Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)

Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)

Представлены результаты по введению в культуру in vitro, регенерации и укоренению побегов у сортообразца Пэрэможець и сорта Мираж рыжика посевного (Camelina sativa). Установлены эффективные концентрации стерилизующих агентов, время обработки ими растительного материала при введении в культуру in vit...

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Дата:2013
Автори: Емец, А.И., Бойчук, Ю.Н., Шиша, Е.Н., Рахметов, Д.Б., Блюм, Я.Б.
Формат: Стаття
Мова:Russian
Опубліковано: Інститут клітинної біології та генетичної інженерії НАН України 2013
Назва видання:Цитология и генетика
Теми:
Оригинальные работы
Онлайн доступ:http://dspace.nbuv.gov.ua/handle/123456789/126570
Теги: Додати тег
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Назва журналу:Digital Library of Periodicals of National Academy of Sciences of Ukraine
Цитувати:Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa) / А.И. Емец, Ю.Н. Бойчук, Е.Н. Шиша, Д.Б. Рахметов, Я.Б. Блюм // Цитология и генетика. — 2013. — Т. 47, № 3. — С. 14-20. — Бібліогр.: 25 назв. — рос.

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Digital Library of Periodicals of National Academy of Sciences of Ukraine
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spelling irk-123456789-1265702017-11-27T03:03:02Z Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa) Емец, А.И. Бойчук, Ю.Н. Шиша, Е.Н. Рахметов, Д.Б. Блюм, Я.Б. Оригинальные работы Представлены результаты по введению в культуру in vitro, регенерации и укоренению побегов у сортообразца Пэрэможець и сорта Мираж рыжика посевного (Camelina sativa). Установлены эффективные концентрации стерилизующих агентов, время обработки ими растительного материала при введении в культуру in vitro, определено соотношение концентраций регуляторов роста, изучено влияние количества сахарозы, тип и возраст экспланта на индукцию образования побегов C. sativa, а также установлена концентрация нафтилуксусной кислоты, вызывающая ризогенез у полученных побегов. Разработан метод Agrobacterium-опосредуемой трансформации рыжика с использованием бинарного вектора pGH217, несущего репортерный ген β-глюкуронидазы (gus) под контролем 35S промотора вируса мозаики цветной капусты и nos-терминатора, а также селективный маркерный ген hpt, обеспечивающий устойчивость к гигромицину у трансформантов. The results on in vitro culture establishment, plantlet regeneration and rooting of Camelina sativa cultivar sample Peremozhets and cultivar Mirazh are presented. Effective concentrations of sterilizing agents and duration of plant material treatment were estimated. Phytohormone ratio, sucrose concentration in nutrient medium that induce effective formation of C. sativa shoots and NAA concentration for plantlet rooting have been established. The method of Agrobacteriummediated transformation of Camelina by using binary vector pGH217 carrying reporter β-glucoronidase (gus) gene driven under 35S CaMV promoter and nos-terminator, and selective marker hpt gene conferring hygromycin-resistance in transgenic plant was elaborated. 2013 Article Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa) / А.И. Емец, Ю.Н. Бойчук, Е.Н. Шиша, Д.Б. Рахметов, Я.Б. Блюм // Цитология и генетика. — 2013. — Т. 47, № 3. — С. 14-20. — Бібліогр.: 25 назв. — рос. 0564-3783 DOI: 10.3103/S0095452713030031 http://dspace.nbuv.gov.ua/handle/123456789/126570 604.6 : 581.143.5 : 582.683.2 ru Цитология и генетика Інститут клітинної біології та генетичної інженерії НАН України
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language Russian
topic Оригинальные работы
Оригинальные работы
spellingShingle Оригинальные работы
Оригинальные работы
Емец, А.И.
Бойчук, Ю.Н.
Шиша, Е.Н.
Рахметов, Д.Б.
Блюм, Я.Б.
Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)
Цитология и генетика
description Представлены результаты по введению в культуру in vitro, регенерации и укоренению побегов у сортообразца Пэрэможець и сорта Мираж рыжика посевного (Camelina sativa). Установлены эффективные концентрации стерилизующих агентов, время обработки ими растительного материала при введении в культуру in vitro, определено соотношение концентраций регуляторов роста, изучено влияние количества сахарозы, тип и возраст экспланта на индукцию образования побегов C. sativa, а также установлена концентрация нафтилуксусной кислоты, вызывающая ризогенез у полученных побегов. Разработан метод Agrobacterium-опосредуемой трансформации рыжика с использованием бинарного вектора pGH217, несущего репортерный ген β-глюкуронидазы (gus) под контролем 35S промотора вируса мозаики цветной капусты и nos-терминатора, а также селективный маркерный ген hpt, обеспечивающий устойчивость к гигромицину у трансформантов.
format Article
author Емец, А.И.
Бойчук, Ю.Н.
Шиша, Е.Н.
Рахметов, Д.Б.
Блюм, Я.Б.
author_facet Емец, А.И.
Бойчук, Ю.Н.
Шиша, Е.Н.
Рахметов, Д.Б.
Блюм, Я.Б.
author_sort Емец, А.И.
title Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)
title_short Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)
title_full Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)
title_fullStr Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)
title_full_unstemmed Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa)
title_sort введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (camelina sativa)
publisher Інститут клітинної біології та генетичної інженерії НАН України
publishDate 2013
topic_facet Оригинальные работы
url http://dspace.nbuv.gov.ua/handle/123456789/126570
citation_txt Введение в культуру in vitro, регенерация и генетическая трансформация рыжика посевного (Camelina sativa) / А.И. Емец, Ю.Н. Бойчук, Е.Н. Шиша, Д.Б. Рахметов, Я.Б. Блюм // Цитология и генетика. — 2013. — Т. 47, № 3. — С. 14-20. — Бібліогр.: 25 назв. — рос.
