In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbec...In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbeck. Observations of the effect of PGRs were carried out using different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D),1-naphthalene acetic acid (NAA) and combinations of 2,4-D and NAA using different basal media. This study found Citrus sinensis (L.) Osbeck exhibited a high frequency of callus induction on MS medium supplemented with 3 mg/L 2,4-D and callus induction frequency was 86.7% ± 3.4% whereas N6 and B5 showed lower callus induction frequency of 83.3% ± 8.8% and 82.2% ± 1.9% respectively compared to that of MS media with supplementation of the same hormone. Among the induced calli, the morphological analysis showed only 40% - 50% was embryogenic calli. Regeneration of plantlets from calli was done using different concentrations and combinations of auxin and cytokinin. The study showed that 3 mg/L 6-benzylaminopurine (BAP) supplemented medium has the maximum potential to promote regeneration of Citrus sinensis (L.) Osbeck from embryogenic calli with the frequency of 89.3% ± 8.8% but no regeneration occurred from the non-embryogenic calli. The regenerated plantlets were rooted on MS medium with supplementation of 5 mg/l NAA. These observations in Citrus sinensis (L.) Osbeck regeneration will be helpful for genetic improvement with desired traits.展开更多
Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the c...Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.展开更多
[Objective] The aim was to identify genetic variation in Citrus sinensis （sweet orange） germplasm from Hunan Province according to the Start Codon Targeted （SCoT） Polymorphism. [Method] The reaction system for SCo...[Objective] The aim was to identify genetic variation in Citrus sinensis （sweet orange） germplasm from Hunan Province according to the Start Codon Targeted （SCoT） Polymorphism. [Method] The reaction system for SCoT amplification from sweet orange was first optimized, and then the SCoT fragments were amplified from 24 sweet orange cultivars collected in Hunan Province and sequenced for genetic variation analysis. [Result] The optimum reaction system for SCoT markers amplification was 2.0 μl containing 80 ng of template DNA, 0.3 mmol/L dNTPs, 0.2 μmol/L primer, 1.6 mmol/L Mg2＋, 1.6 U of Taq DNA polymerase and 10×PCR buffer. By using this reaction system, the PCR products from the sweet orange cultivars produced clear and reproducible bands at 100-2 000 bp through electrophoresis. The SCoT fragments of the 24 sweet orange cultivars were 1 090-1 091 bp, with the homology of 99.84% and nucleotide deletion and substitution. After being sequenced, the SCoT polymorphisms could distinguish 12 sweet orange cultivars. In addition, the BLAST result showed that part of the SCoT fragments coding region shared high homology with ribosomal protein S3 N superfamily. [Conclusion] This study will provide a theoretical basis for breeding sweet orange cultivars.展开更多
The widely known leaves of Camellia sinensis (L.) derive the most consumed beverage globally. All over the world, like in the Democratic Republic of the Congo (DR Congo), they are marketed in the form of packaged and ...The widely known leaves of Camellia sinensis (L.) derive the most consumed beverage globally. All over the world, like in the Democratic Republic of the Congo (DR Congo), they are marketed in the form of packaged and in bulk black tea. The aim of this study was to report the microscopic botanical features, the chromatographic fingerprints, the antioxidant activities, and the peroxidase inhibition of bulk-marketed black teas consumed in DR Congo. The microscopic analysis of powders from unpackaged black tea marketed in DR Congo allowed the identification of specific microscopical features, which are characteristic to C. sinensis (L.) such as numerous asterosclerites, numerous fragments of unicellular trichomes;fragments of abaxial epidermis consisting of cells with slightly wavy walls, numerous anomocytic stomata, and the detection of biological contaminants, e.g. the dust mites. TLC and HPLC analysis indicated that flavonoids (hyperoside, rutin) and phenolic acids (chlorogenic acid, gallic acid) were major phenolic compounds, along with other secondary metabolites. Aqueous extracts from all bulk-marketed black teas exhibited good antioxidant activities at the concentrations range of 1 - 20 μg·mL-1 using in vitro ABTS and DPPH assays. These extracts showed equally inhibitory effects on myeloperoxidase (MPO) and horseradish peroxidase (HRP) activities at the concentration ranges of 1 - 20 μg·mL-1 and 20 - 100 μg·mL-1 respectively. Bulk-marketed black teas consumed in DR Congo possess antioxidant and anti-inflammatory properties comparable to packaged Ceylon tea largely marketed. The observed bioactivities support the health benefits of the consumed black tea by Congolese people, despite the storage conditions to be improved in order to preserve its nutritional and therapeutic assets.展开更多
