идентификация и возможная роль (обзор).

ÑÅËÜÑÊÎÕÎÇßÉÑÒÂÅÍÍÀß ÁÈÎËÎÃÈß, 2015, òîì 50, ¹ 1, ñ. 3-15
Îáçîðû, ïðîáëåìû, èòîãè
ÓÄÊ 633/635:581.4:579.64
doi: 10.15389/agrobiology.2015.1.3rus
ÁÀÊÒÅÐÈÀËÜÍÛÅ ÌÈÊÐÎÎÐÃÀÍÈÇÌÛ, ÀÑÑÎÖÈÈÐÎÂÀÍÍÛÅ
Ñ ÒÊÀÍßÌÈ ÐÀÑÒÅÍÈÉ Â ÊÓËÜÒÓÐÅ in vitro: ÈÄÅÍÒÈÔÈÊÀÖÈß È
ÂÎÇÌÎÆÍÀß ÐÎËÜ
(îáçîð)
Ñ.Å. ÄÓÍÀÅÂÀ1, Þ.Ñ. ÎÑËÅÄÊÈÍ2
Ýôôåêòèâíàÿ ñòåðèëèçàöèÿ ðàñòèòåëüíûõ ýêñïëàíòîâ è ñîáëþäåíèå ïðàâèë àíòèñåïòèêè íå èñêëþ÷àþò ïðèñóòñòâèÿ â in vitro êóëüòóðàõ òàê íàçûâàåìûõ ñêðûòûõ (ýíäîôèòíûõ) áàêòåðèé. Ðîëü ýòèõ áàêòåðèé â êóëüòóðàõ òêàíåé ìàëî èçó÷åíà, îäíàêî åå, íàïðèìåð, ñâÿçûâàþò ñ ðåãåíåðàöèîííîé ñïîñîáíîñòüþ ýêñïëàíòîâ è âîçìîæíîñòüþ òðàíñôîðìèðîâàòü êóëüòèâèðóåìûå in
vitro êëåòêè æèâîòíûõ è ÷åëîâåêà. Áàêòåðèàëüíûå øòàììû, ïàòîãåííûå äëÿ ÷åëîâåêà, ìîãóò óñòîé÷èâî ñîõðàíÿòüñÿ â ïàññèðóåìûõ êóëüòóðàõ è â ðàñòåíèÿõ ex vitro. Ðàñøèðåíèå ñðåäû îáèòàíèÿ áàêòåðèé ñîçäàåò ýêîëîãè÷åñêèå è ãåíåòè÷åñêèå ðèñêè, êîòîðûå îïðåäåëÿþò íåîáõîäèìîñòü
òùàòåëüíîãî ìîíèòîðèíãà ýíäîôèòíûõ ñîîáùåñòâ â ðàñòåíèÿõ, óïîòðåáëÿåìûõ â ïèùó â ñûðîì
âèäå, à òàêæå ïðè ïðèìåíåíèè in vitro òåõíîëîãèé â ïðàêòè÷åñêîì ðàñòåíèåâîäñòâå è äëÿ ïîëó÷åíèÿ ïðîäóêòîâ ïèòàíèÿ. Èäåíòèôèêàöèÿ áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ, êîëîíèçèðóþùèõ in
vitro êóëüòóðû ðàñòåíèé, äàåò âîçìîæíîñòü èçó÷àòü âëèÿíèå áàêòåðèé íà õîçÿèíà, ïðîâîäèòü íàïðàâëåííóþ õèìèîòåðàïèþ, ñîçäàâàòü áàíê äàííûõ ìèêðîîðãàíèçìîâ, àññîöèèðîâàííûõ ñ êóëüòóðîé ðàñòèòåëüíûõ òêàíåé. Íàèáîëåå ðàñïðîñòðàíåíû äâà ìåòîäà èäåíòèôèêàöèè — òðàäèöèîííûé, áîëåå äîñòóïíûé, íî íå ïîçâîëÿþùèé âûÿâëÿòü íåêóëüòèâèðóåìûå ôîðìû (îí îñíîâàí íà
èñïîëüçîâàíèè êóëüòóðàëüíûõ è ìîðôîëîãè÷åñêèõ õàðàêòåðèñòèê, à òàêæå õèìè÷åñêèõ è áèîõèìè÷åñêèõ ðåàêöèé), è ìîëåêóëÿðíî-ãåíåòè÷åñêèé.  ïîñëåäíåì ñëó÷àå ñ èñïîëüçîâàíèåì ìåòàãåíîìíîé ÄÍÊ è ñîîòâåòñòâóþùèõ ñïåöèôè÷åñêèõ ïðàéìåðîâ îïðåäåëÿþò ðàçëè÷íûå ïîñëåäîâàòåëüíîñòè 16S-ðÐÍÊ, èìåþùèå êàê êîíñåðâàòèâíûå ñàéòû, îäèíàêîâûå ó âñåõ ïðîêàðèîòîâ, òàê
è ïðèãîäíûå äëÿ èäåíòèôèêàöèè âèäîñïåöèôè÷íûå ó÷àñòêè. Âíóòðåííèå òðàíñêðèáèðóåìûå ñïåéñåðû (internal transcribed spacers — ITS) ïðåèìóùåñòâåííî ïðèìåíÿþò äëÿ ðàçëè÷åíèÿ ìèêðîîðãàíèçìîâ íà óðîâíå âèäîâ è äàæå øòàììîâ. Òàêñîíîìè÷åñêèé ñîñòàâ áàêòåðèàëüíûõ ýíäîôèòîâ
ñâèäåòåëüñòâóåò îá èõ ðàçíîîáðàçèè è îòñóòñòâèè ñïåöèôè÷åñêîãî íàáîðà â in vitro êóëüòóðàõ
ðàñòåíèé ðàçíîé ñèñòåìàòè÷åñêîé ïðèíàäëåæíîñòè è â ýêñïëàíòàõ, îòíîñÿùèõñÿ ê ðàçíûì îðãàíàì ðàñòåíèÿ. Ñðåäè èäåíòèôèöèðîâàííûõ ýíäîôèòíûõ áàêòåðèé âûÿâëåíû ïîòåíöèàëüíî ïîëåçíûå äëÿ èíòàêòíûõ ðàñòåíèé, â ÷àñòíîñòè Streptomycete, Pantoea agglomerans è äð., à òàêæå ïàòîãåííûå äëÿ ÷åëîâåêà, íàïðèìåð Ralstonia mannitolytica, Staphylococcus epidermidis, Corynebacterium