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fulltext ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 314 Ïðåäñòàâëåíû ðåçóëüòàòû ïî ââåäåíèþ â êóëüòóðó in vitro, ðåãåíåðàöèè è óêîðåíåíèþ ïîáåãîâ ó ñîðòî- îáðàçöà Ïýðýìîæåöü è ñîðòà Ìèðàæ ðûæèêà ïî- ñåâíîãî (Camelina sativa). Óñòàíîâëåíû ýôôåêòèâíûå êîíöåíòðàöèè ñòåðèëèçóþùèõ àãåíòîâ, âðåìÿ îáðà- áîòêè èìè ðàñòèòåëüíîãî ìàòåðèàëà ïðè ââåäåíèè â êóëüòóðó in vitro, îïðåäåëåíî ñîîòíîøåíèå êîíöåí- òðàöèé ðåãóëÿòîðîâ ðîñòà, èçó÷åíî âëèÿíèå êîëè÷å- ñòâà ñàõàðîçû, òèï è âîçðàñò ýêñïëàíòà íà èíäóêöèþ îáðàçîâàíèÿ ïîáåãîâ C. sativa, à òàêæå óñòàíîâëåíà êîíöåíòðàöèÿ íàôòèëóêñóñíîé êèñëîòû, âûçûâàþùàÿ ðèçîãåíåç ó ïîëó÷åííûõ ïîáåãîâ. Ðàçðàáîòàí ìåòîä Agrobacterium-îïîñðåäóåìîé òðàíñôîðìàöèè ðûæèêà ñ èñïîëüçîâàíèåì áèíàðíîãî âåêòîðà pGH217, íåñóùåãî ðåïîðòåðíûé ãåí -ãëþêóðîíèäàçû (gus) ïîä êîíòðî- ëåì 35S ïðîìîòîðà âèðóñà ìîçàèêè öâåòíîé êàïóñòû è nos-òåðìèíàòîðà, à òàêæå ñåëåêòèâíûé ìàðêåðíûé ãåí hpt, îáåñïå÷èâàþùèé óñòîé÷èâîñòü ê ãèãðîìèöèíó ó òðàíñôîðìàíòîâ. Ââåäåíèå. Ðûæèê ïîñåâíîé (Camelina sativa (L.) Crantz) – íåäîñòàòî÷íî èñïîëüçóåìûé ïðåä- ñòàâèòåëü ñåìåéñòâà Brassicaceae, ê êîòîðîìó â ïîñëåäíåå âðåìÿ âîçðîäèëñÿ èíòåðåñ èç-çà óíèêàëüíîãî ñîñòàâà æèðîâ, ñîäåðæàùèõñÿ â åãî ñåìåíàõ. Ðàíåå ðåçóëüòàòû àðõåîëîãè÷å- ñêèõ èññëåäîâàíèé ïîêàçàëè, ÷òî ýòî ðàñòå- íèå êóëüòèâèðîâàëè â Åâðîïå åùå âî âðåìåíà áðîíçîâîãî âåêà [1], ãäå åãî, âåðîÿòíî, èñïîëü- çîâàëè êàê âàæíóþ ìàñëè÷íóþ êóëüòóðó.  îñ- íîâíîì îí áûë ðàñïðîñòðàíåí ïî âñåé òåððè- òîðèè Åâðîïû êàê ñîðíÿê, è äî ñåðåäèíû XX âåêà èñïîëüçîâàëñÿ â ñåëüñêîì õîçÿéñòâå ëèøü â íåçíà÷èòåëüíûõ ìàñøòàáàõ, â òîì ÷èñëå íà Áàëêàíàõ è â Óêðàèíå. Òåïåðü ðûæèê ðàññìà- òðèâàåòñÿ â êà÷åñòâå îäíîãî èç âèäîâ, ê êîòî- ðîìó âîçðîæäàåòñÿ èíòåðåñ êàê ïîòåíöèàëüíîé àëüòåðíàòèâíîé ìàñëè÷íîé êóëüòóðå. Ñîäåðæà- íèå ìàñëà â åãî ñåìåíàõ ñîñòàâëÿåò ïðèáëèçè- òåëüíî 38–43 %, à ïîäàâëÿþùàÿ ÷àñòü æèðíûõ êèñëîò (> 90 %) ïðåäñòàâëåíà íåíàñûùåííû- ìè êèñëîòàìè, âêëþ÷àÿ çíà÷èòåëüíîå êîëè÷å- ñòâî C20:0 ýéêîçàäèåíîâîé êèñëîòû, êîòîðàÿ ñðàâíèòåëüíî ðåäêî âñòðå÷àåòñÿ â ðàñòèòåëü- íûõ ìàñëàõ, à òàêæå ëèíîëåíîâóþ (36,2–39,4 %), îëåèíîâóþ (12,8–14,7 %), ëèíîëåâóþ (16,3–17,2 %) è ýéêîçåíîâóþ (14–15,5 %) êèñëîòû [2].  ñâÿçè ñ ýòèì ðûæèê ÿâëÿåòñÿ î÷åíü ïåðñïåêòèâíîé êóëüòóðîé äëÿ ïîëó÷åíèÿ áèîäèçåëÿ [3]. Ðûæèê ïîñåâíîé èìååò íåñêîëüêî äðóãèõ ïðèâëåêàòåëüíûõ îñîáåííîñòåé â êà÷åñòâå ïî- òåíöèàëüíîé ìàñëè÷íîé êóëüòóðû. Ïî ñðàâ- íåíèþ ñ äðóãèìè êðåñòîöâåòíûìè â áåëêîâîì ñîäåðæèìîì îñòàòî÷íîé ìàññû åãî ñåìÿí ïðè- ñóòñòâóåò î÷åíü ìàëî ãëèêîçèíîëàòîâ [1, 4]. Ñîäåðæàíèå ýðóêîâîé êèñëîòû òàêæå î÷åíü íèçêî – âñåãî 3 % [4], ïîýòîìó ðàñòåíèå èìååò âàæíûé ïîòåíöèàë äëÿ ïèòàíèÿ ÷åëîâåêà è æè- âîòíûõ, à òàêæå êàê èíäóñòðèàëüíàÿ êóëüòóðà.  ïèòàòåëüíûõ öåëÿõ åãî ìîæíî èñïîëüçîâàòü êàê êîðì äëÿ æèâîòíûõ (ïòèöû, ðîãàòîãî ñêî- òà è ðûá), â êà÷åñòâå óäîáðåíèÿ èëè áåëêîâîé ôðàêöèè. Ìàñëî èñïîëüçóþò äëÿ ïðîèçâîäñòâà áèîäèçåëÿ, à òàêæå â ïèùåâîé, êîñìåòè÷åñêîé è ôàðìàöåâòè÷åñêîé ïðîìûøëåííîñòÿõ, èëè æå êàê ñìàçêó.  