vstispСадоводство и виноградарствоHorticulture and viticulture0235-25912618-9003Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»10.31676/0235-2591-2022-2-25-31vstisp-861Research ArticleГЕНЕТИКА, СЕЛЕКЦИЯ, СЕМЕНОВОДСТВОGENETICS, BREEDING, SEED PRODUCTIONГенетическая трансформация формы клонового подвоя косточковых культур 146-2 с использованием репортерного гена зеленого флуоресцентного белкаGenetic transformation of clone rootstock of stone fruits 146-2 using the green fluorescent protein reporter geneМуренецЛ. Ю.MourenetsL. Yu.

Муренец Л. Ю. — младший научный сотрудник

г. Пущино

Mourenets L. Yu., Junior Researcher

Pushchino

ПушинА. С.PushinA. S.

Пушин А. С. — младший научный сотрудник

г. Пущино

A. S. Pushin, Junior Researcher

Pushchino

ДолговС. В.DolgovS. V.

Долгов Сергей Владимирович — доктор биологических наук, главный научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Sergey V. Dolgov, Dr. Sci. (Biol.), Chief Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

dolgov@bibch.ruФилиал Института биоорганической химии им. академиков М. М. Шемякина и Ю. А. Овчинникова Российской академии наукРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesRussian FederationФилиал Института биоорганической химии им. академиков М. М. Шемякина и Ю. А. Овчинникова Российской академии наукРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Nikita Botanical Garden — National Scientifi c Center of the Russian Academy of SciencesRussian FederationФилиал Института биоорганической химии им. академиков М. М. Шемякина и Ю. А. Овчинникова Российской академии наук; Никитский ботанический сад — Национальный научный центр РАН; Федеральный научный селекционно-технологический центр садоводства и питомниководстваРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Nikita Botanical Garden — National Scientifi c Center of the Russian Academy of Sciences; Federal Horticultural Research Center for Breeding, Agrotechnology and NurseryRussian Federation202213052022022531Copyright © Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture», 20222022Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»https://www.sadivin.com/jour/about/submissions#copyrightNoticehttps://www.sadivin.com/jour/article/view/861

Для формы карликового зимостойкого клонового подвоя косточковых культур 146-2 (Prunus pumila L.xP. tomentosa Thunb.), разработана система регенерации и генетической трансформации с использованием зеленого флуоресцентного белка (GFP). Для эффективной регенерации придаточных побегов не требовалось предварительной обработки 6-бензиламино-пурином (BA) и ауксином. Стимуляции регенерации побегов из эксплантов листьев требовала 2-3 недели темного периода. Наилучшая регенерация (более 75 %) наблюдалась при комбинации фитогормонов 3 мг/л BA и 0,75 мг/л IBA. Достигнутая эффективность регенерации позволила разработать протокол генетической трансформации, опосредованной Agrobacterium, для подвоя 146-2. Целые листья из культивированных in vitro побегов использовали в качестве эксплантов для трансформации штаммом A. tumefaciens CBE21, с бинарным вектором pBINmGFP5ER, содержащим ген nptII, кодирующий неомицинфосфотрансферазу II в качестве селектируемого для растений маркера под контролем промотора NOS (нопалинсинтазы) и репортерного гена gfp, кодирующего зеленый флуоресцентный белок под контролем промотора 35S вируса мозаики цветной капусты (Caulifl ower mosaic virus) (CaMV). Интеграцию nptII и gfp в трансгены подтверждали с помощью ПЦР. Экспрессию зеленого флуоресцентного белка наблюдали с помощью флуоресцентной микроскопии. Эффективность трансформации на основе анализа ПЦР независимых линий, устойчивых к канамицину, составила 0,41-0,83 %. Все трансгенные линии показали устойчивость к канамицину при концентрации 40 мг/л. Они были укоренены и акклиматизированы к тепличным условиям. Разработанные протоколы будут использоваться для получения устойчивых к вирусу шарки сливы (PPV) растений.

For the form of dwarf winter-hardy clonal rootstocks of stone crops 146-2 (Prunus pumila L.xP.tomentosa Thunb.), system of regeneration and genetic transformation using green fluorescent protein (GFP) has been developed. For eff ective regeneration of accessory shoots, no pre-treatment with 6-benzylamine-purine (BA) and auxin was required. Stimulation of the regeneration of shoots from leaf explants required 2-3 weeks of a dark period. Th e best percentage of regeneration (greater than 75 %) was observed with a combination of 3 mg/L BA and 0.75 mg/L IBA. The achieved regeneration effi ciency made it possible to develop a protocol for genetic transformation, mediated by Agrobacterium, for rootstock 146-2. Whole leaves from in vitro-cultured shoots were used as explants for transformation by the A. tumefaciens strain CBE21, with the binary vector pBINmGFP5ER containing the nptII encoding neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS promoter (nopalin synthase) and the reporter gfp gene encoding a green fluorescent protein under the control of the cauliflower mosaic virus (CaMV) promoter 35S. Th e integration of nptII and gfp into transgenes was confirmed by PCR. Expression of the green fluorescent protein was observed using fluorescence microscopy. The efficiency of transformation based on PCR analysis of independent lines resistant to kanamycin was 0.41-0.83 %. All transgenic lines showed resistance to kanamycin at a concentration of 40 mg/L. They were rooted and acclimatized to greenhouse conditions. Th e developed protocols will be used to produce Plum pox virus (PPV) resistant plants.

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