amycolatum, Bacillus neonatiensis, Salmonella è Nocaridia spp. Ïðè in vitro êóëüòèâèðîâàíèè ðàñòèòåëüíûõ îáúåêòîâ äëèòåëüíîå áåññèìïòîìíîå ïðèñóòñòâèå áàêòåðèé îáóñëîâëåíî, ñ îäíîé ñòîðîíû, ïîäàâëåíèåì èõ ðîñòà ôàêòîðàìè, ñîïðîâîæäàþùèìè êóëüòèâèðîâàíèå ðàñòèòåëüíûõ ýêñïëàíòîâ (pH, òåìïåðàòóðà íèæå îïòèìóìà äëÿ ðîñòà áàêòåðèé, àêòèâèðîâàíèå çàùèòíûõ ìåõàíèçìîâ), ñ äðóãîé — îäíîâðåìåííûì ïîääåðæàíèåì æèçíåäåÿòåëüíîñòè áàêòåðèé çà ñ÷åò ýêññóäàòîâ, âûäåëÿåìûõ ðàñòèòåëüíûì ýêñïëàíòîì. Áûñòðàÿ ïðîëèôåðàöèÿ áàêòåðèàëüíûõ êëåòîê ìîæåò íàñòóïèòü äàæå ïðè íåáîëüøèõ èçìåíåíèÿõ ïåðâîíà÷àëüíûõ óñëîâèé, ïðè óâåëè÷åíèè êîíöåíòðàöèè ðàñòèòåëüíûõ ýêññóäàòîâ è âñëåäñòâèå ñîáñòâåííî in vitro êóëüòèâèðîâàíèÿ êàê ñòðåññà â îòñóòñòâèå ðåãóëèðóþùåé ðîëè öåëîãî îðãàíèçìà. Ïî ìåðå óâåëè÷åíèÿ ÷èñëà ïàññàæåé äîëÿ
ðàñòèòåëüíûõ êóëüòóð ñî ñêðûòîé áàêòåðèàëüíîé êîíòàìèíàöèåé âîçðàñòàåò. Èìåþòñÿ äàííûå,
÷òî ïðè ýòîì íåêóëüòèâèðóåìûå ýíäîôèòû ìîãóò ïðèîáðåòàòü ñòàòóñ êóëüòèâèðóåìûõ. Ñêðûòûå
áàêòåðèàëüíûå êîíòàìèíàöèè ñïîñîáíû óãíåòàòü ðåãåíåðàöèþ, ìèêðîêëîíàëüíîå ðàçìíîæåíèå,
âûçûâàòü ãèáåëü êóëüòèâèðóåìûõ in vitro ðàñòèòåëüíûõ îáúåêòîâ, ñëóæèòü ïðåïÿòñòâèåì äëÿ âîñïðîèçâîäèìîñòè ïðîòîêîëîâ è èìåòü îòíîøåíèå ê ïîÿâëåíèþ ýïèãåíåòè÷åñêèõ ñîìàêëîíàëüíûõ
âàðèàíòîâ. Òàê, ôèëüòðàòû èçîëÿòîâ Acinetobacter è Lactobacillus plantarum, âûäåëåííûõ èç äåãðàäèðóþùèõ êàëëóñîâ, ïðè èíîêóëÿöèè â ýêñïëàíòû èëè äîáàâëåíèè â ïèòàòåëüíóþ ñðåäó ðåçêî
ñíèæàëè ðåãåíåðàöèþ ïîáåãîâ, à áàêòåðèè Mycobacterium obuense è M. aichiense óãíåòàëè ðàçâèòèå ñåìÿí â êóëüòóðå in vitro. Àêöåíòèðóåòñÿ ïðîáëåìà ïîëó÷åíèÿ ãíîòîáèîòè÷åñêèõ ðàñòèòåëüíûõ
êóëüòóð (â ÷àñòíîñòè, â in vitro êîëëåêöèÿõ ãåíåòè÷åñêèõ áàíêîâ ðàñòåíèé), îáóñëîâëåííàÿ ñëîæíîñòüþ âûÿâëåíèÿ è ýëèìèíàöèè áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ.
Êëþ÷åâûå ñëîâà: êóëüòóðà òêàíåé ðàñòåíèé, áàêòåðèàëüíûå ìèêðîîðãàíèçìû, àíòèáàêòåðèàëüíàÿ òåðàïèÿ.
Ïðè ðàáîòå ñ êóëüòóðîé ðàñòèòåëüíûõ òêàíåé in vitro íàëè÷èå áàêòåðèàëüíîé êîíòàìèíàöèÿ â çíà÷èòåëüíîé ñòåïåíè îïðåäåëÿåòñÿ êà÷åñòâîì
3
ñòåðèëüíîñòè (1, 2). Îäíàêî ýôôåêòèâíàÿ ñòåðèëèçàöèÿ ðàñòèòåëüíûõ ýêñïëàíòîâ è ñîáëþäåíèå ïðàâèë àíòèñåïòèêè íå èñêëþ÷àþò ïðèñóòñòâèÿ â
êóëüòóðàõ in vitro ñêðûòûõ áàêòåðèé (áåç âèçóàëüíî íàáëþäàåìîãî ðîñòà è
ñïåöèôè÷åñêèõ ñèìïòîìîâ) (3-5). Áàêòåðèàëüíûå ìèêðîîðãàíèçìû, åñòåñòâåííûì ìåñòîì îáèòàíèÿ êîòîðûõ ñëóæèò âîçäóøíàÿ ñðåäà, ïî÷âà, ðàñòåíèÿ è ÷åëîâåê, äåòåêòèðóþò è èäåíòèôèöèðóþò ñ ïîìîùüþ ìèêðîáèîëîãè÷åñêèõ, ìîëåêóëÿðíî-ãåíåòè÷åñêèõ è áèîõèìè÷åñêèõ ìåòîäîâ êàê â äëèòåëüíî ïàññèðóåìûõ, òàê è â èíèöèèðóåìûõ in vitro ðàñòèòåëüíûõ êóëüòóðàõ (6-14). Ñêðûòûå áàêòåðèàëüíûå èíôåêöèè, îáîçíà÷àåìûå ìíîãèìè èññëåäîâàòåëÿìè êàê âíóòðåííèå, èëè ýíäîôèòíûå, âûÿâëÿþò â êóëüòèâèðóåìûõ in vitro êàëëóñàõ, ìèêðîðàñòåíèÿõ, à òàêæå â ðàçëè÷íûõ òèïàõ ýêñïëàíòîâ — â âåðõóøêàõ ïîáåãîâ, ïî÷êàõ, ìåðèñòåìàõ (15-22). Áàêòåðèàëüíûå ýíäîôèòû, êîòîðûå âûïîëíÿþò ðÿä ôóíêöèé, âàæíûõ äëÿ ðàñòåíèé,
áûëè è îñòàþòñÿ ïðåäìåòîì ìíîãî÷èñëåííûõ èññëåäîâàíèé (23).  òî æå
âðåìÿ ðîëü ýíäîôèòíûõ áàêòåðèé â êóëüòóðàõ òêàíåé ìåíåå èçó÷åíà, îäíàêî îíà ïðåäñòàâëÿåò èíòåðåñ êàê â ôóíäàìåíòàëüíîì, òàê è â ïðèêëàäíîì
àñïåêòå.  ÷àñòíîñòè, áàêòåðèàëüíûå ýíäîôèòû ðàññìàòðèâàþòñÿ â êà÷åñòâå êëþ÷åâîãî ôàêòîðà, îïðåäåëÿþùåãî ðåãåíåðàöèîííóþ ñïîñîáíîñòü ýêñïëàíòîâ íàðÿäó ñ ãåíîòèïîì è óñëîâèÿìè êóëüòèâèðîâàíèÿ (18). Èõ èçó÷àþò êàê âîçìîæíûé ïåðñïåêòèâíûé èñòî÷íèê íîâûõ êîìïîíåíòîâ äëÿ
