General Anatomical Characteristics Of In Vitro Plants

Generally, anatomical characteristics especially the leaf anatomy of in vitro plants grown in the conventional micropropagation system has been studied intensively in the last few years. Conventionally, micropropagation is carried away using small, relatively airtight culture vessels containing nutrient media with 20-30 g L-1 sucrose (as a carbon source for the plantlets) and under a low PPF of about 30-80 ^mol m-2 s-1. Most of the researches pointed out that under these conditions the anatomical features of the plants are at least to some extend different (abnormal) than the normal in vivo plants, they have a significantly higher content of carbohydrates, mostly in the leaves (Kozai and Zobayed, 2001). Thus the leaves have a poorly developed internal structure and become physiologically abnormal and simply act as a storage organ. It has also been concluded that the poor ventilation or restricted air exchange in the conventional culture vessel can lead to the development of abnormal anatomical features which could prevent or reduce the plant's ability to acclimatize ex vitro (Zobayed et al., 2001a).

4.1. Mesophyll and palisade layer

The mesophyll layer of the leaf consists of parenchymatous tissues situated between the two epidermal layers of the leaf. It usually undergoes differentiation in order to form photosynthetic tissues and thus contains chloroplasts. Another important factor of the mesophyll layer is the presence of well-developed intercellular spaces which facilitates the exchange of gases. Thus for efficient photosynthesis not only the number of chloroplasts but the dimensions of intercellular spaces also play an important role. Zobayed et al. (2001b) showed that leaves of cauliflower and tobacco plants grown photomixotrophically in well sealed vessels exhibited a lack of well defined palisade and spongy mesophyll layers and the cells were more closely packed with smaller intercellular spaces compared to those grown in well aerated vessels (Figure 5). In contrast when grown photomixotrophically in aerated (diffusive and forced) vessels and in vivo both the species showed more structural integrity in the leaves, and had definite palisade and spongy mesophyll layers, the latter with large intercellular spaces. They also suggested that the chloroplast contents of the mesophyll layers in these leaves were greater compared to those of the sealed vessels ones. Similarly when Eucalyptus plants were grown photoautotrophically under forced ventilation had leaves (Figure 6a) that were thicker (723 ^m), with well organized palisade and spongy mesophyll layers and the epidermal cells were well developed with an average depth of 42 ^m. In contrast when grown photomixotrophically the Eucalyptus leaf thickness and epidermal cell thickness were 421 and 28 ^m, respectively compared to those of photoautotrophically grown leaves (Figure 6b). The epidermis had irregular cells (Figure 6b) and smaller, irregular shaped oil cavities compared to that of photoautotrophic ones.

4.2. Wax deposition

Wax is a lipid compound which mainly consists of esters of long-chain fatty acids and long-chain monohydric alcohols. Waxes usually form protective coatings on the epidermis of leaves. The structure and amount of epicuticular wax affects the cuticular permeability and the degree to which a leaf surface can be wetted. The development of epicuticular wax is known to be advantageous for the plantlets

Figure 5. Transverse sections of 3rd and 4th leaves from apex of tobacco (a - e) and cauliflower (f - j) plantlets grown under different types of ventilation and also in vivo for 28 days (160 x). (a, f) Airtight vessel (sealed with silicone rubber bung); (b, g) diffusive ventilation (polypropylene disc); (c, h) slow forced ventilation (flow rate 5 cm3 min1); (d, i) fast forced ventilation (flow rate 10 cm3 min-1); (e, j) in vivo (growth room conditions). Cultures were grown at ca. 25C with 16h photoperiods at PPF 150 ^mol m-2s-1. (after Zobayed et al 2001b ).

