scholarly journals Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey

2021 ◽  
Vol 22 (8) ◽  
pp. 3921
Author(s):  
Sung-il Park ◽  
Hyun-Bin Kim ◽  
Hyun-Ji Jeon ◽  
Hyeran Kim
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Capsicum Annuum ◽  
Gene Editing ◽  
Binary Vector ◽  
Transformation Method ◽  
Bell Pepper ◽  
Hot Pepper ◽  
Genome Sequences ◽  
Agrobacterium Strain ◽  
Cultivated Species

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains—AGL1, EHA101, and GV3101—to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

scholarly journals Evaluasi varietas, sumber eksplan dan strain Agrobacterium terhadap keberhasilan transformasi tebu dengan gen P5CS Evaluation of varieties, explant sources, and Agrobacterium strains for successful sugarcane transformation using P5CS gene

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Hayati MINARSIH ◽  
Dwi SUBIYARTI ◽  
Imron RIYADI ◽  
Soekarno Mismana PUTRA ◽  
Laksmi AMBARSARI
Keyword(s):  
Drought Stress ◽  
Agrobacterium Tumefaciens ◽  
Somatic Embryos ◽  
Saccharum Officinarum ◽  
Transformation Method ◽  
Embryogenic Calli ◽  
Temporary Immersion System ◽  
Agrobacterium Strain ◽  
Solid Media ◽  
P5cs Gene

Abstract Genetic transformation can be used as an alter-native to develop sugarcane (Saccharum officinarum L.) tolerant to drought stress. P5CS gene has a role in biosynthesis of proline, an amino acid that accumulated under drought stress conditions. Transfer of a P5CS gene construct into plant cells in conjunction with regeneration of transgenic plantlets may develop sugarcane tolerant to drought stress. The aim of this research was to obtain an optimal transformation method which includes a suitable strain of Agrobacterium tumefaciens, and the best sugarcane explant and variety. The results showed that transfer of P5CS gene has been successfully carried out on sugarcane explants from solid media-derived calli, embryogenic calli and somatic embryos derived from temporary immersion system (TIS) culture. Whilst Agrobacterium strain LBA4404 was indicated as the most effective transformation vector. The regeneration of Kidang Kencana variety transformants from calli and somatic embryos was better than those of PS 881 and PS 891. The best performance of transformants based on the source of explants obtained from somatic embryos from TIS culture. Moreover, a succesfull Agrobacterium mediated transformation on sugarcane was indicated by transient expression of Gus gene and the ability of the transformants grew in a selection medium containing 50 ppm of kanamycin.Abstrak Transformasi genetik dapat digunakan sebagai upaya untuk merakit tebu (Saccharum officinarum L.) toleran terhadap cekaman kekeringan. Gen P5CS diketahui  berperan  dalam  biosintesis  prolin,  yaitu asam amino yang umumnya terakumulasi ketika tanaman mengalami cekaman kekeringan. Transfor-masi gen P5CS dan regenerasi transgeniknya mungkin dapat menghasilkan tanaman tebu trans-genik yang toleran terhadap cekaman kekeringan. Tujuan penelitian ini adalah untuk mendapatkan metode transformasi yang optimum yang mencakup strain Agrobacterium tumefaciens yang sesuai, sumber eksplan dan varietas tebu terbaik sebagai target transformasi. Hasil penelitian menunjukkan bahwa transformasi gen P5CS telah berhasil dilakukan ke eksplan tebu baik yang berupa kalus asal media padat maupun kalus embriogenik dan embrio somatik asal kultur sistem perendaman sesaat (SPS). Sementara itu strain A. tumefaciens LBA4404 menunjukkan hasil yang paling efektif sebagai vektor transformasi. Pertumbuhan transforman baik pada kalus maupun embrio somatik pada varietas Kidang Kencana terlihat paling baik dibandingkan dengan varietas PS 881 dan PS 891. Sumber eksplan yang paling efektif adalah embrio somatik yang diperoleh dari  kultur SPS. Keberhasilan transformasi tebu me-lalui Agrobacterium ditunjukkan oleh ekspresi transien dari gen GUS dan kemampuan dari trans-forman untuk tumbuh di media yang mengandung    50 ppm kanamisin.

scholarly journals A New Disease, Arboricola Leaf Spot of Bell Pepper, Caused by Xanthomonas arboricola

Plant Disease ◽  
2010 ◽  
Vol 94 (2) ◽  
pp. 271-271 ◽  
Author(s):  
I.-S. Myung ◽  
I. H. Jeong ◽  
S. Y. Moon ◽  
S. W. Lee ◽  
H. S. Shim
Keyword(s):  
Capsicum Annuum ◽  
Leaf Spot ◽  
Bell Pepper ◽  
Leaf Spot Disease ◽  
Identification System ◽  
Hot Pepper ◽  
Bacterial Isolates ◽  
Nucleotide Sequence Data ◽  
Xanthomonas Arboricola ◽  
Reference Strains

