CRISPR Uyumlu Bir Vektör Kullanılarak Marul Genotipi ve Agrobacterium Suşunun Transformasyon Verimliliği Açısından Değerlendirilmesi

Year 2025, Volume: 6 Issue: 2, 101 - 110, 19.10.2025

Fatma Akçakale Kaba , Yüksel Bora Oral , Gülan Ata , Nedim Mutlu , Adem Kaba

https://doi.org/10.70562/tubid.1737836

Abstract

Marul (Lactuca sativa L.), dünya genelinde yetiştirilen ekonomik ve besin değeri yüksek bir sebze türüdür. Ancak, çeşitli biyotik ve abiyotik stres etmenlerine karşı duyarlılığı, biyoteknolojik araçlarla genetik iyileştirmeyi gerekli kılmaktadır. Agrobacterium tumefaciens aracılı transformasyon, bitki biyoteknolojisinde temel bir yöntem olup, marulda genetik modifikasyon çalışmalarında önemli bir rol oynamaktadır. Bu çalışmanın amacı, farklı marul çeşitleri ve Agrobacterium suşları arasında transformasyon verimliliğini karşılaştırmaktır. Dokuz farklı marul çeşidine (Caipira, Emocion, Festival, Funtime, Garone, Maritima, Rüzgar, Sürpriz ve Vaidosa) ait kotiledon eksplantları, iki Agrobacterium suşu (LBA4404 ve EHA105) kullanılarak transformasyona tabi tutulmuştur. Her iki suş da seçici bir belirteç (marker) ve CRISPR ile ilişkili elemanlar içeren bir ikili vektör taşımaktadır. Bu vektör, gen düzenleme amacıyla değil, büyük boyutlu bir plazmidin aktarım verimliliğini değerlendirmek amacıyla kullanılmıştır. Transformasyonun ardından eksplantlar, fosfinotrisin (PPT) içeren seçici MS besiyerine aktarılmıştır. En yüksek sürgün rejenerasyonu Emocion ve Caipira çeşitlerinde gözlemlenmiştir. PCR analizleri, rejenerasyon gösteren bitkilerde transgenin başarıyla entegre olduğunu doğrulamıştır. İstatistiksel analizler, EHA105 suşunun LBA4404’e kıyasla anlamlı derecede daha yüksek transformasyon verimliliği sağladığını göstermiştir (p < 0.05). Test edilen genotipler arasında, Caipira en fazla transgenik sürgünü üretmiş ve en yüksek yanıtı vermiştir. Ayrıca, kotiledon üzerindeki sap (petiyol) bölgesine yakın kısmın sürgün oluşumu için daha elverişli olduğu belirlenmiştir. Bu çalışma, marulda transformasyon verimliliğini artırmaya yönelik protokol optimizasyonuna katkı sağlamakta ve genotip ile Agrobacterium suşu seçiminin önemini ortaya koymaktadır.

Keywords

Bitki biyoteknolojisi , genetik transformasyon , fosfinotrisin seçilimi , kotiledon eksplantı , transgenik sürgün , rejenerasyon verimliliği

Evaluation of Lettuce Genotype and Agrobacterium Strain for Transformation Efficiency Using a CRISPR-Compatible Vector

Abstract

Lettuce (Lactuca sativa L.) is an economically and nutritionally important vegetable crop cultivated worldwide. However, its susceptibility to various biotic and abiotic stresses necessitates genetic improvement through biotechnological tools. Agrobacterium tumefaciens-mediated transformation is a key method in plant biotechnology and plays a vital role in genetic modification studies in lettuce. This study aimed to compare transformation efficiency among different lettuce varieties and Agrobacterium strains. Cotyledon explants from nine lettuce varieties (Caipira, Emocion, Festival, Funtime, Garone, Maritima, Ruzgar, Surpriz, and Vaidosa) were subjected to transformation using two Agrobacterium strains, LBA4404 and EHA105. Both strains carried a binary vector containing a selectable marker and CRISPR-related elements. This vector was not used for gene editing but to assess delivery efficiency of a large-sized plasmid. Following transformation, explants were cultured on phosphinothricin (PPT)-selective MS medium. The highest shoot regeneration was observed in Emocion and Caipira. PCR analysis confirmed successful transgene integration in regenerated plants. Statistical analysis indicated that EHA105 provided significantly higher transformation efficiency than LBA4404 (p < 0.05). Among the tested genotypes, Caipira produced the highest number of transgenic shoots and showed superior responsiveness. Additionally, the region near the petiole on the cotyledon was found to be more favorable for shoot induction. This study contributes to protocol optimization for enhancing transformation efficiency in lettuce based on genotype and Agrobacterium strain selection.

Keywords

plant biotechnology , genetic transformation , phosphinothricin selection , cotyledon explant , transgenic shoot , regeneration efficiency

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