õîäå ìíîãîëåòíèõ èñïûòàíèé, ïðîâåäåííûõ â Ñåâåðíîé Àìåðèêå, óñòàíîâëåíî, ÷òî óðîæàé- íîñòü C. sativa ñðàâíèìà ñ óðîæàéíîñòüþ äðóãèõ ñåìåííûõ ìàñëè÷íûõ êóëüòóð, âêëþ÷àÿ ðàïñ [5]. Ðûæèê ÿâëÿåòñÿ ñàìîîïûëÿþùåéñÿ è äîñòàòî÷- íî íåïðèõîòëèâîé êóëüòóðîé ê óñëîâèÿì âûðà- ùèâàíèÿ. Îò äðóãèõ ïðåäñòàâèòåëåé ñåìåéñòâà êðåñòîöâåòíûõ îí îòëè÷àåòñÿ áîëåå âûñîêîé õîëîäî- è çàñóõîóñòîé÷èâîñòüþ, íå ïîäâåðæåí áîëåçíÿì è áîëåå óñòîé÷èâ ê áëîøêàì êðåñòî- öâåòíûõ ïî ñðàâíåíèþ ñ äðóãèìè âèäàìè Bras- sica; îí òàêæå îáëàäàåò óñòîé÷èâîñòüþ ê ãðèáàì Leptosphaeria maculans è Alternaria brassicae, âû- çûâàþùèì ó êðåñòîöâåòíûõ ôîìîç è àëüòåíà- УДК 604.6 : 581.143.5 : 582.683.2 А.И. ЕМЕЦ 1, Ю.Н. БОЙЧУК 1, Е.Н. ШИША 1, Д.Б. РАХМЕТОВ2, Я.Б. БЛЮМ 1 1 Государственное учреждение «Институт пищевой биотехнологии и геномики НАН Украины», Киев Е-mail: yemets.alla@gmail.com 2 Национальный ботанический сад им. Н.Н. Гришко НАН Украины, Киев ВВЕДЕНИЕ В КУЛЬТУРУ IN VITRO, РЕГЕНЕРАЦИЯ И ГЕНЕТИЧЕСКАЯ ТРАНСФОРМАЦИЯ РЫЖИКА ПОСЕВНОГО (CAMELINA SATIVA) © À.È. ÅÌÅÖ, Þ.Í. ÁÎÉ×ÓÊ, Å.Í. ØÈØÀ, Ä.Á. ÐÀÕÌÅÒÎÂ, ß.Á. ÁËÞÌ, 2013 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3 15 Ââåäåíèå â êóëüòóðó in vitro, ðåãåíåðàöèÿ è ãåíåòè÷åñêàÿ òðàíñôîðìàöèÿ ðûæèêà ïîñåâíîãî ðèîç ñîîòâåòñòâåííî [6]. Ðûæèê èìååò êîðîòêèé âåãåòàöèîííûé ïåðèîä è ìîæåò ïðîèçðàñòàòü íà âñåõ òèïàõ ïî÷â, êðîìå ãëèíèñòûõ, à çàòðàòû íà åãî êóëüòèâèðîâàíèå ñîñòàâëÿþò ïîëîâèíó çàòðàò, íåîáõîäèìûõ ïðè âûðàùèâàíèè ðàïñà [7]. Áëàãîäàðÿ òàêèì êà÷åñòâàì ðûæèê èìååò çíà÷èòåëüíûé ïîòåíöèàë äëÿ âûðàùèâàíèÿ â Óêðàèíå. Óðîæàéíîñòü ýòîé êóëüòóðû ñîñòàâ- ëÿåò ïðèìåðíî 30 ö/ã [8, 9]. Áëàãîïðèÿòíûå óñëîâèÿ äëÿ C. sativa åñòü ïðàêòè÷åñêè âî âñåõ îáëàñòÿõ Óêðàèíû. Êðîìå òîãî, ðàçâèòèå ïîëó- ÷åíèÿ äèçåëüíîãî áèîòîïëèâà èìååò áîëüøóþ ïåðñïåêòèâó â ðàéîíàõ, çàãðÿçíåííûõ ðàäèî- íóêëèäàìè â ðåçóëüòàòå êàòàñòðîôû íà ×åðíî- áûëüñêîé ÀÝÑ, áëàãîäàðÿ ñïîñîáíîñòè ðàñ- òåíèé êàïóñòíûõ êóëüòóð î÷èùàòü ïî÷âó îò ðàäèîíóêëèäîâ, íå íàêàïëèâàÿ èõ â ñåìåíàõ. Õîòÿ ðûæèê è ðàíåå ÿâëÿëñÿ òðàäèöèîííîé êóëüòóðîé ìåëêîòîâàðíîãî ñåëüõîçÿéñòâåííîãî ïðîèçâîäñòâà â Óêðàèíå, ñåé÷àñ åãî êîììåð÷å- ñêàÿ ïðîäóêöèÿ êðàéíå îãðàíè÷åíà, òàê æå êàê è ñåëåêöèîííûå ïðîãðàììû ïî ñîçäàíèþ íî- âûõ âûñîêîïðîäóêòèâíûõ ñîðòîâ ýòîé êóëüòó- ðû. Òåì íå ìåíåå äàëüíåéøåå óñîâåðøåíñòâî- âàíèå êà÷åñòâà çàðîäûøåâîé ïëàçìû ðûæèêà ñ öåëüþ ïîâûøåíèÿ óðîæàéíîñòè, óâåëè÷åíèÿ ðàçìåðà ñåìÿí, ïîâûøåíèÿ ñîäåðæàíèÿ ìàñëà, óëó÷øåíèÿ êà÷åñòâà ïèòàòåëüíûõ õàðàêòåðèñ- òèê ÿâëÿåòñÿ âåñüìà àêòóàëüíîé ïðîáëåìîé. È äëÿ ðåøåíèÿ ýòèõ çàäà÷ çíà÷èòåëüíûé âêëàä ìîã áû áûòü ïðèâíåñåí ïðè ïîìîùè ãåíåòè- ÷åñêîé èíæåíåðèè, ÷òî òðåáóåò ðàçâèòèÿ ìåòî- äîâ, îáåñïå÷èâàþùèõ ìàíèïóëÿöèè ñ ìàòåðèà- ëîì ýòîãî âèäà â óñëîâèÿõ êóëüòóðû in vitro. Ðàíåå â èññëåäîâàíèÿõ ïðîòîïëàñòû ðûæèêà óæå èñïîëüçîâàëè â êà÷åñòâå ïàðòíåðà äëÿ ñëè- ÿíèÿ ïðè ñîìàòè÷åñêîé ãèáðèäèçàöèè ñ äðóãè- ìè âèäàìè Brassica, ãäå âî âñåõ ñëó÷àÿõ öåëüþ ðàáîòû áûëî óëó÷øåíèå ýòèõ âèäîâ [10–13]. Îäíàêî äî ïîñëåäíåãî âðåìåíè îïóáëèêîâàíî ìàëî ðàáîò ïî óñòàíîâëåíèþ êóëüòóðû in vitro ðûæèêà è ðåãåíåðàöèè ðàñòåíèé. Ñîáñòâåííî, ðåøåíèþ ýòîé ïðîáëåìû ïîñâÿùåíî âñåãî ëèøü îäíî èññëåäîâàíèå [14]. Ñîâñåì íåäàâíî îïè- ñàíà ìåòîäèêà ýìáðèîãåíåçà èç ìèêðîñïîð C. sativa [15], ïàðàëëåëüíî ðàçðàáîòàíî íåñêîëüêî ìåòîäèê òðàíñôîðìàöèè ðûæèêà [16–18]. Ïðè ýòîì ñëåäóåò îòìåòèòü, ÷òî ïîäáîð óñ- ëîâèé êóëüòèâèðîâàíèÿ in vitro è îöåíêà ðå- ãåíåðàöèîííîãî ïîòåíöèàëà ïðîâåäåíû âñåãî ëèøü äëÿ íåñêîëüêèõ ãåíîòèïîâ ðûæèêà, êóëü- òèâèðóåìûõ â Ñåâåðíîé Åâðîïå [14]. Ïîýòîìó çàäà÷åé íàñòîÿùåãî èññëåäîâàíèÿ ÿâëÿëñÿ ïîä- áîð îïòèìàëüíûõ óñëîâèé ââåäåíèÿ â êóëüòó- ðó in vitro è ðåãåíåðàöèè ðàñòåíèé èç ðàçíûõ òèïîâ ýêñïëàíòîâ ðûæèêà óêðàèíñêîé ñåëåê- öèè c ïîñëåäóþùåé ðàçðàáîòêîé ýôôåêòèâíîé àãðîáàêòåðèàëüíîé òðàíñôîðìàöèè ýòîãî âèäà. Ìàòåðèàëû è ìåòîäû. Äëÿ ââåäåíèÿ â êóëü- òóðó in vitro â êà÷åñòâå èñõîäíîãî ìàòåðèàëà èñ- ïîëüçîâàëè ñåìåíà C. sativa ñîðòîîáðàçöà Ïý- ðýìîæåöü è ñîðòà Ìèðàæ, ñîçäàííûõ â îòäåëå íîâûõ êóëüòóð Íàöèîíàëüíîãî áîòàíè÷åñêîãî ñàäà èì. Í.