Targeted genome editing using CRISPR/Cas nucleases has become the standard approach for creating mutant plants. Significant progress has been made to enhance the editing efficiencies through optimizing CRISPR/Cas expression, including applying heat stress. In this study, we used heat stress to enhance the Potato virus X (PVX)-mediated CRISPR/Cas9 mutagenesis in Nicotiana benthamiana. We show that heat stress at 4-5 days after PVX inoculation effectively increases the mutagenesis efficiency of Cas9 nuclease. We observed up to a 5-8% increase in mutation efficiency depending on the sgRNA construct when heat stress is applied to the pPVX-Cas9::sgRNA infiltrated samples. Furthermore, analysis of the effect of the heat stress on the pattern of mutation types in the target gene regions showed no obvious changes in CRISPR/Cas9 induced mutagenesis pattern between heat stress treated and no heat stress treated samples. Overall, our experiments demonstrate that heat stress treatment at the optimal time after viral inoculation is most effective in increasing the PVX-mediated CRISPR/Cas9 editing efficiency in plants.

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• Exogenous melatonin enhances heat stress tolerance in sweetpotato by modulating antioxidant defense system, osmotic homeostasis and stomatal traits
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• Development of virus-induced genome editing methods in Solanaceous crops
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• CRISPR/Cas9-gene editing approaches in plant breeding
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The viral disease induced by Tomato yellow leaf curl virus (TYLCV) reduces tomato (Solanum lycopersicum) yield significantly in tropical and subtropical regions. A number of loci, including Ty-1 to Ty-5, conferring resistance to TYLCV have been described and introgressed into modern tomato cultivars. The availability of molecular markers linked to these genes would expedite the introgression of TYLCV resistance into commercial cultivars. In the present study, we developed gene-based markers linked to the Ty-3 gene using a segregating population derived from a cross between the TYLCV-resistant line S. lycopersicum ‘A45’ and the susceptible line S. lycopersicum ‘A39’. Agrobacterium-mediated screening was used to test TYLCV resistance of plants in the segregating population, and the resistance was evaluated by a visual scoring method and polymerase chain reaction analysis. By comparing sequences of the Ty-3 genes of the resistant and susceptible lines, two high-resolution melting (HRM) markers (Ty3-HRM1 and Ty3-HRM2) and one sequence characterized amplified region (SCAR) marker (Ty3-SCAR1) were developed. The HRM markers were based on single nucleotide polymorphisms at the 13th exon and the 15th intron, whereas the SCAR marker was based on a 246-bp deletion in the 16th intron. These gene-based markers will be useful tools for marker-assisted selection in breeding programs to improve TYLCV resistance of tomato.

