vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-26-26vavilov-5035Research ArticleГЕНЕТИЧЕСКАЯ ИНЖЕНЕРИЯOn the 150th anniversary of Gregor Mendel’s report “Experiments in plant hybridization”Роль типа экспланта и применения селективного агента в первичной эффективности трансформации Lens culinaris Medik.The role of explant type and selective agent application in the initial transformation rate of Lens culinaris Medik.ДюбенкоТ. В.DyubenkoT. V.

Санкт-Петербург

St. Petersburg

СмирновК. В.SmirnovK. V.

Пушкин, Санкт-Петербург

Pushkin, St. Petersburg

ТвороговаВ. Е.TvorogovaV. E.

Санкт-Петербург;

Краснодарский край

St. Petersburg;

Sirius Federal Territory, Krasnodar region

krubaza@mail.ruФедеральный исследовательский центр Всероссийский институт генетических ресурсов растений им. Н.И. Вавилова (ВИР)РоссияFederal Research Center the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR)Russian FederationСанкт-Петербургский государственный университет; Всероссийский научно-исследовательский институт сельскохозяйственной микробиологииРоссияSaint Petersburg State University; All-Russia Research Institute for Agricultural MicrobiologyRussian FederationСанкт-Петербургский государственный университет; Научно-технологический университет «Сириус», федеральная территория «Сириус»РоссияSaint Petersburg State University; Sirius University of Science and TechnologyRussian Federation202606042026302233240Copyright © Дюбенко Т.В., Смирнов К.В., Творогова В.Е., 20262026Дюбенко Т.В., Смирнов К.В., Творогова В.Е.Dyubenko T.V., Smirnov K.V., Tvorogova V.E.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/5035

Lens culinaris Medik. (чечевица) – важная сельскохозяйственная культура из семейства бобовых, но модификация ее генома редко используется для создания новых сортов. Вероятно, это связано с низкой эффективностью существующих протоколов трансформации. Как правило, разработка универсальных, независимых от генотипа протоколов получения трансгенных растений зависит, среди прочего, от возможности образования существенного количества трансгенных клеток in vitro. В настоящем исследовании мы предприняли попытку адаптировать разработанный ранее для другого бобового протокол агробактериальной трансформации для получения трансгенной каллусной ткани у L. culinaris. Чтобы оценить эффективность трансформации, мы выбрали две независимые репортерные системы: бета-глюкуронидазу и зеленый флуоресцентный белок. С помощью этих систем мы нашли оптимальный тип экспланта для создания каллусной ткани чечевицы, экспрессирующей рекомбинантную ДНК. Мы также оценили влияние гигромицина, одного из распространенных селективных агентов, на количество трансгенной ткани в развивающихся трансформированных эксплантах L. culinaris. Согласно нашим результатам, протокол трансформации, который обычно применяется для эксплантов листьев Medicago truncatula Gaertn., может быть использован и для получения трансгенных каллусов из верхушек побегов L. culinaris. Экспланты из апексов побегов продемонстрировали более высокую первичную эффективность трансформации по сравнению с эксплантами из корней, стеблей и листьев. Кроме того, экспланты разных типов, культивируемые на среде без гигромицина, образовывали значительно меньше каллусов, экспрессирующих репортерные гены, по сравнению с эксплантами, выращиваемыми на среде с гигромицином. Это подтверждает возможность использования гигромицина в качестве эффективного селективного агента для чечевицы. В ходе нашего анализа мы также обнаружили GUS-подобное окрашивание в каллусах, не содержащих плазмид для экспрессии гена GUS, что можно объяснить так называемой внутренней GUS-подобной активностью, которая была описана в предыдущих исследованиях. Эти данные могут быть полезны для дальнейшей разработки эффективных и универсальных протоколов трансформации и редактирования генома L. culinaris.

Lens culinaris Medik. (lentil) is an agronomically important leguminous species, but its genome modification is rarely used for obtaining new varieties, probably due to a low efficiency of transformation protocols. Development of universal, genotype-independent protocols for obtaining transgenic plants usually relies, among other factors, on the possibility of obtaining a substantial number of transgenic cells in vitro. This study aimed to adapt a previously developed Agrobacteriummediated transformation protocol, used for a related legume, for the production of transgenic callus tissue in L. culinaris. We used two different markers of transgenic tissue, beta-glucuronidase and green fluorescent protein, to find an optimal type of explant for obtaining transgenic tissue in lentil. We also evaluated the impact of hygromycin, a common selective agent, on the amount of transgenic tissue in developing transformed explants of L. culinaris. According to our results, the transformation protocol commonly used for Medicago truncatula Gaertn. leaf explants can also be applied for obtaining transgenic calli from L. culinaris shoot apices. Explants from shoot apices demonstrated higher initial transformation rate in comparison with explants from roots, stems and leaves. Moreover, explants of different types, which were cultivated on medium without hygromycin, developed significantly fewer calli expressing reporter genes than those grown on hygromycin-containing medium, confirming that hygromycin may be used as an effective selection agent for lentil. During our analysis, we noticed GUS-like staining in calli which didn’t contain plasmids for GUS gene expression. This can be explained with so-called intrinsic GUS-like activity, which was described in previous research. These data can be used for further development of effective and universal L. culinaris transformation and genome editing protocols.

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The authors declare that there are no conflicts of interest present.