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Figure 8. Epicuticular wax content of the leaves of sweetpotato plantlets cultured in vitro (day 21) and after transplanting ex vitro (day 7, 14 and 21). Plantlets were cultured photomixotrophically under natural ventilation (PMN) and photoautotrophically under forced ventilation (PAF). Bar represents mean + s.e . of five replicates. (after Zobayed et al., 2000).

The direct correlation between the reduced amount of epicuticular wax and substantially increased water loss of plantlets after transplanting is discussed by Preece and Sutter (1991). In an earlier study Wardle et al. (1983) suggested the possibility of producing in vitro glaucous cauliflower plants with increased amounts of epicuticular wax by reducing the relative humidity using desiccants. The use of a culture vessel in which Tyvek inserts in the lid (Baumgartner Papiers) is shown to reduce the relative humidity from approximately 100% to 94% which resulted in an increased resistance to desiccation in chrysanthemum (Smith et al., 1990) and grapewine (Smith et al., 1992) plantlets. Later Zobayed et al. (2001a) noted that reducing the relative humidity to 84% by introducing forced ventilation in the culture vessel headspace the epicuticular wax content of Eucalyptus leaf was increased 3.4X in photoautotrophic conditions that of the control (photomixotrophic conditions under airtight capping system) (Figure 8).

4.3. Leaf hair

Leaf hair may play a role in insolation and light reflectance (Heide-Jorgensen, 1980). The formation of leaf hair could well be a reflection of the percentage of relative humidity of the plant microclimate, hairs being longest where relative humidity is lowest. Zobayed et al. (2001a) showed that the epidermal hairs were shortest on leaves in sealed vessels and increased in length with increasing efficiency of ventilation (Figure 7F). For instance, the length of hair from the mid-rib region of tobacco leaf was 2.6X as long in well ventilated vessel as those from the sealed vessel.

Figure 9 a) Eucalyptus plantlets grown photomixotrophically in a Magenta vessel with 20gL1 sucrose for 28 days, b) Plantlets grown photoautotrophically in a large vessel with forced ventilation for 28 days; c) Callus (gall) formation on the leaf surface of plants grown photomixotrophically in a Magenta vessel.

Figure 9 a) Eucalyptus plantlets grown photomixotrophically in a Magenta vessel with 20gL1 sucrose for 28 days, b) Plantlets grown photoautotrophically in a large vessel with forced ventilation for 28 days; c) Callus (gall) formation on the leaf surface of plants grown photomixotrophically in a Magenta vessel.

4.4. Hyperhydricity

Hyperhydricity is a phenomenon of plant that occurs as a consequence of plant's response to non-wounding stresses when explants are placed in an unsuitable in vitro environment. The unsuitable environmental conditions include high relative humidity, constant air temperature, accumulation of ethylene, etc. gases, high osmoticity of the culture medium due to the presence of sugar and ammonium, hormonal imbalance in the media, sealing of culture vessels etc. All of these factors are responsible for the morphological and physiological disorders of the in vitro plantlets.

Morphology: Hyperhydrated plantlets are so named because of their "glassy" appearance. They are chiefly characterized by their thick, translucent, and brittle leaves (Ziv, 1991) and thick and easily breakable stems (Park et al., 2004). Potato plantlets grown in conventional airtight vessel with sugar-containing medium were severely hyperhydrated exhibiting typical glassiness; shoot increment after 9 days of culture was only 33% and the shoots remained stunted, reduction in leaf number and significantly lower percent of shoot dry mass were observed (Park et al., 2004).

The low dry weight of the hyperhydrated shoots was attributed to the high water content of the shoots. High concentration of ethylene (0.18 ppm) was accumulated in the airtight vessel compared to 0.04 ppm in the gas permeable one only after 9 days of culture. They suggested that ethylene accumulation in the culture vessel was the major cause of hyperhydricity of the shoots. Other biochemical characteristics of hyperhydrated plants are less lignin deposition, less amount of cellulose which is associated to a low C/N ratio favouring the synthesis of amino acids rather than the sugar units for cellulose, less chlorophyll which causes translucency. Eucalyptus leaves grown photomixotrophically showed callus formation on the leaf surface (Figure 9a and c) which was hyperhydric and friable (Figure 9d). Plantlets grown under photoautotrophic conditions with forced ventilation did not produce any callus on the leaf surface (Figure 9b).

4.5. Stomata

Stomata are microscopic pores, each bounded by two crescent shaped cells known as guard cells. Stomata are usually found on leaf surface and occasionally on stems. They are able to open and close and act as portals for entry of carbon dioxide into the leaf for photosynthesis and an exit for water vapour from the transpiration stream. Their major function is to allow sufficient CO2 to enter the leaf while conserving as much water as possible. The guard cells are usually connected to neighbouring cells via their dorsal walls and because of their relative isolation from the rest of the plant body, stomata are ideally suited for sensing and responding to environmental factors.

The most important anatomical abnormality reported by many researcher observed in a poorly ventilated sugar-containing (heterotrophic or photomixotrophic) medium is the non-functional stomata. As described earlier, the absence or reduction in leaf epicuticular and cuticular waxes, short leaf hair, all of these features combined with non-functional stomata would lead to abnormally and inherently high rates of transpiration, which could not be controlled during a period of acclimatization, an environment characterized by low relative humidity and high light, and which could jeopardize the plant's chances of survival.