èñïîëüçîâàíèÿ â ïðàêòè÷åñêîé ìèêðîáèîëîãèè è ìåäèöèíå (24). Âíèìàíèå
ê áàêòåðèàëüíûì ýíäîôèòàì îáóñëîâëåíî òàêæå íàêîïëåíèåì äàííûõ,
óêàçûâàþùèõ íà óñëîâíîñòü èñòîðè÷åñêè ñëîæèâøåãîñÿ ðàçäåëåíèÿ ìèêðîîðãàíèçìîâ íà ôèòîïàòîãåííûå, ïàòîãåííûå äëÿ æèâîòíûõ (÷åëîâåêà) è
íåïàòîãåííûå (25). Ïîêàçàíî, ÷òî ïàòîãåííûå áàêòåðèàëüíûå øòàììû ÷åëîâåêà ìîãóò óñòîé÷èâî ñîõðàíÿòüñÿ â ïàññèðóåìûõ êóëüòóðàõ è â ðàñòåíèÿõ ex vitro (14), à áàêòåðèè Agrobacterium tumefaciens ñïîñîáíû òðàíñôîðìèðîâàòü êóëüòèâèðóåìûå in vitro êëåòêè ÷åëîâåêà (26) è ýìáðèîíû ìîðñêèõ åæåé (27). Ðàñøèðåíèå ñðåäû îáèòàíèÿ áàêòåðèé ñîçäàåò ýêîëîãè÷åñêèå è ãåíåòè÷åñêèå ðèñêè, êîòîðûå îïðåäåëÿþò íåîáõîäèìîñòü òùàòåëüíîãî ìîíèòîðèíãà ýíäîôèòíûõ ñîîáùåñòâ, îñîáåííî â òåõ ðàñòåíèÿõ,
êîòîðûå ÷åëîâåê óïîòðåáëÿåò â ïèùó â ñûðîì âèäå (14, 28). Ýòà ïðîáëåìà àêòóàëüíà è äëÿ êóëüòóðû òêàíåé ðàñòåíèé, ïîñêîëüêó in vitro òåõíîëîãèè íàõîäÿò øèðîêîå ïðèìåíåíèå â ïðàêòè÷åñêîì ðàñòåíèåâîäñòâå è
ïîëó÷åíèè ïðîäóêòîâ ïèòàíèÿ.
Öåëü íàñòîÿùåãî îáçîðà çàêëþ÷àëàñü â ñáîðå è ñèñòåìàòèçàöèè
äàííûõ, êàñàþùèõñÿ âûÿâëåíèÿ, èäåíòèôèêàöèè, ñîñòàâà, äèíàìèêè, âîçìîæíîé ðîëè è ýëèìèíàöèè ñêðûòûõ áàêòåðèàëüíûõ êîíòàìèíàöèé â
êóëüòóðå òêàíåé ðàñòåíèé.
Áàêòåðèàëüíûå ìèêðîîðãàíèçìû, ïðèñóòñòâèå êîòîðûõ â êóëüòèâèðóåìûõ in vitro ðàñòèòåëüíûõ îáúåêòàõ íå ñîïðîâîæäàåòñÿ âèçóàëüíûìè
ïðîÿâëåíèÿìè è ñïåöèôè÷åñêèìè ñèìïòîìàìè, â ëèòåðàòóðå îáîçíà÷àþò
êàê ëàòåíòíûå, ñêðûòûå, ýíäîãåííûå, âíóòðåííèå, ýíäîôèòíûå, íåðåäêî
èñïîëüçóÿ ýòè òåðìèíû êàê ñèíîíèìû. Íàèáîëåå ÷àñòî òàêèå áàêòåðèàëüíûå ìèêðîîðãàíèçìû â êóëüòóðå òêàíåé ðàñòåíèé íàçûâàþò ëàòåíòíûìè. Â
îäíîé èç ðàáîò (29) ïîä÷åðêèâàåòñÿ, ÷òî òåðìèí «ëàòåíòíûé» çàèìñòâîâàí
èç ôèòîïàòîëîãèè, ãäå îí ïðèìåíÿåòñÿ äëÿ îïèñàíèÿ áåññèìïòîìíûõ ïàòîãåíîâ, â òî âðåìÿ êàê áàêòåðèàëüíûå ìèêðîîðãàíèçìû â êóëüòóðå ðàñòèòåëüíûõ òêàíåé íå îáÿçàòåëüíî ïàòîãåííû (èõ âëèÿíèå ìîæåò áûòü îòðèöàòåëüíûì, ïîëîæèòåëüíûì èëè îòñóòñòâîâàòü). Àâòîð öèòèðóåìîé ðàáîòû
(29) íàðÿäó ñ äðóãèìè èññëåäîâàòåëÿìè (30, 31) ñ÷èòàåò áîëåå öåëåñîîáðàçíûì èñïîëüçîâàòü äëÿ óêàçàííûõ áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ òåðìèí «ñêðûòûå». Ñêðûòûå áàêòåðèàëüíûå ìèêðîîðãàíèçìû ìíîãèå èññëå4
äîâàòåëè íàçûâàþò ýíäîôèòàìè íà îñíîâàíèè èõ íàëè÷èÿ â êóëüòóðå ðàñòèòåëüíûõ îáúåêòîâ, ïðîøåäøèõ ïîâåðõíîñòíóþ ñòåðèëèçàöèþ. Ìû áóäåì
óïîòðåáëÿòü òåðìèí «ýíäîôèòíûå áàêòåðèè» â ñîîòâåòñòâèè ñ åãî ïðèìåíåíèåì àâòîðàìè öèòèðóåìûõ ðàáîò.
Ñîãëàñíî øèðîêî èñïîëüçóåìîìó îïðåäåëåíèþ, ýíäîôèòû — ýòî
ìèêðîîðãàíèçìû, êîòîðûå, íå âûçûâàÿ ñèìïòîìîâ áîëåçíåé, â òå÷åíèå
÷àñòè èëè âñåãî æèçíåííîãî öèêëà îáèòàþò âíóòðè ðàñòåíèÿ (32).  ïðèðîäå îíè ïîñòóïàþò â ðàñòåíèå ÷åðåç óñòüèöà, ïîðàíåíèÿ, êîðíåâóþ ñèñòåìó. Ñóùåñòâåííóþ ðîëü â ôîðìèðîâàíèè ýíäîôèòíîé ìèêðîôëîðû èãðàåò ïåðåäà÷à ìèêðîîðãàíèçìîâ ÷åðåç ñåìåíà, à òàêæå èõ èíòðîäóêöèÿ
âåêòîðíûìè îðãàíèçìàìè — áåñïîçâîíî÷íûìè èëè ãðèáàìè (28, 32). Èíòðîäóöèðîâàííûå ìèêðîîðãàíèçìû ìîãóò âêëþ÷àòüñÿ â ñîñòàâ ìèêðîôëîðû ðàñòåíèÿ â òî÷êå âõîæäåíèÿ è(èëè) ðàñïðîñòðàíÿòüñÿ ïî âñåìó ðàñòåíèþ
(32), ïðè ýòîì îáëèãàòíîñòü íå ñëóæèò îáÿçàòåëüíûì óñëîâèåì (33).