Í. Ãðèøêî ÍÀÍ Óêðàèíû. Äëÿ ýòî- ãî ñåìåíà ñòåðèëèçîâàëè 70%-íûì ýòàíîëîì â òå÷åíèå 1 ìèí, çàòåì îáðàáàòûâàëè ãèïîõëî- ðèòîì íàòðèÿ â êîíöåíòðàöèÿõ 1 è 1,5 % íà ïðîòÿæåíèè 5–10 ìèí [16]. Ïîñëå ñòåðèëè- çàöèè ñåìåíà òðèæäû ïðîìûâàëè ñòåðèëüíîé äèñòèëëèðîâàííîé âîäîé â òå÷åíèå 10 ìèí è ïîìåùàëè â ÷àøêè Ïåòðè íà àãàðèçîâàííóþ áåçãîðìîíàëüíóþ ñðåäó, ñîäåðæàùóþ ïîëîâèí- íûé íàáîð ìàêðî- è ìèêðîñîëåé ÌÑ [19], âè- òàìèíû ÌÑ, 2%-íóþ ñàõàðîçó, 0,8%-íûé àãàð, ðÍ 5,7–5,8. Äëÿ îöåíêè ýôôåêòèâíîñòè ðåãåíåðàöèè ïî- áåãîâ â êà÷åñòâå ýêñïëàíòîâ èñïîëüçîâàëè ñå- ìÿäîëüíûå ÷åðåøêè (ïåòèîëè) è ñåãìåíòû ãè- ïîêîòèëåé 5-, 7-, 9- è 14-äíåâíûõ ïðîðîñòêîâ Ñîñòàâ ïèòàòåëüíûõ ñðåä äëÿ ðåãåíåðàöèè ðàñòåíèé C. sativa ñîðòîîáðàçöà Ïýðýìîæåöü è ñîðòà Ìèðàæ â óñëîâèÿõ in vitro Âàðèàíòû ñðåä Ìàêðî-, ìèêðîñîëè è âèòàìèíû ÌÑ Ñàõàðîçà, ã/ë ÁÀÏ ÍÓÊ ìã/ë 1 2 3 4 5 6 7 8 9 10 11 12 + + + + + + + + + + + + 10 20 10 20 10 20 10 20 10 20 10 20 1 1 2 2 3 3 4 4 1 1 2 2 – – – – – – – – 0,1 0,1 0,1 0,1 16 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3 À.È. Åìåö, Þ.Í. Áîé÷óê, Å.Í. Øèøà è äð. ðûæèêà. Èíäóêöèþ ïîáåãîâ ñ ýêñïëàíòîâ îñó- ùåñòâëÿëè, èñïîëüçóÿ íåñêîëüêèõ âàðèàíòîâ ïè- òàòåëüíûõ ñðåä, îñíîâó êîòîðûõ òàêæå ñîñòàâ- ëÿëà ñðåäà ÌÑ. Ñîñòàâ ïèòàòåëüíûõ ñðåä îò- ëè÷àëñÿ òîëüêî ïî ñîäåðæàíèþ ôèòîãîðìîíîâ è ñàõàðîçû.  ÷àñòíîñòè, èññëåäîâàëè âëèÿíèå ðÿäà êîíöåíòðàöèé áåíçèëàìèíîïóðèíà (ÁÀÏ), à òàêæå íåñêîëüêèõ âàðèàíòîâ ñîîòíîøåíèé ÁÀÏ è íàôòèëóêñóñíîé êèñëîòû (ÍÓÊ). Âñåãî äëÿ èçó÷åíèÿ ðåãåíåðàöèîííîãî ïîòåíöèàëà C. sativa ïðîòåñòèðîâàëè 12 âàðèàíòîâ ïèòàòåëü- íûõ ñðåä (òàáëèöà). Ýêñïëàíòû èíêóáèðîâàëè ïðè 22–24 ºÑ è 16-÷àñîâîì ôîòîïåðèîäå. ×åðåç êàæäûå òðè íåäåëè ìàòåðèàë ïàññè- ðîâàëè íà ñâåæåïðèãîòîâëåííûå ïèòàòåëüíûå ñðåäû. Èíäóöèðîâàííûå ïîáåãè îòäåëÿëè îò ýêñïëàíòîâ è ïåðåñàæèâàëè äëÿ äàëüíåéøåãî ðîñòà è óêîðåíåíèÿ íà ñðåäó ÌÑ ëèáî íà åå âàðèàíòû, ñîäåðæàùèå ÍÓÊ â êîíöåíòðàöèè 0,1; 0,5 èëè 1 ìã/ë.  ýêñïåðèìåíòàõ ïî ãåíåòè÷åñêîé òðàíñôîð- ìàöèè èñïîëüçîâàëè øòàìì Agrobacterium tume- faciens LBA4404, ñîäåðæàùèé ïëàçìèäó pGH217 ñ ðåïîðòåðíûì ãåíîì -ãëþêóðîíèäàçû (gus) ïîä êîíòðîëåì 35S ïðîìîòîðà âèðóñà ìîçàèêè öâåòíîé êàïóñòû (ÂÌÖÊ) è nos-òåðìèíàòîðà, à òàêæå ñåëåêòèâíûé ìàðêåðíûé ãåí hpt, îáå- ñïå÷èâàþùèé óñòîé÷èâîñòü ê ãèãðîìèöèíó ó òðàíñôîðìàíòîâ. Ïëàçìèäà ëþáåçíî ïðåäîñòàâ- ëåíà ä-ðîì Â.Â. Ðàä÷óêîì (Èíñòèòóò ãåíåòèêè ðàñòåíèé è èññëåäîâàíèé êóëüòóðíûõ ðàñòå- íèé, Ãàòåðñëåáåí, Ãåðìàíèÿ) (ðèñ. 1). Íî÷íóþ êóëüòóðó àãðîáàêòåðèè âûðàùèâà- ëè â 20 ìë æèäêîé ñðåäû LB, äîïîëíåííîé 100 ìã/ë êàíàìèöèíà è 100 ìã/ë ðèôàìïèöèíà, ïðè òåìïåðàòóðå 28 °Ñ è ïîñòîÿííîì ïåðåìå- øèâàíèè íà îðáèòàëüíîì øåéêåðå. Çàòåì àãðî- áàêòåðèþ î÷èùàëè îñàæäåíèåì ïðè öåíòðè- ôóãèðîâàíèè (4000 îá/ìèí) â òå÷åíèå 5 ìèí. Ñóïåðíàòàíò óäàëÿëè, îñàäîê ïåðåä èíîêóëÿ- öèåé ðàçáàâëÿëè æèäêîé ñðåäîé ÌÑ äî äîñòè- æåíèÿ îïòè÷åñêîé ïëîòíîñòè â èíòåðâàëå îò OD600= 0,01 äî OD600= 0,5.  