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• Hybrid analysis for ToLCV resistance in tomato (Solanum lycopersicum L.) andmolecular validation of Ty-3 gene
  R Shanmugabhavatharani, T Saraswathi, M Kavitha, N Manivannan, N Seenivasan, S Harish
  Journal of Horticultural Sciences.2025;[Epub]     CrossRef
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  Molecular Biotechnology.2025; 67(6): 2576.     CrossRef
• The genetic diversity and certain disease assessments of wild tomato genetic resources and integration into breeding
  Hasan Can
  Frontiers in Life Sciences and Related Technologies.2025; 6(3): 154.     CrossRef
• Evaluation of processing tomato resistance to tomato yellow leaf curl virus (TYLCV) and detecting Ty-2 and Ty-3 resistance genes with SCAR-markers
  S. Earsakul, N. Pornsopin, S. Techawongstien, C. Lapjit, N. Jeeatid, P. Suwor, P. Phimchan, C. Sangrit, W. Nawae, T. Tarinta
  Acta Horticulturae.2024; (1404): 967.     CrossRef
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  Deepak Maurya, Arnab Mukherjee, Bhagyashree, Surabhi Sangam, Randhir Kumar, Shirin Akhtar, Tirthartha Chattopadhyay
  Physiology and Molecular Biology of Plants.2023; 29(1): 121.     CrossRef
• Progress in Marker-Assisted Selection to Genomics-Assisted Breeding in Tomato
  Jagesh Kumar Tiwari, Suresh Reddy Yerasu, Nagendra Rai, Dhananjaya P. Singh, Achuit K. Singh, Suhas G. Karkute, Prabhakar M. Singh, Tusar K. Behera
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• Gene-Based Allele Specific Marker for Resistance to Phytophthora sojae in Soybean (Glycine max L.)
  Young Eun Jang, Sungwoo Lee
  Plant Breeding and Biotechnology.2021; 9(2): 164.     CrossRef
• Validation of molecular markers for multiple disease resistance in tomato (Solanum lycopersicum)
  ZAKIR HUSSAIN, SUMAN LATA, MANISHA MANGAL, B S TOMAR, R K YADAV, GOKUL GOSAVI, ASHWANI KUMAR, PAWAN YADAV, MONIKA MONIKA, S K YADAV
  The Indian Journal of Agricultural Sciences.2019; 89(6): 964.     CrossRef
• qPCR analysis of Ty-2 and Ty-3 gene pyramided lines of tomato for resistance to tomato leaf curl New Delhi virus (ToLCNDV)
  SUMAN LATA, ZAKIR HUSSAIN, MANISHA MANGAL, R K YADAV, VINUTHA T, GOGRAJ SINGH JAT, GOKUL GOSAVI, PAWAN KUMAR, SHELLY PERVEEN, B S TOMAR
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  A T Sadashiva, Peter Hanson, M Krishna Reddy, K V Ravishankar, Manoj Prasad, H C Prasanna, K Madhavi Reddy, T H Singh, R K Saritha, Zakir Hussain, J B Mythili, K S Shivashankara, R M Bhatt, R H Laxman, R B Tiwari, V Sridhar, V Sowmya, N P Kumar, Manmohan
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Capsinoids are the class of secondary metabolites identified in non-pungent peppers exhibiting the same bioactive properties as capsaicinoid. Previously, it has been demonstrated that capsinoid production is controlled by the capsaicin synthase (CS) gene and the putative-aminotransferase (pAMT) gene. In this study, we report that C. chinense ‘SNU11-001’ containing high levels of capsinoid has an early stop codon in pAMT resulted from 403 bp and 8 bp insertions deletion in the third and sixth exons. In order to know whether CS expression is correlated with the level of capsinoid, CS and pAMT expressions were determined using SNU11-001 and four Capsicum accessions with different pungency level. RT-PCR analysis showed higher transcription levels of CS in pungent accessions but no clear differences in pAMT expression. To investigate the effect of the substitution of the pAMT allele of C. chinense ‘Habanero’ with the dysfunctional pAMT allele of SNU11-001, an F₂ population was constructed by a cross between aforementioned parental lines. Molecular markers were developed to distinguish CS and pAMT genotypes of SNU11-001 and Habanero and F₂ plants were genotyped. All F₂ plants having the pAMT genotype of SNU11-001 contained high levels of capsinoid while very low levels of capsaicinoid. There was no significant difference in levels of capsinoid among the F₂ plants regardless of CS genotypes. This may be due to strong CS expression of both parental lines. In conclusion, our results show that it is possible to develop a new Habanero cultivar with high capsinoid content by introducing a dysfunctional pAMT allele.

