaves, low chlorophyll content which decreased after 15-18 days of culture. Under these conditions the lowest dry-matter percent production and the highest ethylene synthesis were also observed. In a recent study, Park et al. (2004) observed that the chlorophyll content of the hyperhydrated shoots of potato plantlets was significantly lower in the completely sealed vessel than those of the normal shoots of gas permeable vessels. A fivefold increase in chlorophyll content was also observed in normal shoots of carnation plantlets than those of hyperhydrated one (Jo et al., 2002).

3.6. Photosynthetic enzymes: Rubisco

Photosynthetic enzymes such as Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is the primary carboxylating enzyme used by C3 plants during photosynthesis to incorporate CO2 into sugars needed for growth and development. Even C4 and CAM plants, which use PEP-carboxylase as their primary carboxylating enzyme, utilize Rubisco during subsequent secondary CO2 assimilation events. As a carboxylase, it is involved in CO2 fixation to the five-carbon sugar ribulose-1,5-bisphosphate to form two molecules of 3-phosphoglycerate which proceed to sugar production. Conventional photomixotrophic micropropagation involves the production of plants associated with low activities of Rubisco and the high net photosynthetic rate generally observed in photoautotrophic grown plants are perhaps due to enhanced Rubisco activities as explained by Desjardins et al. (1995). As pointed out by Roberts et al.

(1994) that cauliflower plants grown photomixotrophically under low light irradiance exhibited low net photosynthetic rate which was attributed to low levels of chlorophyll and ribulose bisphosphate carboxylase activity.

3.7. Transpiration

Transpiration rate is generally low in conventional in vitro plantlets and thus the low uptake rate of water and minerals are common. The prolonged exposure of the plantlets to high relative humidity, low CO2 concentration and accumulated ethylene in the headspace of the vessel often causes the lack of development of a cuticular layer, or functional stomata (Figure 3) to control the transpiration or water loss (Figure 4) of the plantlets under low relative humidity (or high vapor pressure deficit) conditions. As described earlier, the water environment inside the vessel is affected by that of the culture room, transpiration from the plantlets, evaporation from the medium or condensed water, and condensation on inner surfaces of the vessel or on the medium. The work of Sallanon and Maziere (1992) illustrates the vapor pressure deficit and water potential of the plantlets in the vessels under different relative humidity conditions. Slight changes in water vapor deficit in vitro resulted in significant differences in growth and morphology of the plantlets (Sallanon and Maziere, 1992).

Under lower vapor pressure deficit, plantlets had higher multiplication ratios, larger leaf area, and longer shoot length. More axillary buds developed from the explant base under low vapor pressure deficit conditions while development occurred on the upper part of the explant under high vapor pressure deficit. The culture conditions examined were based on a conventional system (containing sugar in the medium).

Under photoautotrophic conditions especially under forced ventilation, in vitro transpiration rate can be increased due to i) comparatively low relative humidity in the culture vessel, ii) higher air current speed around the leaf and iii) functional stomata. Increased transpiration can stimulate plant growth by enhancing acropetal transport of dissolved nutrients and evaporation from the cuticle draws wax precursors to the leaf surface (Roberts et al., 1994). Transpiration or control of water loss is also important during the early stage of ex vitro acclimatization. A photoautotrophic culture system with forced ventilation can significantly improve the development of normal functional stomata, and the formation of large amounts of epicuticular wax which contribute to better control of transpiration and result in less water loss after transplanting ex vitro, thereby conferring a high survival percentage compared to conventional in vitro (Zobayed et al., 2000).

Figure 3. Stomata on the abaxial surface of fully expanded 3rd/4th leaves from the apex of 21 days old sweetpotato plantlets cultured photomixotrophically under natural ventilation (a, c, e, g) and photoautotrophic ally under forced ventilation (b, d, f, h). Photographs were taken 1 (a, b), 2 (c, d), 30 (e, f) and 60 min. (g, h) after transplanting ex vitro. (after Zobayed et al., 2000)

Figure 3. Stomata on the abaxial surface of fully expanded 3rd/4th leaves from the apex of 21 days old sweetpotato plantlets cultured photomixotrophically under natural ventilation (a, c, e, g) and photoautotrophic ally under forced ventilation (b, d, f, h). Photographs were taken 1 (a, b), 2 (c, d), 30 (e, f) and 60 min. (g, h) after transplanting ex vitro. (after Zobayed et al., 2000)

Figure 4. Percent water loss of excised (A) and attached (B) leaves of sweetpotato plantlets immediately after transplanting ex vitro. Plantlets were cultured photomixotrophically under natural ventilation (O) and photoautotrophically under forced ventilation (k.). Each point represents mean + s.e. of five replicates. (after Zobayed et al., 2000).

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