The study of the water status and activity of catalase in Chinese tea plants (Camellia sinensis (L.) O. Kuntze) and hazelnut (Corylus pontica C. Koch.). The indicators, which not only describe the physiological state ...The study of the water status and activity of catalase in Chinese tea plants (Camellia sinensis (L.) O. Kuntze) and hazelnut (Corylus pontica C. Koch.). The indicators, which not only describe the physiological state of plants in the stressful period, but also allow it to identify more adaptive varieties within a given area was determined. Analysis of catalase activity data in Chinese tea and hazelnut revealed the presence of similar patterns: there is a change in enzymatic activity in response to hydrothermal factors. It is established that the stable varieties Camellia sinensis (L.) O. Kuntze and Corylus pontica C. Koch. characterized by a more active catalase, which is of particular importance during droughts. The water regime parameters variance scale for Camellia sinensis was developed, which allows differentiating varieties into groups of varying degrees of stability. It is shown that all methods that are used to estimate stability of plant species to extreme environmental conditions are based on several basic principles connected with peculiarities of adaptation mechanisms. The result was a selection of diagnostic indicators proposed for assessing adaptability: the water content of the leaf tissue, water-holding capacity of leaves, and concentration of cellular juice sprouts and activity of catalase.展开更多
Two new acetylated flavonoid diglycosides, scutellarein-5-O-[α-L-rhamnopyranosyl-（1 → 4）-β-D-6-O-acetylglucopyranoside]（1）, apigenin-5-O-[α-L-rhamnopyranosyl-（1→ 2）-β-D-3,6-di-O-acetylglucopyranoside] （2...Two new acetylated flavonoid diglycosides, scutellarein-5-O-[α-L-rhamnopyranosyl-（1 → 4）-β-D-6-O-acetylglucopyranoside]（1）, apigenin-5-O-[α-L-rhamnopyranosyl-（1→ 2）-β-D-3,6-di-O-acetylglucopyranoside] （2）, were isolated from the leaves of Cephalotaxus sinensis. Their structures were determined by chemical and spectroscopic methods including HR-FAB-MS,^1H-^1H COSY, HSQC and HMBC experiments.展开更多
This paper reports on the effects of organic ingredients in facilitating direct shoot regeneration from nodal explants of Hibiscus rosa-sinensis L. This paper also compares the sterilization conditions for 3 types of ...This paper reports on the effects of organic ingredients in facilitating direct shoot regeneration from nodal explants of Hibiscus rosa-sinensis L. This paper also compares the sterilization conditions for 3 types of explants (node, internode, and shoot tip) harvested from an open field. The optimized sterilization conditions for the explants were 40% Clorox- 20 min exposure, 10% Clorox-15 min exposure, and 5% Clorox-40 min exposure for the node, internode and shoot tip, respectively. In the direct shoot regeneration using the nodal explants, we found MS medium containing 40 g/L sucrose, 0.3% (w/v) activated charcoal, and supplementations with myo-inositol, thiamine and nicotinic acid were suitable. The in vitro shoot survival rate was 30% with a mean leaf numbers of 2.68 produced, and a mean leaf length of 1.71 cm achieved after 5 weeks of culture on the modified medium.展开更多
The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 culti...The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 cultivars were divided into two groups by PCA(Principal Component Analysis)clustering analysis of their carotenoid content,and OPLS-DA(Orthogonal projections to latent structures)indicated that the levels ofβ-carotene(VIP=2.89)and lutein(VIP=2.30)were responsible for much of the variation between the two groups.Interestingly,theβ-carotene toβ-ionone conversion rates in Group 1 were higher than in Group 2,while theβ-carotene content was significantly lower in Group 1 than in Group 2.Theβ-ionone content was significantly higher in Group 1.Pearson Correlation Coefficient calculation between the transcription level of candidate genes(CsCCD1 and CsCCD4)and the accumulation ofβ-ionone indicated that CsCCD1 may involve in the formation ofβ-ionone in 13 cultivars.Prokaryotic expression and in vitro enzyme activity assays showed that‘Chuanhuang 1’had an amino acid mutation in carotenoid cleavage dioxygenases 1(CsCCD1)compared with‘Shuchazao’,resulting in a significantly higherβ-ionone content in‘Chuanhuang 1’.Sequence analysis showed that‘Chuanhuang 1’and‘Huangdan’had different CsCCD1 promoter sequences,leading to significantly higher CsCCD1 expression andβ-ionone accumulation in‘Chuanhuang 1’.These results indicated that the promoter and coding sequence diversity of CsCCD1 might contribute to the differential accumulation ofβ-ionone in different tea cultivars.展开更多
Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor....Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.展开更多
文摘In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbeck. Observations of the effect of PGRs were carried out using different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D),1-naphthalene acetic acid (NAA) and combinations of 2,4-D and NAA using different basal media. This study found Citrus sinensis (L.) Osbeck exhibited a high frequency of callus induction on MS medium supplemented with 3 mg/L 2,4-D and callus induction frequency was 86.7% ± 3.4% whereas N6 and B5 showed lower callus induction frequency of 83.3% ± 8.8% and 82.2% ± 1.9% respectively compared to that of MS media with supplementation of the same hormone. Among the induced calli, the morphological analysis showed only 40% - 50% was embryogenic calli. Regeneration of plantlets from calli was done using different concentrations and combinations of auxin and cytokinin. The study showed that 3 mg/L 6-benzylaminopurine (BAP) supplemented medium has the maximum potential to promote regeneration of Citrus sinensis (L.) Osbeck from embryogenic calli with the frequency of 89.3% ± 8.8% but no regeneration occurred from the non-embryogenic calli. The regenerated plantlets were rooted on MS medium with supplementation of 5 mg/l NAA. These observations in Citrus sinensis (L.) Osbeck regeneration will be helpful for genetic improvement with desired traits.
文摘Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.
基金Supported by National Key Technology Research and Development Program(2006BAD01A1702)~~
文摘[Objective] The aim was to identify genetic variation in Citrus sinensis （sweet orange） germplasm from Hunan Province according to the Start Codon Targeted （SCoT） Polymorphism. [Method] The reaction system for SCoT amplification from sweet orange was first optimized, and then the SCoT fragments were amplified from 24 sweet orange cultivars collected in Hunan Province and sequenced for genetic variation analysis. [Result] The optimum reaction system for SCoT markers amplification was 2.0 μl containing 80 ng of template DNA, 0.3 mmol/L dNTPs, 0.2 μmol/L primer, 1.6 mmol/L Mg2＋, 1.6 U of Taq DNA polymerase and 10×PCR buffer. By using this reaction system, the PCR products from the sweet orange cultivars produced clear and reproducible bands at 100-2 000 bp through electrophoresis. The SCoT fragments of the 24 sweet orange cultivars were 1 090-1 091 bp, with the homology of 99.84% and nucleotide deletion and substitution. After being sequenced, the SCoT polymorphisms could distinguish 12 sweet orange cultivars. In addition, the BLAST result showed that part of the SCoT fragments coding region shared high homology with ribosomal protein S3 N superfamily. [Conclusion] This study will provide a theoretical basis for breeding sweet orange cultivars.
文摘The widely known leaves of Camellia sinensis (L.) derive the most consumed beverage globally. All over the world, like in the Democratic Republic of the Congo (DR Congo), they are marketed in the form of packaged and in bulk black tea. The aim of this study was to report the microscopic botanical features, the chromatographic fingerprints, the antioxidant activities, and the peroxidase inhibition of bulk-marketed black teas consumed in DR Congo. The microscopic analysis of powders from unpackaged black tea marketed in DR Congo allowed the identification of specific microscopical features, which are characteristic to C. sinensis (L.) such as numerous asterosclerites, numerous fragments of unicellular trichomes;fragments of abaxial epidermis consisting of cells with slightly wavy walls, numerous anomocytic stomata, and the detection of biological contaminants, e.g. the dust mites. TLC and HPLC analysis indicated that flavonoids (hyperoside, rutin) and phenolic acids (chlorogenic acid, gallic acid) were major phenolic compounds, along with other secondary metabolites. Aqueous extracts from all bulk-marketed black teas exhibited good antioxidant activities at the concentrations range of 1 - 20 μg·mL-1 using in vitro ABTS and DPPH assays. These extracts showed equally inhibitory effects on myeloperoxidase (MPO) and horseradish peroxidase (HRP) activities at the concentration ranges of 1 - 20 μg·mL-1 and 20 - 100 μg·mL-1 respectively. Bulk-marketed black teas consumed in DR Congo possess antioxidant and anti-inflammatory properties comparable to packaged Ceylon tea largely marketed. The observed bioactivities support the health benefits of the consumed black tea by Congolese people, despite the storage conditions to be improved in order to preserve its nutritional and therapeutic assets.