Recent researches revealed that stomatal characteristics and leaf anatomy could be largely improved by using photoautotrophic micropropagation. For instance,

Figure 10. Stomata on lower epidermis of fresh 3rd or 4th leaf from apex collected during photoperiod from shoots of cauliflower plantlets after 28 days. Cultures were grown at ca. 25C with 16 h photoperiods at PPF 150 ^mol m-2s-1; RH: 26-32%. Plantlets were grown under: (A, B) airtight vessel (sealed with silicone rubber bung); (C, D) diffusive ventilation (polypropylene disc); (E, F) slow forced ventilation (flow rate 5 cm3 min-1); (G, H) fast forced ventilation (flow rate 10 cm3 min-1). Note the degree of stomatal closing under different conditions. (after Zobayed et al, 2001b).

Figure 10. Stomata on lower epidermis of fresh 3rd or 4th leaf from apex collected during photoperiod from shoots of cauliflower plantlets after 28 days. Cultures were grown at ca. 25C with 16 h photoperiods at PPF 150 ^mol m-2s-1; RH: 26-32%. Plantlets were grown under: (A, B) airtight vessel (sealed with silicone rubber bung); (C, D) diffusive ventilation (polypropylene disc); (E, F) slow forced ventilation (flow rate 5 cm3 min-1); (G, H) fast forced ventilation (flow rate 10 cm3 min-1). Note the degree of stomatal closing under different conditions. (after Zobayed et al, 2001b).

stomatal density of the photoautotrophic potato plantlets increased twofold compared to that of the photomixotrophic plantlets (Zobayed et al., 1999b).

Stomatal density is found to be increased significantly under photoautotrophic conditions with CO2 enrichment (Kirdmanee et al., 1995). In Eucalyptus plantlets grown photoautotrophically, stomata opened during the light period and closed in the dark period, while in many of the photomixotrophic plants stomata remained widely open in both light and dark periods indicating abnormal functioning of stomata. The reduction of stomatal density has been reported in the hyperhydric leaves of regenerated carnarion (Olmos and Hellin, 1998). Lowering the relative humidity in the culture microclimate by using forced ventilation may greatly contribute towards the normal functioning of these stomata (Figure 10). Cauliflower plantlets grown in a well aerated vessel (forced ventilation) also showed functional stomata (Figure 10; Zobayed et al., 2001b). The following table (Table 1 and Figure 11) summarizes the major characteristics of stomata from leaves of photoautotrophic and photomixotrophic plantlets.

Table 1. Major characteristics of stomata of leaves of plants grown under photoautotrophic and photomixotrophic conditions.

Stomata of the leaves of plants grown Stomata of the leaves of plants grown photoautotrophically photomixotrophically

Functional stomata

High density

Smaller in size

Low stomatal conductance

High ability of conserving water after transplanting ex vitro

Non functional stomata

Low density

Generally larger in size

High stomatal conductance

Poor or low ability of conserving water after transplanting ex vitro

Figure 11. Stomata of the leaves of sweetpotato plantlets under dark condition a) Photoautotrophic. Note the high density and closed state of stomata and b) Photomixotrophic. Note the low density and opened state of stomata.

4.6. Root Formation

Improved root system is essential for plant growth in vitro not only because it plays a role in water and nutrient uptake but also replace water loss by the shoots especially during the acclimatization stage. An excellent study into the effects of different growth substrates (Florialite, vermiculite, sorbarods) and gelled media (gellangum and agar) on root formation and a mean of improving the root and /or shoot growth was that of Afreen et al. (2000). Among other things they concluded that selection of supporting material, in addition to controlling the culture microclimate, is very important for achieving better growth. in their case it was the use of Florialite (a mixture of vermiculite and cellulose fibre). in Florialite grown sweetpotato plantlets the main adventitious root gave rise to dense growth of fine lateral roots which could have resulted in a higher nutrient absorption capacity and can also be a major source of oxygen efflux. Thus the extensive root system was indirectly involved in the enhancement of growth, a higher net photosynthetic rate and higher dry mass accumulation and especially higher percent survival ex vitro. in contrast when grown in agar medium the main adventitious root produced sparse, short laterals, which explained the poor shoot growth of the plantlets in vitro followed by the poor survival percentage ex vitro. in both cases the plantlets were grown photoautotrophically. chrysanthemum plantlets produced better root system when grown in cellulose plugs saturated with nutrient solution instead of gelling agents (Roberts et al., 1994).

Figure 12. Root growth of sweetpotato plantlets cultured photoautotrophically for 21 days in a) Florialite and b) Agar matrix. (after Afreen et al., 2000).

Transpiration High CO2 High PPF

Ventilation

(High number of air exchange)

Low Relative Humidity

Ventilation

(High number of air exchange)

Low Relative Humidity

. Transpiration Extensive root formation

Functional stomata B yf

Wax Formation

. Transpiration Extensive root formation

Functional stomata B yf

High oxygen efflux

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