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• Impact of Low-Intensity Exercise and Capsinoid Supplementation on Blood Lipids in Overweight and Obese Women
  Tae Gyu Kim, Hyun Joo Kang
  The Asian Journal of Kinesiology.2026; 28(1): 73.     CrossRef
• Molecular mechanisms and genomic strategies for enhancing stress resilience in pepper crop
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• Combined Exercise and HabaNova^TM (NET-2201, Capsicum chinense L. cv.) Supplementation Potentiates Improvements in Body Composition and Anthropometric Measures in Overweight/Obese Women: A Double-Blind Randomized Controlled Pilot Study
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• Recent Understanding of the Biosynthesis of Capsaicinoids and Low-pungent Analogs towards Quality Improvement of Chili Pepper
  Yoshiyuki Tanaka
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• Effects of NET-2201 (Capsicum chinense L. cv.) on brown adipose tissue activation and white adipose tissue browning in high-fat-diet-induced obese mice
  Yoon-Young Han, Ha-Neul Jo, Bo-Mi Kim, Jae-Sun Lee, Ji-Min Kim, Dae-Ho Ryu, Dong-Hee Kim, Chan-Sung Park, Byoung-Cheorl Kang, Yong-Wook Lee
  Food Science and Biotechnology.2025; 34(3): 769.     CrossRef
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  Tanyarat Tarinta, Saksit Chanthai, Eiji Nawata, Suchila Techawongstien
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• Update on the genetic and molecular regulation of the biosynthetic pathways underlying pepper fruit color and pungency
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• Breeding Capsicum chinense Lines with High Levels of Capsaicinoids and Capsinoids in the Fruit
  Siyoung Jang, Minjeong Park, Do-Gyeong Lee, Jung-Hyun Lim, Ji-Won Jung, Byoung-Cheorl Kang
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• Factors affecting the capsaicinoid profile of hot peppers and biological activity of their non-pungent analogs (Capsinoids) present in sweet peppers
  Virgílio Gavicho Uarrota, Marcelo Maraschin, Ângela de Fátima M. de Bairros, Romina Pedreschi
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• Influence of Fruit Ripening on the Total and Individual Capsaicinoids and Capsiate Content in Naga Jolokia Peppers (Capsicum chinense Jacq.)
  Mercedes Vázquez-Espinosa, José Arturo Olguín-Rojas, Oreto Fayos, Ana V. González-de-Peredo, Estrella Espada-Bellido, Marta Ferreiro-González, Carmelo G. Barroso, Gerardo F. Barbero, Ana Garcés-Claver, Miguel Palma
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  Mercedes Vázquez-Espinosa, Oreto Fayos, Ana V. González-de-Peredo, Estrella Espada-Bellido, Marta Ferreiro-González, Miguel Palma, Ana Garcés-Claver, Gerardo F. Barbero
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  Mercedes Vázquez-Espinosa, Oreto Fayos, Ana V. González-de-Peredo, Estrella Espada-Bellido, Marta Ferreiro-González, Miguel Palma, Ana Garcés-Claver, Gerardo F. Barbero
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• Positional differences of intronic transposons in pAMT affect the pungency level in chili pepper through altered splicing efficiency
  Yoshiyuki Tanaka, Takaya Asano, Yorika Kanemitsu, Tanjuro Goto, Yuichi Yoshida, Kenichiro Yasuba, Yuki Misawa, Sachie Nakatani, Kenji Kobata
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• Quantitation of capsiate and dihydrocapsiate and tentative identification of minor capsinoids in pepper fruits (Capsicum spp.) by HPLC-ESI-MS/MS(QTOF)
  Oreto Fayos, María Savirón, Jesús Orduna, Gerardo F. Barbero, Cristina Mallor, Ana Garcés-Claver
  Food Chemistry.2019; 270: 264.     CrossRef
• Assessment of Capsaicinoid and Capsinoid Accumulation Patterns during Fruit Development in Three Chili Pepper Genotypes (Capsicum spp.) Carrying Pun1 and pAMT Alleles Related to Pungency
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• Synthesis of (±)-3,4-dimethoxybenzyl-4-methyloctanoate as a novel internal standard for capsinoid determination by HPLC-ESI-MS/MS(QTOF)
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  Open Chemistry.2018; 16(1): 87.     CrossRef
• Identification of a Novel Mutant pAMT Allele Responsible for Low-pungency and Capsinoid Production in Chili Pepper: Accession ‘No. 4034’ (Capsicum chinense)
  Yoshiyuki Tanaka, Shiho Fukuta, Sota Koeda, Tanjuro Goto, Yuichi Yoshida, Ken-ichiro Yasuba
  The Horticulture Journal.2018; 87(2): 222.     CrossRef
• QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum
  Koeun Han, Hea‐Young Lee, Na‐Young Ro, On‐Sook Hur, Joung‐Ho Lee, Jin‐Kyung Kwon, Byoung‐Cheorl Kang
  Plant Biotechnology Journal.2018; 16(9): 1546.     CrossRef
• Screening old peppers (Capsicum spp.) for disease resistance and pungency-related traits
  Cristina Silvar, Carlos A. García-González
  Scientia Horticulturae.2017; 218: 249.     CrossRef
• Marker-assisted backcross breeding for development of pepper varieties (Capsicum annuum) containing capsinoids
  Hyeon-Seok Jeong, Siyoung Jang, Koeun Han, Jin-Kyung Kwon, Byoung-Cheorl Kang
  Molecular Breeding.2015;[Epub]     CrossRef

Cytoplasmic male sterility (CMS) is a phenomenon specific to plant reproduction that has been extensively exploited for hybrid seed production. Orf456 was previously identified as a candidate gene mediating male sterility in pepper. To gain further insight of this candidate gene, we carried out experiments to transform Chinese cabbage (Brassica rapa L.). About 30 T₁ transgenic lines were obtained and approximately 50 % of T₁ transgenic Chinese cabbage lines showed male-sterility. To evaluate pollen viability, three different approaches including plasmolysis test, staining pollen and in vitro germination assay were used. Analysis of the CMS transgenic lines showed that trasgenic Chinese cabbages produced aberrant pollen development while some were unable to produce pollen. In conclusion, the mitochondrial orf456 gene could induce partial male sterility in transgenic Chinese cabbage. Nevertheless, how the orf456 gene precisely functions to induce male sterility and its biochemical function remains to be discovered.

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• Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea
  Caitao Chang, Deling Sun, Kana Hondo, Fumika Kakihara, A.‐M. Chevre
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