Ýíäîôèòíûå áàêòåðèè îáíàðóæåíû â öèòîïëàçìå êëåòîê, ìåæêëåòî÷íîì ïðîñòðàíñòâå (34) è â ñîñóäèñòîé ñèñòåìå (35) ðàñòåíèé.  ðÿäå
ðàáîò ïðèñóòñòâèå ýíäîôèòíûõ ìèêðîîðãàíèçìîâ â êóëüòèâèðóåìûõ in vitro ðàñòèòåëüíûõ îáúåêòàõ ïîêàçàíî ñ ïîìîùüþ ñâåòîâîé è ýëåêòðîííîé
ìèêðîñêîïèè, â òîì ÷èñëå ñ èñïîëüçîâàíèåì ìåòîäà in situ ãèáðèäèçàöèè
(15, 16, 21, 36-38).
È ñ ò î ÷ í è ê è á à ê ò å ð è à ë ü í î é ì è ê ð î ô ë î ð û. Ýíäîôèòíûå
áàêòåðèè ïðîèñõîäÿò îò ýïèôèòíûõ áàêòåðèàëüíûõ àññîöèàöèé ðèçîñôåðû
è ôèòîñôåðû ðàñòåíèÿ. Îñíîâíîé èñòî÷íèê ýíäîôèòíîé áàêòåðèàëüíîé
ìèêðîôëîðû â êóëüòóðàõ ðàñòèòåëüíûõ òêàíåé — ýêñïëàíòû èñõîäíûõ ðàñòåíèé. Àñåïòè÷åñêèå ýêñïëàíòû äëÿ êóëüòóðû in vitro ñëîæíî ïîëó÷èòü îò
ðàñòåíèé ñ ðîçåòî÷íîé ôîðìîé ðîñòà, äðåâåñíûõ, ìíîãîëåòíèõ (12, 38),
ïðîèçðàñòàþùèõ â ñûðûõ ìåñòàõ, ñîáðàííûõ â ïåðèîä âëàæíîé è òåïëîé
ïîãîäû, áîëüíûõ ðàñòåíèé (21, 39), à òàêæå ïðè èñïîëüçîâàíèè ïîäçåìíûõ
îðãàíîâ (êîðíåé, êîðíåâèù, êëóáíåëóêîâèö) (40, 41), ïî÷åê (èç-çà ïëîòíîãî è ìíîãîñëîéíîãî ëèñòîâîãî ôóòëÿðà), ýïèäåðìàëüíûõ òêàíåé, îñîáåííî
ñ âîëîñêàìè íà ïîâåðõíîñòè (42, 43). Ïðè ñòåðèëèçàöèè ðàñòèòåëüíîãî ìàòåðèàëà íåêîòîðàÿ ÷àñòü áàêòåðèàëüíûõ ýïèôèòîâ ìîæåò îñòàâàòüñÿ â íåäîñòóïíûõ äëÿ äåçèíôèöèðóþùèõ ðàñòâîðîâ ìåñòàõ, òàêèõ êàê çàêðûòûå
óñòüèöà, ñêëàäêè ïîâåðõíîñòè êîðíåâûõ ÷åðåíêîâ, ïðîñòðàíñòâà ìåæäó
ýïèäåðìàëüíûìè êëåòêàìè (5, 9).
Ê èñòî÷íèêàì ñèñòåìíîé áàêòåðèàëüíîé èíôåêöèè â êóëüòóðå òêàíåé ðàñòåíèé ìîãóò áûòü îòíåñåíû òàêæå ðàáî÷åå ìåñòî, îïåðàòîð, èíñòðóìåíòû, ïîñóäà (2, 44). Ñïîðû íåêîòîðûõ áàêòåðèé ñîõðàíÿþòñÿ ïðè àâòîêëàâèðîâàíèè (36) è äîëãî îñòàþòñÿ æèçíåñïîñîáíûìè â ýòèëîâîì
ñïèðòå (37).
Ì å ò î ä û â û ÿ â ë å í è ÿ è è ä å í ò è ô è ê à ö è è á à ê ò å ð è é. Äëÿ
âûÿâëåíèÿ è èäåíòèôèêàöèè ñêðûòûõ áàêòåðèàëüíûõ êîíòàìèíàöèé èñïîëüçóþò ïèòàòåëüíûå ñðåäû è ôèçèîëîãè÷åñêèå òåñòû, ìåòîäû, îñíîâàííûå íà ïðèìåíåíèè ôàãîâ, ïðîôèëèðîâàíèè æèðíûõ êèñëîò è áåëêîâ, à
òàêæå òèïèðîâàíèå ãåíîòèïîâ ñ ïîìîùüþ MALDI TOF (Matrix assisted laser desorption/ionization time of flight) ìàññ-ñïåêòðîìåòðèè è ìîëåêóëÿðíîãåíåòè÷åñêèõ ìàðêåðîâ (RAPD-PCR — random amplified polymorphic DNA
polymerase chain reaction, REP-PCR — repetitive extragenic palindromic polymerase chain reaction, AFLP — amplified fragment length polymorphism,
ARDRA — amplified ribosomal DNA restriction analysis, 16S-rRNA). Óêàçàííûå ïðèåìû ñïåöèôè÷íû ïðè ìîíèòîðèíãå íà ðàçíîì òàêñîíîìè÷åñêîì
óðîâíå. Òàê, áîëüøèíñòâî èç íèõ ïîçâîëÿåò îïðåäåëèòü áàêòåðèàëüíûå
ìèêðîîðãàíèçìû íà óðîâíå ñåìåéñòâà, ðîäà è âèäà, íî äëÿ àòòðèáóòèðîâà5
íèÿ ïîäâèäîâ, áèîâàðîâ è øòàììîâ ïðåäïî÷òèòåëüíåå èñïîëüçîâàòü ñîâðåìåííûå áèîõèìè÷åñêèå è ìîëåêóëÿðíî-ãåíåòè÷åñêèå ìåòîäû (45).