In June 2007, a leaf spot disease was observed on seedlings of bell pepper (Capsicum annuum L. var. angulosum) in a commercial greenhouse in Iksan City, Korea. Symptoms on leaves included small, irregularly shaped, brown lesions with yellow halos and marginal necrosis. Four bacterial isolates, BC2526, BC2527, BC2528, and BC2529, were obtained from the diseased plants. The isolates were gram-negative aerobic rods with a single flagellum. On peptone sucrose agar, colonies were yellow and raised with smooth margins. Pathogenicity was confirmed by spraying cell suspensions containing 106 CFU/ml onto seedlings of bell pepper (cv. Fieste), tomato (Solanum lycopersicon cv. Seokwang), and hot pepper (Capsicum annuum cv. Daekwang) in a greenhouse maintained at 26 ± 3°C. The isolates induced symptoms, spots, and margin blights on leaves of bell pepper, tomato, and hot pepper 2 weeks after inoculation. No symptoms were noted on the control plants inoculated with sterilized distilled water. The identity of the bacteria was confirmed with the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA). The gyrB region was partially sequenced to aid in identification of four isolates using PCR primers reported by Parkinson et al. (1). A 701-bp fragment of the gyrB region from the isolates was compared with sequences of the reference strains of Xanthomonas available in the DDBL/EMBL/GenBank databases (4). The bacterial isolates clustered with Xanthomonas arboricola pathovars in a phylogenetic tree generated with the neighbor-joining method in MEGA (version 4.1) (3). The sequence of the gyrB from the isolates had distance indexes of 0.016, 0.014, 0.016, 0.013, 0.037, and 0.019 as determined by the Jukes-Cantor model (2) with sequences of the reference strains of X. arboricola pvs. pruni (EU498953), celebensis (EU498984), corylina (EU499002), juglandis (EU 498951), populi (EU 499035), and a X. arboricola strain from bell pepper (EU 499039) (4), respectively. To our knowledge, this is the first report of a leaf disease on bell pepper caused by X. arboricola. We propose the name arboricola leaf spot for the disease. Further studies are required for determining pathovar status of the strain. Nucleotide sequence data reported are available under Accession Nos. GQ502678, GQ502679, GQ502680, and GQ502681 for gyrB of BC2626, BC2527, BC2528, and BC2923, respectively. The disease is expected to have a significant economic impact on tomato and pepper production in Korea. References: (1) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009. (2) N. Saitou and M. Nei. Mol. Biol. Evol. 4:406, 1987. (3) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007. (4) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

scholarly journals Stable Production of Transgenic Pepper Plants Mediated by Agrobacterium tumefaciens

HortScience ◽  
2007 ◽  
Vol 42 (6) ◽  
pp. 1425-1430 ◽  
Author(s):  
Moon Kyung Ko ◽  
Hyunchul Soh ◽  
Kyung-Moon Kim ◽  
Young Soon Kim ◽  
Kyunghoan Im
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Segregation Ratio ◽  
Transformation Method ◽  
Selection Strategy ◽  
Northern Hybridization ◽  
Hot Pepper ◽  
Hygromycin Selection ◽  
Transgenic Lines ◽  
Transgenic Pepper ◽  
Pepper Plants

The aim of this study was to establish a stable transformation method for hot pepper using the hygromycin phosphotransferase (hpt)/hygromycin selection strategy. Explants from aseptic pepper seedlings were inoculated with Agrobacterium tumefaciens carrying pCAMBIA1301. A number of calli were developed on the medium containing hygromycin to discriminate the induction of “false-positive buds,” and then shoots were successfully regenerated from the hygromycin-resistant calli. Southern and Northern hybridization analysis indicated that the hpt gene was integrated and expressed in the transgenic pepper plants (T0) and transmitted to the progeny (T1) without genetic modification. Most T1 progenies derived from self-pollination revealed a 3:1 segregation ratio for hygromycin resistance, indicating that one copy of the T-DNA was integrated into the respective transgenic lines. Both uidA and hpt genes were stably expressed in the T1 generation and coinherited in the progenies. Finally, homozygous progenies were identified in the T1 generation of the transgenic peppers, and the homozygous state was maintained in all progenies tested (T2). The results show the reliability and stability of the hpt/hygromycin selection protocol for pepper transformation.

scholarly journals ATTITUDES OF SERBIAN CONSUMERS TOWARDS DIFFERENT PEPPER FRUITS

2021 ◽  
Author(s):  
Dario Đ Danojević ◽  
Svetlana Glogovac ◽  
Sladjana Medic-Pap ◽  
Djordje Moravcevic
Keyword(s):  
Capsicum Annuum ◽  
Bell Pepper ◽  
Hot Pepper ◽  
Fruit Colour ◽  
Fruit Type ◽  
Capsicum Annuum L ◽  
Lack Of Information ◽  
Pepper Fruit ◽  
The World ◽  
Hot Peppers