êà÷åñòâå ýêñïëàíòîâ äëÿ òðàíñôîðìàöèè èñïîëüçîâàëè ïåòèîëè è ãèïîêîòèëè 5- èëè 7- äíåâíûõ ïðîðîñòêîâ. Âðåìÿ êîêóëüòèâèðîâà- íèÿ ýêñïëàíòîâ ñ àãðîáàêòåðèåé ñîñòàâëÿëî îò 15 ìèí äî 1 ÷. Çàòåì ýêñïëàíòû ïåðåíîñèëè íà àãàðèçîâàííóþ ñðåäó äëÿ äàëüíåéøåãî êîêóëü- òèâèðîâàíèÿ, ïðåäâàðèòåëüíî ïðîìîêíóâ èõ ñòåðèëüíîé ôèëüòðîâàëüíîé áóìàãîé äëÿ óäà- ëåíèÿ èçëèøêîâ àãðîáàêòåðèè. ×åðåç 2–3 äíÿ òðàíñôîðìèðîâàííûå ýêñïëàíòû ïîìåùàëè íà 7 äíåé â âîññòàíîâèòåëüíóþ ñðåäó ÌÑ, ñîäåð- æàùóþ ôèòîãîðìîíû è 400 ìã/ë öåôîòàêñèìà äëÿ ýëèìèíàöèè àãðîáàêòåðèè, à çàòåì – íà àíàëîãè÷íóþ ïî ñîñòàâó ñðåäó, íî ñ äîáàâëå- íèåì ãèãðîìèöèíà â êîíöåíòðàöèè 5 ìã/ë äëÿ ñåëåêöèè òðàíñãåííûõ ëèíèé. ×òîáû âûáðàòü îïòèìàëüíóþ ñåëåêòèâíóþ êîíöåíòðàöèþ ãè- ãðîìèöèíà, çàðàíåå ïðîâåëè ýêñïåðèìåíòû ïî èçó÷åíèþ âëèÿíèÿ ðàçíûõ êîíöåíòðàöèé (1– 10 ìã/ë) ýòîãî ñåëåêòèâíîãî àãåíòà íà æèçíåñïî- ñîáíîñòü ýêñïëàíòîâ. Äëÿ ãèñòîõèìè÷åñêîãî îïðåäåëåíèÿ ýêñïðåñ- ñèè gus(uid A)-ãåíà (ãåíà -ãëþêóðîíèäàçû) â Ðèñ. 1. Ñõåìà êîíñòðóêöèè pGH217: LB è RB – ëåâàÿ è ïðàâàÿ ãðàíèöû Ò-ÄÍÊ, P35S – 35S ïðîìîòîð ÂÌÖÊ, GUS – ãåí -ãëþêóðîíèäàçû, nos – íîïàëèíîâûé òåðìèíàòîð, hpt – ãåí óñòîé÷èâîñòè ê ãèãðîìèöèíó Ðèñ. 2. Ýôôåêòèâíîñòü ïðîðàñòàíèÿ ñåìÿí ïðè îá- ðàáîòêå 1,5%-íûì ðàñòâîðîì ãèïîõëîðèòà íàòðèÿ: ïî âåðòèêàëè – êîëè÷åñòâî ïðîðîñøèõ ñåìÿí, %; ïî ãîðèçîíòàëè – âðåìÿ ñòåðèëèçàöèè, ìèí 17ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3 Ââåäåíèå â êóëüòóðó in vitro, ðåãåíåðàöèÿ è ãåíåòè÷åñêàÿ òðàíñôîðìàöèÿ ðûæèêà ïîñåâíîãî òêàíÿõ ðàñòåíèé C. sativa ïîñëå àãðîáàêòåðè- àëüíîé òðàíñôîðìàöèè èñïîëüçîâàëè 5-áðîì- 4-õëîð-3-èíäîëèëãëþêîðîíèä (Õ-Gluc).  ðå- çóëüòàòå ðåàêöèè ñ ýòèì ñóáñòðàòîì â îáëàñòè ëîêàëèçàöèè ôåðìåíòà â òðàíñãåííûõ êëåòêàõ îáðàçóåòñÿ ãîëóáîé îñàäîê [20]. Ðåçóëüòàòû èññëåäîâàíèé è èõ îáñóæäåíèå.  íà÷àëå íàøèõ èññëåäîâàíèé áûëè ïîäîáðàíû îïòèìàëüíûå óñëîâèÿ äëÿ ââåäåíèÿ â êóëüòóðó in vitro èñõîäíîãî ðàñòèòåëüíîãî ìàòåðèàëà, â ÷àñòíîñòè, îïðåäåëåíû è óñòàíîâëåíû ýôôåê- òèâíûå êîíöåíòðàöèè ñòåðèëèçóþùèõ àãåíòîâ è âðåìÿ îáðàáîòêè èìè ñåìÿí C. sativa. Ïðè èçó÷åíèè ýôôåêòèâíîñòè ñòåðèëèçàöèè ñåìÿí C. sativa êàê äëÿ ñîðòîîáðàçöà Ïýðýìîæåöü, òàê è äëÿ ñîðòà Ìèðàæ óñòàíîâëåíî, ÷òî ïðè èñ- ïîëüçîâàíèè ãèïîõëîðèòà íàòðèÿ â êîíöåí- òðàöèè 1 % íåçàâèñèìî îò âðåìåíè îáðàáîòêè ñåìÿí íà 5-é äåíü íàáëþäàëè êîíòàìèíàöèþ ìàòåðèàëà. Êîíòàìèíàöèÿ ïðèñóòñòâîâàëà ïðè îáðàáîòêå ñåìÿí 1,5%-íûì ãèïîõëîðèòîì íà- òðèÿ â òå÷åíèå 3 ìèí ó ñîðòîîáðàçöà Ïýðýìî- æåöü è â òå÷åíèå 5 ìèí – ó ñîðòà Ìèðàæ. Àíàëèç âëèÿíèÿ óñëîâèé ñòåðèëèçàöèè íà ïðîðàñòàíèå ñåìÿí ïîçâîëèë óñòàíîâèòü, ÷òî íàèáîëåå îïòèìàëüíûì âàðèàíòîì äëÿ ñîðòîî- áðàçöà Ïýðýìîæåöü ÿâëÿåòñÿ 5–7-ìèíóòíàÿ îá- ðàáîòêà 1,5%-íûì ãèïîõëîðèòîì íàòðèÿ. Ïðè ýòîì 100%-íîå ïðîðàñòàíèå ñåìÿí â óñëîâèÿõ in vitro íàáëþäàëè ïîñëå 5-ìèíóòíîé îáðàáîò- êè è 97%-íîå ïðè 6- è 7-ìèíóòíîé. Òàêèì îá- ðàçîì, ñåìåíà ñîðòà Ìèðàæ íåîáõîäèìî ñòå- ðèëèçîâàòü 1,5%-íûì ãèïîõëîðèòîì íàòðèÿ â òå÷åíèå 6–7 ìèí, òàê êàê ïðè ýòîì íå íàáëþ- äàåòñÿ çàðàæåíèå, è ïðîöåíò ïðîðàñòàííÿ ñå- ìÿí ñîñòàâëÿåò 100 % (ðèñ. 2). Ïðè èçó÷åíèè ïðîöåññà ðåãåíåðàöèè ïîáå- ãîâ ðûæèêà â êà÷åñòâå íàèáîëåå îïòèìàëüíûõ òèïîâ ýêñïëàíòîâ äëÿ èíèöèàöèè îðãàíîãåíåçà in vitro ó ìíîãèõ âèäîâ ðàñòåíèé ñåìåéñòâà êðå- ñòîöâåòíûõ îáû÷íî èñïîëüçóþò ñåìÿäîëüíûå è ãèïîêîòèëüíûå ýêñïëàíòû [21, 22] (ðèñ. 3, á, â, ñì. âêëåéêó â êîíöå íîìåðà).  