Íàèáîëåå øèðîêî ïðèìåíÿþò äâà ñïîñîáà âûÿâëåíèÿ è èäåíòèôèêàöèè áàêòåðèé. Îäèí èç íèõ, òðàäèöèîííûé, îñíîâàí íà èñïîëüçîâàíèè
êóëüòóðàëüíûõ è ìîðôîëîãè÷åñêèõ õàðàêòåðèñòèê, à òàêæå õèìè÷åñêèõ è
áèîõèìè÷åñêèõ ðåàêöèé (46). Îí áîëåå äîñòóïåí è íå òðåáóåò äîðîãîñòîÿùåãî îáîðóäîâàíèÿ, îäíàêî íå ïîçâîëÿåò âûÿâèòü íåêóëüòèâèðóåìûå, òî
åñòü íå ìåòàáîëèçèðóþùèå ñóáñòðàò ïèòàòåëüíûõ ñðåä, ôîðìû áàêòåðèé.
Âòîðîé ìåòîä, ìîëåêóëÿðíî-ãåíåòè÷åñêèé, áàçèðóåòñÿ íà àíàëèçå ãåíîâ,
êîäèðóþùèõ ýâîëþöèîííî êîíñåðâàòèâíûå ðèáîñîìàëüíûå ÐÍÊ, ïîñêîëüêó ýòè ãåíû ïðèñóòñòâóþò âî âñåõ áàêòåðèàëüíûõ êëåòêàõ è ðîäîñïåöèôè÷íû äëÿ áîëüøèíñòâà ìèêðîîðãàíèçìîâ (23). Äëÿ èäåíòèôèêàöèè ñåêâåíèðóþò ïîñëåäîâàòåëüíîñòè ðèáîñîìàëüíûõ ãåíîâ, êîäèðóþùèõ 23SðÐÍÊ áîëüøîé ñóáúåäèíèöû (äëèíà ~3000 ï.í.) è 16S-ðÐÍÊ ìàëîé ñóáúåäèíèöû (~1500 ï.í.), à òàêæå ó÷àñòêè âíóòðåííèõ òðàíñêðèáèðóåìûõ ñïåéñåðîâ (internal transcribed spacers — ITS) (47). ×àñòî îïðåäåëÿþò ðàçëè÷íûå
ïîñëåäîâàòåëüíîñòè 16S-ðÐÍÊ, èìåþùèå êàê êîíñåðâàòèâíûå ñàéòû, îäèíàêîâûå ó âñåõ ïðîêàðèîòîâ, òàê è ïðèãîäíûå äëÿ èäåíòèôèêàöèè âèäîñïåöèôè÷íûå ó÷àñòêè (48, 49). Åùå áîëåå èíôîðìàòèâíû ïîñëåäîâàòåëüíîñòè ðèáîñîìàëüíûõ ñïåéñåðîâ 16S—23S-ðÐÍÊ, çíà÷èòåëüíî áîëåå âàðèàáåëüíûå ïî ðàçìåðàì è ñòðóêòóðå ïî ñðàâíåíèþ ñ ðàéîíàìè ñàìèõ ãåíîâ.
Ïîýòîìó âíóòðåííèå òðàíñêðèáèðóåìûå ñïåéñåðû ïðåèìóùåñòâåííî èñïîëüçóþò äëÿ ðàçëè÷åíèÿ ìèêðîîðãàíèçìîâ íà óðîâíå âèäîâ è äàæå øòàììîâ (50). Äëÿ àìïëèôèêàöèè ôðàãìåíòîâ ãåíîâ 16S-ðÐÍÊ è ITS ðåãèîíîâ
îñóùåñòâëÿþò ïîëèìåðàçíóþ öåïíóþ ðåàêöèþ (ÏÖÐ) ñ ìåòàãåíîìíîé
ÄÍÊ è ñîîòâåòñòâóþùèìè ñïåöèôè÷åñêèìè ïðàéìåðàìè (13, 51, 52). Ïîëó÷åííûå ïðîäóêòû ñåêâåíèðóþò è ïðîâîäÿò ïîèñê ãîìîëîãè÷íûõ ïîñëåäîâàòåëüíîñòåé ñ ïîìîùüþ áàçû äàííûõ GenBank (53) äëÿ òàêñîíîìè÷åñêîé èäåíòèôèêàöèè áàêòåðèé â èññëåäóåìîì ìàòåðèàëå.  ïóáëèêàöèè
À.Â. Ïèíåâè÷à (54) óïîìèíàåòñÿ î ñåêâåíèðîâàíèè ãåíîìîâ 60 âèäîâ áàêòåðèé, â òî âðåìÿ êàê îáùåå ÷èñëî îïèñàííûõ áàêòåðèàëüíûõ âèäîâ ñîñòàâëÿåò 5007.
È ä å í ò è ô è ê à ö è ÿ á à ê ò å ð è é. Èäåíòèôèêàöèÿ ìèêðîîðãàíèçìîâ, êîëîíèçèðóþùèõ in vitro êóëüòóðû ðàñòåíèé, äàåò âîçìîæíîñòü èçó÷àòü âëèÿíèå áàêòåðèé íà õîçÿèíà, ïðîâîäèòü íàïðàâëåííóþ õèìèîòåðàïèþ, ñîçäàâàòü áàíê äàííûõ ïî ìèêðîîðãàíèçìàì, àññîöèàòèâíûì ñ êóëüòóðîé ðàñòèòåëüíûõ òêàíåé.  ðàííèõ ðàáîòàõ äëÿ èäåíòèôèêàöèè â îñíîâíîì ïðèìåíÿëè òåñòû êëàññè÷åñêîé ìèêðîáèîëîãèè — îïðåäåëåíèå
ñïîñîáíîñòè áàêòåðèàëüíîé êóëüòóðû ê ðîñòó íà ïèòàòåëüíûõ ñðåäàõ ðàçíîãî ñîñòàâà, îêðàñêà ïî Ãðàìó, ìîðôîëîãèÿ è öâåò êîëîíèé (4, 6, 9, 10,
46). Ñîâðåìåííûå ìåòîäû ïîçâîëÿþò ñóùåñòâåííî äîïîëíèòü èíôîðìàöèþ
î òàêñîíîìè÷åñêîì ðàçíîîáðàçèè áàêòåðèé, àññîöèðîâàííûõ ñ êóëüòóðîé
òêàíåé ðàñòåíèé. Áàçà äàííûõ ïî òàêèì ìèêðîîðãàíèçìàì ñòðåìèòåëüíî
óâåëè÷èâàåòñÿ.  òàáëèöå ïðèâåäåí òàêñîíîìè÷åñêèé ñîñòàâ áàêòåðèàëüíûõ ýíäîôèòîâ â in vitro êóëüòóðàõ äëÿ ñðàâíèòåëüíî îãðàíè÷åííîé âûáîðêè èññëåäîâàííûõ ðàñòåíèé, ñâèäåòåëüñòâóþùèé î ðàçíîîáðàçèè ôîðì,
ñïîñîáíûõ êîëîíèçèðîâàòü òêàíè â êóëüòóðå in vitro — íèøå, â êîòîðîé
óñëîâèÿ ðåçêî îòëè÷àþòñÿ îò ïðèðîäíûõ. Ïðè ýòîì îòìå÷àåòñÿ îòñóòñòâèå
ñïåöèôè÷åñêîãî ñîñòàâà ýíäîôèòíûõ áàêòåðèé â in vitro êóëüòóðàõ ðàñòåíèé ðàçíîé ñèñòåìàòè÷åñêîé ïðèíàäëåæíîñòè è â ýêñïëàíòàõ, îòíîñÿùèõñÿ ê ðàçíûì îðãàíàì ðàñòåíèÿ. Òàêîé æå âûâîä ìîæíî ñäåëàòü, àíàëèçèðóÿ äàííûå ïî èäåíòèôèêàöèè áàêòåðèé â êóëüòóðàõ òêàíåé, ïîëó÷åííûå â
áîëåå ðàííèõ èññëåäîâàíèÿõ (4, 6, 9, 10).