文摘The study of the water status and activity of catalase in Chinese tea plants (Camellia sinensis (L.) O. Kuntze) and hazelnut (Corylus pontica C. Koch.). The indicators, which not only describe the physiological state of plants in the stressful period, but also allow it to identify more adaptive varieties within a given area was determined. Analysis of catalase activity data in Chinese tea and hazelnut revealed the presence of similar patterns: there is a change in enzymatic activity in response to hydrothermal factors. It is established that the stable varieties Camellia sinensis (L.) O. Kuntze and Corylus pontica C. Koch. characterized by a more active catalase, which is of particular importance during droughts. The water regime parameters variance scale for Camellia sinensis was developed, which allows differentiating varieties into groups of varying degrees of stability. It is shown that all methods that are used to estimate stability of plant species to extreme environmental conditions are based on several basic principles connected with peculiarities of adaptation mechanisms. The result was a selection of diagnostic indicators proposed for assessing adaptability: the water content of the leaf tissue, water-holding capacity of leaves, and concentration of cellular juice sprouts and activity of catalase.
文摘Two new acetylated flavonoid diglycosides, scutellarein-5-O-[α-L-rhamnopyranosyl-（1 → 4）-β-D-6-O-acetylglucopyranoside]（1）, apigenin-5-O-[α-L-rhamnopyranosyl-（1→ 2）-β-D-3,6-di-O-acetylglucopyranoside] （2）, were isolated from the leaves of Cephalotaxus sinensis. Their structures were determined by chemical and spectroscopic methods including HR-FAB-MS,^1H-^1H COSY, HSQC and HMBC experiments.
文摘This paper reports on the effects of organic ingredients in facilitating direct shoot regeneration from nodal explants of Hibiscus rosa-sinensis L. This paper also compares the sterilization conditions for 3 types of explants (node, internode, and shoot tip) harvested from an open field. The optimized sterilization conditions for the explants were 40% Clorox- 20 min exposure, 10% Clorox-15 min exposure, and 5% Clorox-40 min exposure for the node, internode and shoot tip, respectively. In the direct shoot regeneration using the nodal explants, we found MS medium containing 40 g/L sucrose, 0.3% (w/v) activated charcoal, and supplementations with myo-inositol, thiamine and nicotinic acid were suitable. The in vitro shoot survival rate was 30% with a mean leaf numbers of 2.68 produced, and a mean leaf length of 1.71 cm achieved after 5 weeks of culture on the modified medium.
基金financially supported by National Natural Science Foundation of China(Grant Nos.31961133030,31870678,32022076)Science Fund for Distinguished Young Scientists of Anhui Province(Grant No.1908085J12).
文摘The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 cultivars were divided into two groups by PCA(Principal Component Analysis)clustering analysis of their carotenoid content,and OPLS-DA(Orthogonal projections to latent structures)indicated that the levels ofβ-carotene(VIP=2.89)and lutein(VIP=2.30)were responsible for much of the variation between the two groups.Interestingly,theβ-carotene toβ-ionone conversion rates in Group 1 were higher than in Group 2,while theβ-carotene content was significantly lower in Group 1 than in Group 2.Theβ-ionone content was significantly higher in Group 1.Pearson Correlation Coefficient calculation between the transcription level of candidate genes(CsCCD1 and CsCCD4)and the accumulation ofβ-ionone indicated that CsCCD1 may involve in the formation ofβ-ionone in 13 cultivars.Prokaryotic expression and in vitro enzyme activity assays showed that‘Chuanhuang 1’had an amino acid mutation in carotenoid cleavage dioxygenases 1(CsCCD1)compared with‘Shuchazao’,resulting in a significantly higherβ-ionone content in‘Chuanhuang 1’.Sequence analysis showed that‘Chuanhuang 1’and‘Huangdan’had different CsCCD1 promoter sequences,leading to significantly higher CsCCD1 expression andβ-ionone accumulation in‘Chuanhuang 1’.These results indicated that the promoter and coding sequence diversity of CsCCD1 might contribute to the differential accumulation ofβ-ionone in different tea cultivars.
基金supported by the Fundamental Research Funds for the Central Universities (Grant No. XDJK 2018B016)the National Natural Sciences Foundation of China (Grant No. 31972393)+1 种基金he earmarked fund for China Agriculture Research System (Grant No. CARS-26)the Natural Science Foundation of Chongqing (Grant No. cstc2020jcyj-msxmX1064)。
文摘Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.
