Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicu... more Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicum L.) volatile aroma compounds derived from oxygenation of unsaturated fatty acids. Chilling and heating may suppress production of these C6 volatiles. The objective of this research was to determine the effects of chilling or heating on gene expression and enzyme activity of lipoxygenase (LOX), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH), which catalyze key steps in C6 volatile production via the oxylipin pathway. 'Tasti-Lee' and 'Sanibel' tomatoes, harvested at different stages of fruit development, were ripened to full (red) ripe stage at 20 • C. Fruit were then treated by either chilling (5 • C for 5 d) or heating (52 • C hot water for 15 min), then cooled with 23 • C tap water to 25 • C and placed at 20 • C for 4 d, or held continuously at 20 • C as the control. Both chilling and heating reduced C6 aldehyde and alcohol aroma volatiles immediately after treatment, and the levels of aldehydes did not fully recover after 4 d at 20 • C. Chilling down-regulated expression of TomloxA, B, and C, but not D; however, it increased total LOX activity. Chilling also down-regulated HPL and ADH expression immediately after treatment, but, after 4 d at 20 • C, both genes were up-regulated compared to the control. HPL activity in chilled tomatoes was reduced, but recovered to control levels after 4 d at 20 • C. ADH activity in chilled fruit decreased after 4 d at 20 • C. On the other hand, heating greatly up-regulated TomloxB and C expression, even after 4 d at 20 • C, and slightly down-regulated TomloxA and D, while increasing overall LOX activity. Heating up-regulated both HPL and ADH, and that effect persisted for 4 d at 20 • C. However, heating reduced the activities of HPL for 4 d at 20 • C and ADH immediately after treatment. The results indicate that heating and chilling regulate C6 volatile production by different mechanisms. Chilling-induced inhibition of C6 volatile production may be due to down-regulation of gene expression, and subsequent reduction of HPL and ADH enzyme activities in the oxylipin pathway. Heating-inhibition of C6 volatile production, however, does not appear to be due to down-regulation of gene expression, but HPL and ADH activities were briefly suppressed.
Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Dec... more Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Decaisne, and Catharanthus roseus (L.) G. Don, or from leaves of Vinca minor L. Protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. Hybrid combinations were Rauwolfia + Vinca, Rauwolfia + Catharanthus, Rauwolfia + Rhazya, and Catharanthus + Vinca. For hybrid isolation, the physiological complementation method was utilized. Analyses of the material obtained included a cytogenetic study of the chromosomes, a study of multiple molecular forms of transferases and esterases, and the blot hybridization of restricted nuclear DNA using ribosomal DNA as a probe. Hybrids were identified in all species' combinations tried. A ten-fold increase in the accumulation of raucaffricine (relative to the parental Rauwolfia strain) was observed in one cell line of the Rauwolfia + Vinca hybrid. Our studies indicated the genetic stability of the great majority of the hybrid cell lines over a period of more than 20 months of in vitro growth. No shoot morphogenesis has so far been observed in this material.
Many orchids take several years to flower. We have been able to induce early flowering in the tem... more Many orchids take several years to flower. We have been able to induce early flowering in the temperate orchid Cymbidium niveo-marginatum Mak in vitro. The combined treatment of cytokinin (6benzylaminopurine), restricted nitrogen supply with phosphorus enrichment, and root excision (pruning) induced transition of the Cymbidium shoot from a vegetative to a reproductive stage. Nearly 100% of the plants flowered within 90 days only when the combined treatment was applied. When root excision and/or 6benzylaminopurine were omitted from the combined treatments, flower induction was significantly reduced. The auxin transport inhibitor, 2,3,5-triiodobenzoic acid prevented flowering of Cymbidium in vitro, although auxin (a-naphthaleneacetic acid) itself did not induce flowering. Gibberellic acid markedly delayed flowering in C. niveo-marginatum even when the flowerpromoting treatment was applied. Paclobutrazol, an anti-gibberellin agent, totally blocked the inductive effects of either cytokinin or pruning. These observations suggest that concerted actions of auxin, cytokinin, and gibberellin, as well as nutrient concentration and putative promoting/suppressing agents, determine the timing of Cymbidium orchid transition from the vegetative to reproductive stage.