6
Ñðåäè èäåíòèôèöèðîâàííûõ ýíäîôèòíûõ áàêòåðèé âûÿâëåíû ïîòåíöèàëüíî ïîëåçíûå äëÿ èíòàêòíûõ ðàñòåíèé, â ÷àñòíîñòè Streptomycete, Pantoea agglomerans è äð., à òàêæå ïàòîãåííûå äëÿ ÷åëîâåêà, íàïðèìåð Ralstonia
mannitolytica, Staphylococcus epidermidis, Corynebacterium amycolatum, Bacillus
neonatiensis, Salmonella è Nocaridia spp. (13).
Ä è í à ì è ê à ý ê ñ ï ð å ñ ñ è è á à ê ò å ð è é. Äëèòåëüíîå áåññèìïòîìíîå ïðèñóòñòâèå áàêòåðèé â in vitro ðàñòèòåëüíûõ êóëüòóðàõ îáåñïå÷èâàåòñÿ äâóìÿ ïðîòèâîïîëîæíûìè ïðîöåññàìè — îãðàíè÷åíèåì ðîñòà è ïîääåðæàíèåì æèçíåäåÿòåëüíîñòè ýòèõ ìèêðîîðãàíèçìîâ. Ðîñò ïîäàâëÿåòñÿ
ôàêòîðàìè, ñîïðîâîæäàþùèìè êóëüòèâèðîâàíèå ðàñòèòåëüíûõ îáúåêòîâ: pH
(ïîäêèñëåíèå ïèòàòåëüíîé ñðåäû), òåìïåðàòóðà (25 °Ñ) íèæå îïòèìóìà äëÿ
ðîñòà áàêòåðèé, âîçìîæíîå àêòèâèðîâàíèå ìåõàíèçìîâ, îáóñëîâëèâàþùèõ
óñòîé÷èâîñòü ðàñòèòåëüíûõ êóëüòóð ê áàêòåðèÿì (55).  òî æå âðåìÿ æèçíåñïîñîáíîñòü áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ â êóëüòóðå ðàñòèòåëüíûõ
òêàíåé ïîääåðæèâàåòñÿ çà ñ÷åò ýêññóäàòîâ, âûäåëÿåìûõ ýêñïëàíòîì, òàê
êàê áîëüøèíñòâî áàêòåðèé, íåñìîòðÿ íà ãåòåðîòðîôíûé òèï ïèòàíèÿ, íå
ìîãóò íàäåæíî ñóùåñòâîâàòü â îòñóòñòâèå ðàñòèòåëüíîãî ìàòåðèàëà (12,
55).  ðåçóëüòàòå ñîõðàíÿåòñÿ íåêîòîðûé íåâûñîêèé òèòð áàêòåðèé, àññîöèèðîâàííûõ ñ êóëüòóðîé òêàíåé ðàñòåíèé, ñ ÷åì ñâÿçàíà áåññèìïòîìíîñòü è òðóäíî èñêîðåíÿåìîå äëèòåëüíîå ïðèñóòñòâèå ìèêðîîðãàíèçìîâ
(ïåðñèñòåíòíîñòü).
Áûñòðàÿ ïðîëèôåðàöèÿ áàêòåðèàëüíûõ êëåòîê ìîæåò íàñòóïèòü äàæå ïðè íåáîëüøèõ èçìåíåíèÿõ ïåðâîíà÷àëüíûõ óñëîâèé — âñëåäñòâèå ïîâûøåíèÿ òåìïåðàòóðû îêðóæàþùåãî âîçäóõà, èçìåíåíèÿ êèñëîòíîñòè èëè
ñîñòàâà ïèòàòåëüíîé ñðåäû, íàïðèìåð â ðåçóëüòàòå ïîñòóïëåíèÿ äîïîëíèòåëüíîãî àçîòà èç îòìèðàþùèõ òêàíåé ýêñïëàíòà (55, 56), à òàêæå ïðè ñóáêóëüòèâèðîâàíèè ñòàðûõ êóëüòóð íà ïèòàòåëüíîé ñðåäå ñ âûñîêîé êîíöåíòðàöèåé öèòîêèíèíîâ (8, 57). Ðàçìíîæåíèå áàêòåðèé èíäóöèðóåòñÿ óâåëè÷åíèåì êîíöåíòðàöèè ýêññóäàòîâ, âûäåëÿåìûõ ðàñòèòåëüíûìè êóëüòóðàìè
in vitro, êîòîðîå, â ñâîþ î÷åðåäü, ìîæåò ñòèìóëèðîâàòüñÿ ïîâûøåíèåì
òåìïåðàòóðû îêðóæàþùåé ñðåäû, óâåëè÷åíèåì ïëîòíîñòè in vitro êóëüòóð,
ïåðåíîñîì èõ íà ïèòàòåëüíóþ ñðåäó äëÿ óêîðåíåíèÿ (55). Â ðåçóëüòàòå áûñòðîãî ðàçìíîæåíèÿ áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ âîçìîæíî ïîÿâëåíèå âèäèìûõ ñèìïòîìîâ â in vitro êóëüòóðàõ è(èëè) âèäèìîãî ðîñòà íà
ñðåäå êóëüòèâèðîâàíèÿ ýêñïëàíòîâ (3). Âûõîä áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ èç êóëüòèâèðóåìûõ òêàíåé â òîëùó ïèòàòåëüíîé ñðåäû, êàê ïðàâèëî, ñîïðîâîæäàåòñÿ îáðàçîâàíèåì â íåé ìóòíîãî îðåîëà, îòìå÷åííîãî ìíîãèìè èññëåäîâàòåëÿìè (7, 10, 11, 58, 59).  îòñóòñòâèå ðåãóëèðóþùåé ðîëè
öåëîãî îðãàíèçìà áåñêîíòðîëüíîå ðàçìíîæåíèå ýíäîôèòîâ â êóëüòóðå òêàíåé ðàñòåíèé ìîæåò ñòèìóëèðîâàòüñÿ ñîáñòâåííî in vitro êóëüòèâèðîâàíèåì êàê ôàêòîðîì ñòðåññà (16).
