Light microscopy structure of Dianthus giganteus subsp. banaticus leaflet; control sample; wild plants. Ob. X 40 and 100. 

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... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

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... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

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... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

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... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

Context 5

... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

Context 6

... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

Context 7

... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

Context 8

... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

Context 9

... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...

Context 10

... concentration with the atmosphere (0.03%) Dianthus giganteus D’Urv subsp . (Cristea, 2000). The lack of saccharose in banaticus (Heuff.) Tutin is a species of the nutritive medium of photoautotrophic Dianthus genus which is endemic in SW of cultures means less primary and secondary the Carpathians, considered vulnerable/rare infections (Cristea, 2000). In vitro classical in Romania. Ex situ multiplication of some cultures are heterotrophic, the carbon species and re-inclusion in the natural source (saccharose, generally) being habitats may help to maintenance, necessary in the nutritive medium. consolidation and even extension of the Photoautotrophic cultures may natural populations. The photoautotrophic replace in vitro classical cultures or may cultures are the same as in vitro classical represent a stage before vitroplantlets ones, but the nutritive medium has no acclimatization. The role of this stage is to carbon source (saccharose). get ready plants for an autotrophic nutrition, This is the reason for which to develop the photosynthetic apparatus, photoautotrophic cultures need CO 2 which is less developed to in vitro classical supplementation of vessel atmosphere, but culture plants. Besides this, because of there are also in vitro photoautotrophic gases exchanges, the humidity inside cultures which develop at an equal CO 2 culture vessel will be lower than in vitro 331 classical cultures, and this will function as a solution 0.1M phosphate buffer, pH 7.2, for buffer to the impact of transferring plants 2 hours, at 4oC. Samples were washed in 4 from in vitro to in vivo (Kozai and Kubota, successive 0.15M phosphate buffer baths, 2005). Besides water stress during pH 7.2, at 4oC, for 1 hour each. Then, they acclimatization, an important role is played were postfixed in 2 % osmium acid in by the photosynthetic factor in plantlets 0.15M phosphate buffer for 1 hour and surviving during the transfer from one washed in 2 baths of phosphate buffer. The medium to another (Lee et al., 1985). samples were dehydrated in successive Structural differences in leaves caused by acetone baths of growing concentrations the culture type were also observed in Beta (50%, 60%, 70%, 80%, 90%, and 100%), vulgaris var. saccharifera (Cachi ţă -Cosma, 30 minutes in each bath. Leaf tissue et al, 2008), the changes consisting in larger fragments were infiltrated and included in intercellular spaces in the leaf mesophyll, Epon 812 synthetic resin, encapsulated and wide opening of stomata ostioles in the maintained for 48 h at 60oC for resin inferior epidermis, deformed and gap polymerization. Semi-thin sections of 200 – assimilatory parenchyma cells. These are 500 nm were obtained on Leica UC6 reversible processes in most of the cases, ultramicrotome, which were stained with a during vitroplantlets acclimatization the special stain for epoxidic resins - Epoxy cells revert to the initial form (Cachi ţă - Tissue Stain. The stained sections were Cosma, et al , 2008). examined by Olympus BX 51 light microscope having a CCD Camera for Material and methods recording the images. Plant material was sampled from Dianthus giganteus subsp. banaticus plants, Results and discussion from wild (i), from in vitro classical culture In case of wild plants, the study of plants (ii), and from photoautotrophic cross sections in leaves reveals the entire culture plants (iii). In vitro culture was structure of mesophyll, with the upper initiated from seeds sampled from wild epidermis covered by a wall with small an individuals, which were sterilized and rare crests and the inferior epidermis with inoculated on MS culture medium numerous and large crests (Fig. 1 A), which (Mourasig&Skog, 1996). After 30 days, are more visible in magnified images (Fig. apical explants were obtained and 1 D). transferred on two types of MS media with The mesophyll contains numerous 0.1 mg/l NAA, and 0.5 mg/l K; for in vitro cells, which cross-sectioned appear to be classical