íàøåì ýêñ- ïåðèìåíòå äëÿ ïîëó÷åíèÿ ýêñïëàíòîâ èñïîëü- çîâàëè 5-, 7-, 9- è 14-äíåâíûå ïðîðîñòêè C. sa- tiva, êîòîðûå ðàçìåùàëè íà ñðåäàõ, îïèñàííûõ â «Ìàòåðèàëàõ è ìåòîäàõ». Èç ëèòåðàòóðíûõ äàííûõ èçâåñòíî, ÷òî ó ðÿäà ïðåäñòàâèòåëåé ñåìåéñòâà êðåñòîöâåòíûõ ôîðìèðîâàíèå ïîáåãîâ ïðîèñõîäèò â ïðèñóò- ñòâèè êàê öèòîêèíèíîâ, êîòîðûå ñïîñîáíû âûçûâàòü äåëåíèå êëåòîê è èíèöèèðîâàòü äèô- ôåðåíöèàöèþ ïîáåãîâ, òàê è ïðè ñîâìåñòíîì äîáàâëåíèè â ñðåäó ðàçëè÷íûõ êîìáèíàöèé ðå- ãóëÿòîðîâ öèòîêèíèíîâîé è àóêñèíîâîé ïðè- ðîäû [21–24]. Áîëåå ýôôåêòèâíî ðåãåíåðàöèÿ ïîáåãîâ ðûæèêà ïðîèñõîäèò íà ñðåäàõ, ñîäåð- æàùèõ öèòîêèíèíû â êîìáèíàöèè ñ àóêñèíà- ìè [14]. Òàêèì îáðàçîì, äëÿ èçó÷åíèÿ ïðîöåññîâ ðå- ãåíåðàöèè ïîáåãîâ ó ñîðòîîáðàçöà Ïýðýìîæåöü è ñîðòà Ìèðàæ íàìè ïðîòåñòèðîâàí ðÿä êîí- öåíòðàöèé ÁÀÏ, à òàêæå êîìáèíàöèè ÁÀÏ ñ ÍÓÊ (òàáëèöà). Íåîáõîäèìî îòìåòèòü, ÷òî íå- êîòîðîå âëèÿíèå íà ýôôåêòèâíîñòü êàëëóñî- ãåíåçà è äàëüíåéøóþ ðåãåíåðàöèþ ïîáåãîâ ñïîñîáíà îêàçûâàòü òà èëè èíàÿ êîíöåíòðà- Ðèñ. 4. Ýôôåêòèâíîñòü ðåãåíåðàöèè ïîáåãîâ íà ñðå- çàõ ÷åðåøêîâ ñåìÿäîëåé ïðîðîñòêîâ ðûæèêà â ðàç- íûõ âàðèàíòàõ ñðåä: à – 5-äíåâíûå; á – 7-äíåâíûå; â – 9-äíåâíûå ïðîðîñòêè; ïî âåðòèêàëè – êîëè÷åñòâî ïîáåãîâ, øò.; ïî ãîðèçîíòàëè – íîìåðà âàðèàíòîâ ñðåä (òàáëèöà) 18 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3 À.È. Åìåö, Þ.Í. Áîé÷óê, Å.Í. Øèøà è äð. öèÿ ñàõàðîçû â ïèòàòåëüíîé ñðåäå.  ðåçóëü- òàòå íàøèõ èññëåäîâàíèé áûëè ïðîòåñòèðî- âàíû 12 âàðèàíòîâ ñðåä, îòëè÷àþùèõñÿ íå òîëü- êî ïî ñîñòàâó ðåãóëÿòîðîâ ðîñòà, íî è ïî ñîäåðæàíèþ ñàõàðîçû. Äëÿ ýòîãî ðàñòèòåëüíûé ìàòåðèàë êàæäûå òðè íåäåëè ïàññèðîâàëè íà ñâåæåïðèãîòîâëåííûå ïèòàòåëüíûå ñðåäû. Ïîñ- ëå òðåõ-÷åòûðåõ íåäåëü êóëüòèâèðîâàíèÿ íà ïåòèîëÿõ è ñåãìåíòàõ ãèïîêîòèëåé 5-, 7- è 9-äíåâíûõ ïðîðîñòêîâ ðûæèêà íàáëþäàëè ôîð- ìèðîâàíèå æåëòî-çåëåíîãî èëè çåëåíîãî êàëëó- ñà ñ õîðîøî çàìåòíûìè òî÷êàìè èíèöèàöèè áóäóùèõ ïîáåãîâ íà âñåõ âàðèàíòàõ ñðåä. Íà ýêñïëàíòàõ 14-äíåâíûõ ïðîðîñòêîâ ôîðìèðî- âàíèå êàëëóñíûõ ñòðóêòóð ïðîèñõîäèëî áîëåå ìåäëåííî, à ôîðìèðîâàíèå çà÷àòêîâ ïîáåãîâ íà âñåõ ñðåäàõ âîîáùå îòñóòñòâîâàëî. Îòñþäà ñëåäóåò, ÷òî äëÿ ðåãåíåðàöèè ïîáåãîâ ðûæè- êà â óñëîâèÿõ in vitro ýôôåêòèâíåå èñïîëüçî- âàòü áîëåå ìîëîäûå êëåòêè è òêàíè. Íåîáõî- äèìî îòìåòèòü, ÷òî ýôôåêòèâíîñòü ðåãåíåðà- öèè ïîáåãîâ íà ñðåäàõ, ñîäåðæàùèõ ÁÀÏ è ÍÓÊ, áûëà âûøå, ÷åì íà ñðåäàõ, ñîäåðæàùèõ òîëü- êî ÁÀÏ. Òàêàÿ òåíäåíöèÿ ïðîñëåæèâàåòñÿ äëÿ âñåõ òèïîâ ýêñïëàíòîâ, ïîëó÷åííûõ êàê èç 5-, 7-, òàê è 9-äíåâíûõ ïðîðîñòêîâ. Ïðè ýòîì áîëüøåå êîëè÷åñòâî ïîáåãîâ èíäóöèðîâàíî èç ïåòèîëåé è ýêñïëàíòîâ ãèïîêîòèëåé 5- è 7- äíåâíûõ ïðîðîñòêîâ (ðèñ. 4 è 5). Óñòàíîâëåíî òàêæå, ÷òî ïðè ñîäåðæàíèè â ñðåäå 10 ã/ë ñàõàðîçû ôîðìèðîâàíèå êàëëóñà ïðîèñõîäèëî ìåíåå èíòåíñèâíî, ÷åì ïðè êîí- öåíòðàöèè 20 ã/ë, îäíàêî íà ôîðìèðîâàíèå ïî- áåãîâ ýòî ñóùåñòâåííî íå âëèÿëî. Òàêèì îáðà- çîì, â õîäå íàøèõ ýêñïåðèìåíòîâ èçó÷åíî äåé- ñòâèå ðÿäà êîíöåíòðàöèé ôèòîãîðìîíîâ ÁÀÏ, ñîîòíîøåíèå íåñêîëüêèõ âàðèàíòîâ êîíöåíòðà- öèé ôèòîãîðìîíîâ ÁÀÏ è ÍÓÊ, à òàêæå äåé- ñòâèå äâóõ êîíöåíòðàöèé ñàõàðîçû (10 è 20 ã/ë) íà ïðîöåññ ôîðìèðîâàíèÿ ïîáåãîâ èç ïå- òèîëåé è ñåãìåíòîâ ãèïîêîòèëåé 5-, 7-, 9- è 14-äíåâíûõ ïðîðîñòêîâ. Íà ðèñ. 3, ã ïîêàçà- íû ñôîðìèðîâàâøèåñÿ ïîáåãè íà