Pepper (Capsicum annuum L.) is one of the major vegetable species in the world. In Balkan cuisines, as well as in Serbia, pepper has a very diverse use. Knowledge about consumers habits and preferences is of great importance for a breeding process as well as in the market-orientated production. Because of the lack of information about consumers preferences towards pepper types, in the Serbian market, the present research was conducted. Four hundred and two participants, classified into groups, according to gender, age and education, answered the survey questions. According to this research, the most preferred pepper type in Serbia is kapia, while the bell pepper is the second chosen type. Also, obtained results showed that the most favourite colour of pepper fruit is red. There is a tendency in higher importance of fruit type than fruit colour. The highest percentage of hot pepper consumers prefer medium hot peppers. The obtained trend shows that women generally prefer less spicy pepper fruits than men.

scholarly journals A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Hyeran Kim ◽  
Jisun Choi ◽  
Kang-Hee Won
Keyword(s):  
Genome Editing ◽  
Capsicum Annuum ◽  
Gene Editing ◽  
Critical Time ◽  
Sweet Pepper ◽  
Hot Pepper ◽  
Sequencing Analysis ◽  
Guide Rna ◽  
Guide Rnas

Abstract Background DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops. Results This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum ‘CM334’ and C. annuum ‘Dempsey’. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs. Conclusions Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.

scholarly journals Evaluasi varietas, sumber eksplan dan strain Agrobacterium terhadap keberhasilan transformasi tebu dengan gen P5CS Evaluation of varieties, explant sources, and Agrobacterium strains for successful sugarcane transformation using P5CS gene

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Hayati MINARSIH ◽  
Dwi SUBIYARTI ◽  
Imron RIYADI ◽  
Soekarno Mismana PUTRA ◽  
Laksmi AMBARSARI
Keyword(s):  
Drought Stress ◽  
Agrobacterium Tumefaciens ◽  
Somatic Embryos ◽  
Saccharum Officinarum ◽  
Transformation Method ◽  
Embryogenic Calli ◽  
Temporary Immersion System ◽  
Agrobacterium Strain ◽  
Solid Media ◽  
P5cs Gene

Abstract Genetic transformation can be used as an alter-native to develop sugarcane (Saccharum officinarum L.) tolerant to drought stress. P5CS gene has a role in biosynthesis of proline, an amino acid that accumulated under drought stress conditions. Transfer of a P5CS gene construct into plant cells in conjunction with regeneration of transgenic plantlets may develop sugarcane tolerant to drought stress. The aim of this research was to obtain an optimal transformation method which includes a suitable strain of Agrobacterium tumefaciens, and the best sugarcane explant and variety. The results showed that transfer of P5CS gene has been successfully carried out on sugarcane explants from solid media-derived calli, embryogenic calli and somatic embryos derived from temporary immersion system (TIS) culture. Whilst Agrobacterium strain LBA4404 was indicated as the most effective transformation vector. The regeneration of Kidang Kencana variety transformants from calli and somatic embryos was better than those of PS 881 and PS 891. The best performance of transformants based on the source of explants obtained from somatic embryos from TIS culture. Moreover, a succesfull Agrobacterium mediated transformation on sugarcane was indicated by transient expression of Gus gene and the ability of the transformants grew in a selection medium containing 50 ppm of kanamycin.Abstrak Transformasi genetik dapat digunakan sebagai upaya untuk merakit tebu (Saccharum officinarum L.) toleran terhadap cekaman kekeringan. Gen P5CS diketahui  berperan  dalam  biosintesis  prolin,  yaitu asam amino yang umumnya terakumulasi ketika tanaman mengalami cekaman kekeringan. Transfor-masi gen P5CS dan regenerasi transgeniknya mungkin dapat menghasilkan tanaman tebu trans-genik yang toleran terhadap cekaman kekeringan. Tujuan penelitian ini adalah untuk mendapatkan metode transformasi yang optimum yang mencakup strain Agrobacterium tumefaciens yang sesuai, sumber eksplan dan varietas tebu terbaik sebagai target transformasi. Hasil penelitian menunjukkan bahwa transformasi gen P5CS telah berhasil dilakukan ke eksplan tebu baik yang berupa kalus asal media padat maupun kalus embriogenik dan embrio somatik asal kultur sistem perendaman sesaat (SPS). Sementara itu strain A. tumefaciens LBA4404 menunjukkan hasil yang paling efektif sebagai vektor transformasi. Pertumbuhan transforman baik pada kalus maupun embrio somatik pada varietas Kidang Kencana terlihat paling baik dibandingkan dengan varietas PS 881 dan PS 891. Sumber eksplan yang paling efektif adalah embrio somatik yang diperoleh dari  kultur SPS. Keberhasilan transformasi tebu me-lalui Agrobacterium ditunjukkan oleh ekspresi transien dari gen GUS dan kemampuan dari trans-forman untuk tumbuh di media yang mengandung    50 ppm kanamisin.

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