culture 0.3% saccharose was dense packed with small and rare added and the vessels were covered with intercellular spaces (Fig. 1 A and B). Even transparent foil. the gap space has few aquiferous spaces The vessels were covered with (Fig. 1 A, C, and D), that are more obvious transparent foil with ′′ sun cap ′′ system, in the inferior area, close to epidermis (Fig. which allows gases exchange with the 1 E and F), where it can be also observed outside atmosphere, for the the presence of stomata in ′′ close ′′ position photoautotrophic culture. This way, the (Fig. 1 E). CO 2 supplement was assured, having the Towards the superior epidermis, the same concentration as the atmosphere, cells have a palisade parenchyma made up about 0.03 ppm. Both types of cultures by numerous cells, parietal arranged, with were maintained at the same temperature, many lenticular chloroplasts, almost the photoperiod being 16h light/8h dark, without starch (Fig. 1 B). These cells are and the light intensity of 90 μ Mol m -2 s -1 . elongated and more visible in longitudinal Leaves fragments from fresh plants sections (Fig. 1 C). were prefixed in 2.7% glutaraldehyde 332 The cells have less chloroplasts between the two situations above towards the inferior epidermis (Fig. 1 A and mentioned, meaning that the epidermal cells D). of the upper and inferior parts, as well as The cells from both epidermis are those of the photosynthetic palisade tissue large, arranged in a single row and lack have a very similar structure to that of the chloroplasts (Fig. 1 A, C, and F). control (Fig. 3 A, C and G). In the middle of the mesophyll there The epidermal cells are arranged in can be observed the xylem and phloem a single row and are deformed only here vessels, well developed and obvious in and there (Fig. 3 A, C, D, E and F). In cross cross sections (Fig. 1 A and G) as well as in sections, the palisade cells are densely longitudinal sections (Fig. 1 C and H). packed and have many chloroplasts with In case of in vitro classical culture little starch (Fig. 3 A, C and E). In plants, all the cells, including those of longitudinal sections, the cells are epidermis and of the whole mesophyll, elongated, as in case of the control (Fig. 3 B seem to undergo a hyperhidroses process, and E). because they are swelled, many of them The mesophyll’s median zone has having distorted, sinuous and flexible cell cells with slightly deformed walls, and with walls, and the chloroplasts are few, but moderately large intercellular spaces (Fig. 3 have starch grains (Fig. 2 A-F). A and E). It is worthy to mention that the On the contrary, in some parts cells nearby inferior epidermis have towards the inferior epidemis, the cells are relatively numerous chloroplasts (Fig. 3 F smaller, deformed and have many and G), similar to the situation of the chloroplasts (Fig. 2 G), suggesting that, at control samples. this level, an exoosmosis happened, by Noted that the cuticle of the inferior loosing the water into intercellular spaces, epidermis has only rare crests (Fig. 3 B, E which are very large in this area. and G), and the stomata are in the ′′ open ′′ It is worthy to note that the cuticle position (Fig. 3 G). The veins have vessels of the inferior epidermis has rare crests with normal structure (Fig. 3 H). being smoother, similar to the upper epidermis (Fig. 2 B, F, and G). The palisade Conclusion tissue has the cells arranged in a single row, Following light microscopy the cells being deformed and having just a investigations, it was observed that there few chloroplasts (Fig. 2 A, B, E). In some are many changes in the structure of the leaf cells, the tonoplast is torn and the blade in the three types of plant cultures. chloroplasts have an altered structure and After a period of maintaining the float in the vacuolar system (Fig. 2 D and plant in photoautotrophic culture, these F). changes will attenuate. This can be At the veins level, vessels cells are observed in the similarities which appear fewer and their structure is slightly altered between the sections of the wild plants (Fig. 2 A, C, H). compared to those obtained in We mention that very similar photoautotrophic in vitro culture. changes were reported by Cachi ţă et al ., (2008) in beet leaflet grown in vitro . The authors also mention that such dysfunctions References which lead to major alterations do not Cachi ţă -Cosma, D., Petru ş , C.M., Petru ş -Vancea, A., Cr ă ciun, C., Fenomenul de hiperhidrie manifestat necessary cause the death of in vitro la nivelul vitroculturilor de sfecla de zahar ( Beta cultured plant material. vulgaris L. var. sachhharifera ). I. Aspecte de The structure analysis of plant microscopie optica surprinse in ţ esuturile leaflets obtained by photoautotrophic limburilor frunzuli ţ elor normale ş i a celor culture represents an intermediary stage hiperhidrice, Analele SNBC, XIII, 101-112, 2008. ...