 ðÿäå ðàáîò ïîêàçàíî, ÷òî ïî ìåðå óâåëè÷åíèÿ ÷èñëà ïàññàæåé äîëÿ ðàñòèòåëüíûõ êóëüòóð ñ âèäèìîé áàêòåðèàëüíîé êîíòàìèíàöèåé óìåíüøàåòñÿ, ñî ñêðûòîé — âîçðàñòàåò. Ïðè ýòîì ìîæåò ïðîèñõîäèòü èçìåíåíèå
ñîñòàâà ìèêðîîðãàíèçìîâ, óâåëè÷åíèå ÷èñëà ãðàìïîëîæèòåëüíûõ ôîðì è
ñïîñîáíîñòè áàêòåðèé ê êóëüòèâèðîâàíèþ (4, 13, 21). Òàê, â ïàññèðóåìîé
êóëüòóðå èíäèâèäóàëüíûõ ìèêðîðàñòåíèé áàíàíà ïîêàçàíî ñóùåñòâîâàíèå
áàêòåðèàëüíûõ ýíäîôèòîâ, êîòîðûå â ïåðâûõ òðåõ ïàññàæàõ áûëè íåêóëüòèâèðóåìûìè è âûÿâèëèñü òîëüêî ìåòîäîì ñåêâåíèðîâàíèÿ ãåíà 16SðÐÍÊ (áàêòåðèè VBNC — viable but nonculturable). Îäíàêî â ïîñëåäóþùèõ
ïàññàæàõ (ñ 4-ãî äî 18-ãî) â ýòèõ æå ìèêðîðàñòåíèÿõ âûÿâèëèñü êóëüòèâèðóåìûå áàêòåðèè (13, 21). Àâòîðû öèòèðóåìûõ ðàáîò ïîëàãàþò, ÷òî ïî ìåðå âîçðàñòàíèÿ ÷èñëà ïàññàæåé ðàñòèòåëüíûõ êóëüòóð ñîäåðæàùèåñÿ â íèõ
7
VBNC ýíäîôèòû ìîãóò ïðèîáðåòàòü ñòàòóñ êóëüòèâèðóåìûõ.
Âëèÿíèå áàêòåðèé íà êîëîíèçèðóåìûå èìè ðàñòèò å ë ü í û å ê ó ë ü ò ó ð û in vitro. Áàêòåðèàëüíûå ìèêðîîðãàíèçìû, àññîöèèðîâàííûå ñ êóëüòóðîé ðàñòèòåëüíûõ òêàíåé, ñïîñîáíû îòðèöàòåëüíî âëèÿòü íà ðåãåíåðàöèîííóþ ñïîñîáíîñòü êàëëóñà, êëåòî÷íûõ ñóñïåíçèé è
ïðîòîïëàñòîâ (56, 38), óãíåòàòü ìèêðîêëîíàëüíîå ðàçìíîæåíèå, ðîñò è
óêîðåíåíèå ïîáåãîâ, âûçûâàòü ãèáåëü êóëüòèâèðóåìûõ in vitro è ex vitro
ðàñòèòåëüíûõ îáúåêòîâ (3, 11, 13, 21, 29, 36, 37, 57, 60). Ñêðûòûå áàêòåðèàëüíûå êîíòàìèíàöèè â ðàñòèòåëüíûõ êóëüòóðàõ ìîãóò ñëóæèòü ïðåïÿòñòâèåì äëÿ âîñïðîèçâîäèìîñòè ïðîòîêîëîâ (29) è èìåòü îòíîøåíèå ê ïîÿâëåíèþ ýïèãåíåòè÷åñêèõ ñîìàêëîíàëüíûõ âàðèàíòîâ (61). Îäíà èç âåðîÿòíûõ ïðè÷èí íåãàòèâíîãî âëèÿíèÿ ñêðûòûõ áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ — ïîâûøåíèå èõ òèòðà. Òàêàÿ âîçìîæíîñòü ðàññìàòðèâàåòñÿ íåêîòîðûìè àâòîðàìè â ñâÿçè ñ ãèáåëüþ ðàñòèòåëüíûõ êóëüòóð ïîñëå 2-ãî èëè 3-ãî
ñóáêóëüòèâèðîâàíèÿ (18, 62).  ñâîèõ èññëåäîâàíèÿõ ìû òàêæå îòìå÷àëè,
÷òî 2-é è 3-é ïàññàæè — êðèòè÷åñêèå äëÿ ìèêðîðàçìíîæåíèÿ ââåäåííûõ â
êóëüòóðó in vitro ýêñïëàíòîâ ìàëèíû (59). Óãíåòàþùåå äåéñòâèå áàêòåðèé
íà in vitro ðàñòèòåëüíûå îáúåêòû ìîæåò áûòü òàêæå ñâÿçàíî ñ èçìåíåíèåì
êèñëîòíîñòè ïèòàòåëüíîé ñðåäû èëè åå ñîñòàâà ïîä âëèÿíèåì áàêòåðèé
(ïîòðåáëåíèå ñàõàðîçû, îáðàçîâàíèå ãåðáèöèäíûõ ñóáñòàíöèé) (63).
Îñîáûé èíòåðåñ ïðåäñòàâëÿþò ðàáîòû ïî èçó÷åíèþ âëèÿíèÿ àêñåíè÷åñêèõ áàêòåðèàëüíûõ êóëüòóð íà in vitro ðàñòèòåëüíûå îáúåêòû. Ïîêàçàíî, ÷òî ôèëüòðàòû êóëüòóð Acinetobacter è Lactobacillus plantarum, âûäåëåííûõ èç äåãðàäèðóþùèõ êàëëóñîâ ðàñòåíèé, ïðè èíîêóëèðîâàíèè â ðàñòèòåëüíûå ýêñïëàíòû èëè äîáàâëåíèè â ïèòàòåëüíóþ ñðåäó ðåçêî ñíèæàëè
ðåãåíåðàöèþ ïîáåãîâ ó êàëëóñîâ (56, 63). Áàêòåðèè Mycobacterium obuense è
M. aichiense óãíåòàëè ðàçâèòèå ñåìÿí â êóëüòóðå in vitro (38).
Ïîëåçíóþ ðîëü ýíäîôèòíûõ ìèêðîîðãàíèçìîâ â in vitro êóëüòóðàõ
ðàñòåíèé íà÷àëè èññëåäîâàòü ïîçäíåå, è ýòè äàííûå ìåíåå ìíîãî÷èñëåííû.  ðÿäå ðàáîò îòìå÷åí ïîçèòèâíûé ýôôåêò áàêòåðèé ðîäà Methylobacterium íà èíäóêöèþ îðãàíîãåíåçà è ýìáðèîãåíåçà (15, 17, 64-68). Ïðåäïîëàãàåòñÿ, ÷òî âûÿâëåííûå ìåòîäîì in situ ãèáðèäèçàöèè â êóëüòóðå òêàíåé
ñîñíû ýíäîôèòû Mycobacterium sp., Methylobacterium spp., Pseudomonas spp.,
Rhodotorula minuta ìîãóò èìåòü ïîëîæèòåëüíîå âëèÿíèå íà ìîðôîãåíåç â
êóëüòóðå in vitro (17) ïî àíàëîãèè ñ äåéñòâèåì áàêòåðèé íà ðàçâèâàþùèåñÿ
òêàíè æèâîòíûõ (69). Ïîêàçàíî ñòèìóëèðóþùåå âëèÿíèå Bacillus circulans
íà ñîìàòè÷åñêèé ìîðôîãåíåç ó ãåðàíè (Pelargonium ½ hortorum Bailey) (70).