Figure 5. Transverse sections of 3rd and 4th leaves from apex of tobacco (a - e) and cauliflower (f - j) plantlets grown under different types of ventilation and also in vivo for 28 days (160 x). (a, f) Airtight vessel (sealed with silicone rubber bung); (b, g) diffusive ventilation (polypropylene disc); (c, h) slow forced ventilation (flow rate 5 cm3 min1); (d, i) fast forced ventilation (flow rate 10 cm3 min-1); (e, j) in vivo (growth room conditions). Cultures were grown at ca. 25C with 16h photoperiods at PPF 150 ^mol m-2s-1. (after Zobayed et al 2001b ).

Figure 6. Transverse section of the 4th leaf from apex of 28 days old Eucalyptus plantlets grown (a) photoautotrophically in a scaled-up vessel under forced ventilation and (b) photomixotrophically in a Magenta vessel (control). oc, oil cavity; as, air space. (after Zobayed et al., 2001a).

especially during the acclimatization period (Grout, 1975; Sutter and Langhans, 1982) as it helps the plants from desiccation (Zobayed et al., 2001b), it also reduce the damage to photosynthesis and heat load of leaves by reflecting the light (McClendon, 1984). The degree of wax formation depends on the environmental conditions to which a plant is exposed. Lack of epicuticular wax formation was noticed in the leaves of cauliflower (Figure 7) and Eucalyptus (Figure 8) when grown in well sealed and poorly aerated vessels. On the contrary the plants from well aerated vessels and greenhouse showed intense epicuticular wax development which appeared as white powdery coating under the microscope (Zobayed et al., 2001b). They also noticed the formation of cuticular wax in well aerated and greenhouse grown plants.

Figure 7. Transverse sections of upper epidermis of fresh 3rd or 4<h leaf from apex of 28 days old cauliflower plantlets; sections were stained in 0.02% aqueous auramine and photographed under blue light to show waxes fluorescing yellow (white in this photograph; 688 x). Cultures were grown at ca. 25C with 16 hphotoperiods atPPF150 ^mol m'2s-1; RH: 26-32%. Plantlets were grown under: (A) airtight vessel (sealed with silicone rubber bung); (B) diffusive ventilation (polypropylene disc); (C) slow forced ventilation (flow rate 5 cm3 min'1); (D) fast forced ventilation (flow rate 10 cm3 min -1) and (E) in vivo (growth room conditions). Note in (A) and (B) the cuticles appeared thinner and fluoresced to a smaller degree than in (C, D, E). The lack of fluorescence was particularly obvious in (B). (F) Features of upper epidermis of fresh 3d or 4th leaf from apex of 28 days old tobacco plantlets grown under forced ventilation (fast flow; rate -10 cm3 min'1). Specimens were stained in 0.02% aqueous auramine and photographed under blue light to show waxes fluorescing yellow. Note the epidermal hairs have waxy walls and globular tips (150x). (after Zobayed et al., 2001b).

Figure 7. Transverse sections of upper epidermis of fresh 3rd or 4<h leaf from apex of 28 days old cauliflower plantlets; sections were stained in 0.02% aqueous auramine and photographed under blue light to show waxes fluorescing yellow (white in this photograph; 688 x). Cultures were grown at ca. 25C with 16 hphotoperiods atPPF150 ^mol m'2s-1; RH: 26-32%. Plantlets were grown under: (A) airtight vessel (sealed with silicone rubber bung); (B) diffusive ventilation (polypropylene disc); (C) slow forced ventilation (flow rate 5 cm3 min'1); (D) fast forced ventilation (flow rate 10 cm3 min -1) and (E) in vivo (growth room conditions). Note in (A) and (B) the cuticles appeared thinner and fluoresced to a smaller degree than in (C, D, E). The lack of fluorescence was particularly obvious in (B). (F) Features of upper epidermis of fresh 3d or 4th leaf from apex of 28 days old tobacco plantlets grown under forced ventilation (fast flow; rate -10 cm3 min'1). Specimens were stained in 0.02% aqueous auramine and photographed under blue light to show waxes fluorescing yellow. Note the epidermal hairs have waxy walls and globular tips (150x). (after Zobayed et al., 2001b).

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