ñðåçàõ ÷å- ðåøêà ñåìÿäîëè, íà ðèñ. 3, ä – íà ñåãìåíòå ãèïîêîòèëÿ. Åñëè ðåãåíåðàöèÿ êðåñòîöâåòíûõ ðàñòåíèé â óñëîâèÿõ in vitro ïðîèñõîäèò ïîä âëèÿíèåì öèòîêèíèíîâ èëè èõ êîìáèíàöèé ñ àóêñè- íàìè, òî óêîðåíåíèå ïîëó÷åííûõ ðåãåíåðàí- òîâ – ïðè äîáàâëåíèè â ñðåäó àóêñèíîâ [25]. Èñõîäÿ èç ýòîãî, ñëåäóþùèì ýòàïîì íàøèõ èññëåäîâàíèé áûëî ïðîâåäåíèå ýêñïåðèìåíòîâ ïî èçó÷åíèþ äåéñòâèÿ àóêñèíîâ, â ÷àñòíîñòè ðÿäà êîíöåíòðàöèé ÍÓÊ, íà ïðîöåññ ðèçî- ãåíåçà ó ïîëó÷åííûõ ïîáåãîâ. Ñôîðìèðîâàâ- øèéñÿ è îòäåëåííûé îò ýêñïëàíòà ïîáåã ïðåä- ñòàâëåí íà ðèñ. 3, å. Ñëåäóåò îòìåòèòü, ÷òî ïåðåä íà÷àëîì ýòèõ èññëåäîâàíèé ðåãåíåðè- ðîâàâøèå ïîáåãè (ðèñ. 3, ã–å) âûñàæèâàëè íà ñðåäû ÌÑ è íà 1/2 ÌÑ, îäíàêî îáðàçîâàíèå êîðíåé ó íèõ íå ïðîèñõîäèëî. Ïðè äîáàâëå- íèè ÍÓÊ (0,1; 0,5; 1 ìã/ìë) â ñðåäó ÌÑ îò- ìå÷åíà èíèöèàöèÿ îáðàçîâàíèÿ êîðíåé ëèøü ïðè êîíöåíòðàöèè ýòîãî ôèòîãîðìîíà 1 ìã/ìë. Òàêèì îáðàçîì, íàìè óñòàíîâëåíî, ÷òî äëÿ èíäóêöèè ïîáåãîâ ó Ñ. sativa ëó÷øå âñåãî èñ- ïîëüçîâàòü 5- èëè 7-äíåâíûå ïðîðîñòêè, ïî- ñêîëüêó ýôôåêòèâíîñòü ðåãåíåðàöèè íà èõ ýêñïëàíòàõ âûøå, ÷åì ó 9-äíåâíûõ. ×òî êà- ñàåòñÿ òèïà ýêñïëàíòà, òî â îäèíàêîâîé ìåðå Ðèñ. 5. Ýôôåêòèâíîñòü ðåãåíåðàöèè ïîáåãîâ èç ýêñïëàíòîâ ãèïîêîòèëåé ïðîðîñòêîâ ðûæèêà â ðàç- íûõ âàðèàíòàõ ñðåä: à – 5-äíåâíûå; á – 7-äíåâíûå; â – 9-äíåâíûå ïðîðîñòêè; ïî âåðòèêàëè – êîëè- ÷åñòâî ïîáåãîâ, øò.; ïî ãîðèçîíòàëè – íîìåðà âàðèàíòîâ ñðåä (òàáëèöà) 19ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3 Ââåäåíèå â êóëüòóðó in vitro, ðåãåíåðàöèÿ è ãåíåòè÷åñêàÿ òðàíñôîðìàöèÿ ðûæèêà ïîñåâíîãî äëÿ ðåãåíåðàöèè ïîáåãîâ ìîæíî èñïîëüçîâàòü è ïåòèîëè, è ñåãìåíòû ãèïîêîòèëåé. Ýôôåê- òèâíîñòü ðåãåíåðàöèè ðàñòåíèé íà ïåòèîëÿõ è ãèïîêîòèëüíûõ ýêñïëàíòàõ ó ñîðòîîáðàçöà Ïýðýìîæåöü íåçíà÷èòåëüíî âûøå ïî ñðàâíå- íèþ ñ ñîðòîì Ìèðàæ íà íåñêîëüêèõ âàðèàíòàõ ñðåä (ðèñ. 4 è 5). Òåì íå ìåíåå äëÿ ðåãåíåðàöèè ïîáåãîâ íàìè âûáðàíà ñðåäà ÌÑ êàê íàèáîëåå ýôôåêòèâíàÿ, ñîäåðæàùàÿ â êà÷åñòâå ôèòîãîð- ìîíîâ ÁÀÏ (äî 4 ìã/ìë) è ÍÓÊ (0,1 ìã/ìë), à äëÿ èõ óêîðåíåíèÿ – ñðåäà ÌÑ ñ 1 ìã/ìë ÍÓÊ. Êðîìå òîãî, íàìè ðàçðàáîòàí ìåòîä Agro- bacterium-îïîñðåäóåìîé òðàíñôîðìàöèè C. sa- tiva. Äëÿ ýòîãî èñïîëüçîâàëè âåêòîðíóþ êîí- ñòðóêöèþ pGH217, ñîäåðæàùóþ ðåïîðòåðíûé ãåí -ãëþêóðîíèäàçû (gus) ïîä êîíòðîëåì 35S ïðîìîòîðà ÂÌÖÊ è nos-òåðìèíàòîðà, à òàêæå ñåëåêòèâíûé ìàðêåðíûé ãåí hpt, êîòîðûé îáå- ñïå÷èâàåò óñòîé÷èâîñòü ê ãèãðîìèöèíó ó îòî- áðàííûõ òðàíñãåííûõ ðàñòåíèé. Óïîìÿíóòûé âåêòîð ñ gus-ãåíîì ïîçâîëÿåò áûñòðî (óæå ÷åðåç 1 ñóò) âèçóàëèçèðîâàòü ýêñïðåññèþ ïåðåíåñåí- íîãî ÷óæåðîäíîãî ãåíà â ðàñòèòåëüíûõ êëåòêàõ ïî íàëè÷èþ ãîëóáîãî îêðàøèâàíèÿ â òêàíÿõ òðàíñãåííûõ ëèíèé [20]. Ïîñêîëüêó â êà÷åñòâå ýêñïëàíòîâ äëÿ òðàíñôîðìàöèè èñïîëüçîâàëè ïåòèîëè è ãè- ïîêîòèëè 5- èëè 7-äíåâíûõ ïðîðîñòêîâ C. sa- tiva, ñíà÷àëà áûëè ïðîâåäåíû ýêñïåðèìåíòû ïî èçó÷åíèþ âëèÿíèÿ ãèãðîìèöèíà íà èõ âû- æèâàåìîñòü ñ öåëüþ óñòàíîâëåíèÿ åãî ñåëåê- òèâíîé êîíöåíòðàöèè ïðè îòáîðå òðàíñôîð- ìàíòîâ. Ãèãðîìèöèí òåñòèðîâàëè â êîíöåí- òðàöèÿõ îò 1 äî 10 ìã/ë, äîáàâëÿÿ åãî â ñðå- äó äëÿ ðåãåíåðàöèè ïîáåãîâ, è ÷åðåç 30 äíåé îöåíèâàëè ñïîñîáíîñòü òêàíåâûõ ýêñïëàíòîâ âûæèâàòü è ðåãåíåðèðîâàòü ïðè òàêèõ óñëî- âèÿõ.  