Somatic hybridization of higher plants is known to result in the creation of new "synthetic" cell... more Somatic hybridization of higher plants is known to result in the creation of new "synthetic" cells whose genomes contain inherited determinants from both parents; the genomes of the hybrid cells and plants regenerated from them often show significant reconstructions, whose extent and direction can only poorly, if at all, be predicted. There are also significant changes in the secondary metabolism of the reconstructed cells and tissues.
A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacte... more A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand-held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand-held injection. Results from inoculations with the automated device are in accord with the results with the hand-held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ÔHamlinÕ sweet orange are susceptible.
Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter ta... more Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.
The effects of 2 consecutive years of annual defoliation during the harvest season on fruit size,... more The effects of 2 consecutive years of annual defoliation during the harvest season on fruit size, yield, juice quality, leaf size and number were examined in trees of the midseason cultivarHamlin'and the late-season cultivarValencia'orange [Citrus sinensis (L. ...
1-MCP is a gaseous ethylene binding inhibitor that controls or delays ethylene-related postharves... more 1-MCP is a gaseous ethylene binding inhibitor that controls or delays ethylene-related postharvest problems in a range of horticultural commodities. Our previous work demonstrated that exposure of calamondin to 1-MCP 16 hours before canopy sprays of ...
Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicu... more Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicum L.) volatile aroma compounds derived from oxygenation of unsaturated fatty acids. Chilling and heating may suppress production of these C6 volatiles. The objective of this research was to determine the effects of chilling or heating on gene expression and enzyme activity of lipoxygenase (LOX), hydroperoxide lyase (HPL), and alcohol
The anthracnose fungus, Colletotrichum gloeospoioides, rnteracts incompatibly with the ripe fruit... more The anthracnose fungus, Colletotrichum gloeospoioides, rnteracts incompatibly with the ripe fruit of pepper (Capsicum annuum).It interacts compatibly with the unripe-mature fruit. We isolated a defensin gene,7/-1, and a thionin-like gene, PepThi, expressed in the incompatible interaction by using an mRNA differential display method.
The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompati... more The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripemature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of Pep-CYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.
Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicu... more Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicum L.) volatile aroma compounds derived from oxygenation of unsaturated fatty acids. Chilling and heating may suppress production of these C6 volatiles. The objective of this research was to determine the effects of chilling or heating on gene expression and enzyme activity of lipoxygenase (LOX), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH), which catalyze key steps in C6 volatile production via the oxylipin pathway. 'Tasti-Lee' and 'Sanibel' tomatoes, harvested at different stages of fruit development, were ripened to full (red) ripe stage at 20 • C. Fruit were then treated by either chilling (5 • C for 5 d) or heating (52 • C hot water for 15 min), then cooled with 23 • C tap water to 25 • C and placed at 20 • C for 4 d, or held continuously at 20 • C as the control. Both chilling and heating reduced C6 aldehyde and alcohol aroma volatiles immediately after treatment, and the levels of aldehydes did not fully recover after 4 d at 20 • C. Chilling down-regulated expression of TomloxA, B, and C, but not D; however, it increased total LOX activity. Chilling also down-regulated HPL and ADH expression immediately after treatment, but, after 4 d at 20 • C, both genes were up-regulated compared to the control. HPL activity in chilled tomatoes was reduced, but recovered to control levels after 4 d at 20 • C. ADH activity in chilled fruit decreased after 4 d at 20 • C. On the other hand, heating greatly up-regulated TomloxB and C expression, even after 4 d at 20 • C, and slightly down-regulated TomloxA and D, while increasing overall LOX activity. Heating up-regulated both HPL and ADH, and that effect persisted for 4 d at 20 • C. However, heating reduced the activities of HPL for 4 d at 20 • C and ADH immediately after treatment. The results indicate that heating and chilling regulate C6 volatile production by different mechanisms. Chilling-induced inhibition of C6 volatile production may be due to down-regulation of gene expression, and subsequent reduction of HPL and ADH enzyme activities in the oxylipin pathway. Heating-inhibition of C6 volatile production, however, does not appear to be due to down-regulation of gene expression, but HPL and ADH activities were briefly suppressed.
Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Dec... more Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Decaisne, and Catharanthus roseus (L.) G. Don, or from leaves of Vinca minor L. Protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. Hybrid combinations were Rauwolfia + Vinca, Rauwolfia + Catharanthus, Rauwolfia + Rhazya, and Catharanthus + Vinca. For hybrid isolation, the physiological complementation method was utilized. Analyses of the material obtained included a cytogenetic study of the chromosomes, a study of multiple molecular forms of transferases and esterases, and the blot hybridization of restricted nuclear DNA using ribosomal DNA as a probe. Hybrids were identified in all species' combinations tried. A ten-fold increase in the accumulation of raucaffricine (relative to the parental Rauwolfia strain) was observed in one cell line of the Rauwolfia + Vinca hybrid. Our studies indicated the genetic stability of the great majority of the hybrid cell lines over a period of more than 20 months of in vitro growth. No shoot morphogenesis has so far been observed in this material.
Many orchids take several years to flower. We have been able to induce early flowering in the tem... more Many orchids take several years to flower. We have been able to induce early flowering in the temperate orchid Cymbidium niveo-marginatum Mak in vitro. The combined treatment of cytokinin (6benzylaminopurine), restricted nitrogen supply with phosphorus enrichment, and root excision (pruning) induced transition of the Cymbidium shoot from a vegetative to a reproductive stage. Nearly 100% of the plants flowered within 90 days only when the combined treatment was applied. When root excision and/or 6benzylaminopurine were omitted from the combined treatments, flower induction was significantly reduced. The auxin transport inhibitor, 2,3,5-triiodobenzoic acid prevented flowering of Cymbidium in vitro, although auxin (a-naphthaleneacetic acid) itself did not induce flowering. Gibberellic acid markedly delayed flowering in C. niveo-marginatum even when the flowerpromoting treatment was applied. Paclobutrazol, an anti-gibberellin agent, totally blocked the inductive effects of either cytokinin or pruning. These observations suggest that concerted actions of auxin, cytokinin, and gibberellin, as well as nutrient concentration and putative promoting/suppressing agents, determine the timing of Cymbidium orchid transition from the vegetative to reproductive stage.
Somatic hybridization of higher plants is known to result in the creation of new "synthetic" cell... more Somatic hybridization of higher plants is known to result in the creation of new "synthetic" cells whose genomes contain inherited determinants from both parents; the genomes of the hybrid cells and plants regenerated from them often show significant reconstructions, whose extent and direction can only poorly, if at all, be predicted. There are also significant changes in the secondary metabolism of the reconstructed cells and tissues.
A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacte... more A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand-held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand-held injection. Results from inoculations with the automated device are in accord with the results with the hand-held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ÔHamlinÕ sweet orange are susceptible.
Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter ta... more Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.
The effects of 2 consecutive years of annual defoliation during the harvest season on fruit size,... more The effects of 2 consecutive years of annual defoliation during the harvest season on fruit size, yield, juice quality, leaf size and number were examined in trees of the midseason cultivarHamlin'and the late-season cultivarValencia'orange [Citrus sinensis (L. ...
1-MCP is a gaseous ethylene binding inhibitor that controls or delays ethylene-related postharves... more 1-MCP is a gaseous ethylene binding inhibitor that controls or delays ethylene-related postharvest problems in a range of horticultural commodities. Our previous work demonstrated that exposure of calamondin to 1-MCP 16 hours before canopy sprays of ...
Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicu... more Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicum L.) volatile aroma compounds derived from oxygenation of unsaturated fatty acids. Chilling and heating may suppress production of these C6 volatiles. The objective of this research was to determine the effects of chilling or heating on gene expression and enzyme activity of lipoxygenase (LOX), hydroperoxide lyase (HPL), and alcohol
The anthracnose fungus, Colletotrichum gloeospoioides, rnteracts incompatibly with the ripe fruit... more The anthracnose fungus, Colletotrichum gloeospoioides, rnteracts incompatibly with the ripe fruit of pepper (Capsicum annuum).It interacts compatibly with the unripe-mature fruit. We isolated a defensin gene,7/-1, and a thionin-like gene, PepThi, expressed in the incompatible interaction by using an mRNA differential display method.
The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompati... more The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripemature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of Pep-CYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.
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