ðåçóëüòàòå ýêñïåðèìåíòîâ óñòàíîâèëè, ÷òî íàèáîëåå ýôôåêòèâíîé äëÿ ñåëåêöèè ÿâ- ëÿåòñÿ êîíöåíòðàöèÿ ãèãðîìèöèíà 5 ìã/ë, ÷òî ñîâïàäàåò ñ äàííûìè, îïèñàííûìè ðàíåå [16]. Ïîñëå êîêóëüòèâèðîâàíèÿ ñ àãðîáàêòåðèåé ýêñïëàíòû ïåðåíîñèëè ñíà÷àëà íà âîññòàíîâè- òåëüíóþ ñðåäó ÌÑ, ñîäåðæàùóþ ôèòîãîðìî- íû è 400 ìã/ë öåôîòàêñèìà äëÿ ýëèìèíàöèè àãðîáàêòåðèè, à çàòåì íà àíàëîãè÷íóþ ïî ñî- ñòàâó ñðåäó, íî ñ äîáàâëåíèåì ãèãðîìèöèíà â êîíöåíòðàöèè 5 ìã/ë äëÿ ñåëåêöèè òðàíñãåí- íûõ ëèíèé (ðèñ. 6, à–â, ñì. âêëåéêó â êîíöå íîìåðà). Ïåðâîå òåñòèðîâàíèå ïðîòðàíñôîð- ìèðîâàííûõ ýêñïëàíòîâ íà GUS-ýêñïðåññèþ ïðîâîäèëè ÷åðåç òðè äíÿ ïîñëå ïåðåíåñåíèÿ èõ íà ñåëåêòèâíóþ ñðåäó. Ðåçóëüòàòû ýòîãî GUS-òåñòà ïðåäñòàâëåíû íà ðèñ. 6, ã è ä. Íà ñåëåêòèâíîé ñðåäå ÷åðåç 2–3 íåäåëè ïîñëå ïðî- âåäåíèÿ òðàíñôîðìàöèè ïî êðàÿì ñðåçîâ íà íåêîòîðûõ ïåòèîëÿõ è ñåãìåíòàõ ãèïîêîòèëåé íàáëþäàëè ôîðìèðîâàíèå êàëëóñà, êîòîðûé äà- âàë íà÷àëî ïîáåãàì (ðèñ. 6, á). Ñïóñòÿ 2–3 ìå- ñÿöà íà ñåëåêòèâíîé ñðåäå îòîáðàëè ïåðâûå òðàíñãåííûå ðàñòåíèÿ C. sativa. Ñ ïîìîùüþ ãèñ- òîõèìè÷åñêîãî àíàëèçà êàëëóñà è ëèñòèêîâ ðå- ãåíåðèðîâàâøèõ ïîáåãîâ îáíàðóæèëè ýêñïðåñ- ñèþ ïåðåíåñåííîãî gus-ãåíà, ÷òî ïîäòâåðæäàåò òðàíñãåííóþ ïðèðîäó ïîëó÷åííûõ ëèíèé. Òàêèì îáðàçîì, ðàçðàáîòàííûå íàìè ìå- òîäû ââåäåíèÿ â êóëüòóðó in vitro è ðåãåíåðà- öèè ðàñòåíèé C. sativa, à òàêæå Agrobacterium- îïîñðåäóåìîé òðàíñôîðìàöèè ìîãóò ñëóæèòü îñíîâîé äàëüíåéøåãî áèîòåõíîëîãè÷åñêîãî óñîâåðøåíñòâîâàíèÿ ýòîãî âèäà äëÿ óëó÷øåíèÿ êà÷åñòâà ïîëó÷àåìîãî èç íåãî áèîäèçåëÿ. Ðàáîòà âûïîëíåíà ïðè ôèíàíñîâîé ïîääåðæêå ïðîåêòà «Ââåäåíèå â êóëüòóðó in vitro è ãåíåòè÷å- ñêàÿ òðàíñôîðìàöèÿ ðûæèêà ñ öåëüþ óëó÷øåíèÿ åãî ïðîäóêòèâíûõ õàðàêòåðèñòèê äëÿ ïðîèçâîä- ñòâà áèîäèçåëÿ» öåëåâîé êîìïëåêñíîé ïðîãðàììû íàó÷íûõ èññëåäîâàíèé ÍÀÍ Óêðàèíû «Áèîìàññà êàê òîïëèâíîå ñûðüå» («Áèîòîïëèâî»). A.I. Yemets, Yu.N. Boychuk, E.N. Shysha, D.B. Rakhmetov, Ya.B. Blume Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, Kyiv Å-mail: yemets.alla@gmail.com National Botanical Garden named M.M. Gryshko, NAS of Ukraine ESTABLISHMENT OF IN VITRO CULTURE, PLANT REGENERATION AND GENETIC TRANSFORMATION OF CAMELINA SATIVA The results on in vitro culture establishment, plantlet regeneration and rooting of Camelina sativa cultivar sample Peremozhets and cultivar Mirazh are presented. Effective concentrations of sterilizing agents and du- ration of plant material treatment were estimated. Phytohormone ratio, sucrose concentration in nutrient medium that induce effective formation of C. sativa shoots and NAA concentration for plantlet rooting have been established. The method of Agrobacterium- mediated transformation of Camelina by using binary vector pGH217 carrying reporter -glucoronidase (gus) ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 320 À.È. Åìåö, Þ.Í. Áîé÷óê, Å.Í. Øèøà è äð. gene driven under 35S CaMV promoter and nos-ter- minator, and selective marker hpt gene conferring hygro- mycin-resistance in transgenic plant was elaborated. ÑÏÈÑÎÊ ËÈÒÅÐÀÒÓÐÛ 1. Schuster A., Friedt W. Camelina sativa : Old face – new prospects // Crucifer. News. – 1995. – 17. – P. 6–8. 2. Gugel R.K., Falk K.C. Agronomic and seed quality evaluation of Camelina sativa in western Canada // Can. J. Plant Sci. – 2006. – 86. – P. 1047–1058. 3. Pilgeram A.L., Sands D.C., Boss D., Dale N., Wichman D., Lamb P., Lu C., Barrows R., Kirkpatrick M., Thomp- son B., Johnson D.L. 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