Òàêèì îáðàçîì, áàêòåðèàëüíûå ìèêðîîðãàíèçìû, àññîöèèðîâàííûå
ñ òêàíÿìè ðàñòåíèé â êóëüòóðå in vitro, ìîãóò îêàçûâàòü íà íèõ êàê
ïîçèòèâíûå âîçäåéñòâèÿ, òàê è èãðàòü ðîëü ôàêòîðîâ, ëèìèòèðóþùèõ ðîñò
è æèçíåäåÿòåëüíîñòü.
 ãåíåòè÷åñêèõ áàíêàõ ðàñòåíèé îñíîâíîå òðåáîâàíèå ê in vitro è
êðèîêîëëåêöèÿì ðàñòåíèé çàêëþ÷àåòñÿ â îòñóòñòâèè âíóòðåííåé ìèêðîôëîðû. Ãíîòîáèîòè÷åñêèé ñòàòóñ ñåðòèôèöèðîâàííîãî ðàñòèòåëüíîãî ìàòåðèàëà ïîçâîëÿåò èçáåæàòü ïåðåíîñà ñêðûòûõ èíôåêöèé ïðè èñïîëüçîâàíèè òåõíîëîãèé ìèêðîðàçìíîæåíèÿ è ñëóæèò êðèòåðèåì íàäåæíîãî ñîõðàíåíèÿ ãåíîòèïîâ â êîíòðîëèðóåìûõ óñëîâèÿõ ñðåäû. Äëÿ ñåðòèôèêàöèè
ðàñòèòåëüíîãî ìàòåðèàëà â íåì ïðîâîäÿò âûÿâëåíèå, èäåíòèôèêàöèþ è
ýëèìèíàöèþ îñíîâíûõ âèðóñîâ, ìèêîïëàçì è âñåé áàêòåðèàëüíîé ìèêðîôëîðû (39). Ðàçíîîáðàçèå, ìíîãî÷èñëåííîñòü è äèíàìè÷åñêîå ñîñòîÿíèå
áàêòåðèé â in vitro ðàñòèòåëüíûõ êóëüòóðàõ ñîçäàþò íåîáõîäèìîñòü ïåðèîäè÷åñêîãî òåñòèðîâàíèÿ ðàñòèòåëüíîãî ìàòåðèàëà íà ïðèñóòñòâèå áàêòåðèé
8
Ýíäîôèòíûå áàêòåðèè, âûÿâëåííûå â in vitro êóëüòóðå òêàíåé ðàñòåíèé
Ðîä, âèä
(ñîðò ðàñòåíèÿ)
Chrysanthemum
(Arka Swarna)
Pinus sylvestris
Pinus sylvestris
Prunus cerasus
(Montmorency)
Bactris gasipaes
Musa sp.
Òèï êóëüòóð (ïðîäîëæèòåëüíîñòü
êóëüòèâèðîâàíèÿ in vitro)
Ìèêðîðàñòåíèÿ (1-7 ïàññàæåé)
Êàëëóñíàÿ êóëüòóðà
Êàëëóñíàÿ è ñóñïåíçèîííàÿ
êóëüòóðû
Ìèêðîðàñòåíèÿ
Ìèêðîðàñòåíèÿ
Ìèêðîðàñòåíèÿ (äëèòåëüíîå
ìèêðîêëîíèðîâàíèå)
Musa sapientum
(Chini champa)
Âåðõóøêè ïîáåãîâ
(1-2 íåä êóëüòèâèðîâàíèÿ)
Larix, Picea
Ñóñïåíçèîííàÿ êóëüòóðà
(6-8 íåä êóëüòèâèðîâàíèÿ)
Ìèêðîðàñòåíèÿ
Chrysanthemum
(Arka Ravi)
Rubus idaeus,
Fragaria ananassa,
Ñerasus vulgaris,
Ribes nigrum
Ìèêðîðàñòåíèÿ èç êîëëåêöèè
in vitro
Jatropha curcas
Ëèñòîâûå ýêñïëàíòû
Fragaria ananassa
Ìåðèñòåìà
(Camarosa, Sweet
Charlie, Oso-Grande)
Musa sp.
Âåðõóøêè ïîáåãîâ
Ðîä, âèä áàêòåðèé
(÷àñòîòà îáíàðóæåíèÿ, %)
Ðàçëè÷íûå ìîðôîòèïû Curtobacterium
citreum
Hormonema dematioides (isolates L, M),
Methylobacterium extorquens (isolate F),
Pseudomonas synxantha (isolates G, H, J),
Pseudomonas sp. (isolates K, N),
Rhodotorula minuta (isolate T)
Methylobacterium extorquens
Ññûëêà
(8)
(16)
(18)
Pseudomonas aeruginosa
(19)
Brevibacillus sp., Moraxella sp.
Alcaligenes, Bacillus spp.,
Brachybacterium, Brevibacterium,
Brevundimonas, Corynebacterium,
Enterobacter, Klebsiella, Kocuri,
Methylobacterium,
Microbacterium, Oceanobacillus,
Ochrobactrum, Pantoea, Pseudomonas,
Ralstonia, Staphylococcus, Tetrasphaer spp.
Ãðàìïîëîæèòåëüíûå: Bacillus megaterium,
Cellulomona uda, C. flavigena,
Corynebacterium paurometabolum
Ãðàìîòðèöàòåëüíûå: Erwinia cypripedii,
Klebsiell sp., Pseudomonas sp.
Acinetobacter
(20)
(21)
Enterobacter, Methylobacterium spp.,
Ralstonia
Arthrobacter (23,5 %), Bacillus (51,5 %)
(57)
Ðåæå âñòðå÷àþòñÿ Agrobacterium,
Bacterium, Brevibacterium, Flavobacterium,
Micrococcus, Mycobacterium,
Pseudomonas
Enterobacter ludwigii
17 áàêòåðèàëüíûõ øòàììîâ èç ðîäîâ
Bacillus, Sphingopyxis, Virgibacillus
Bacillus, Brevibacillus, Paenibacillus,
Staphylococcus spp., Virgibacillus;
(22)
(56)
(58)
(62)
(64)
(51)
Actinobacteria (Cellulomonas, Micrococcus,
Corynebacterium, Kocuria spp.);
α-proteobacteria (Paracoccus sp.);
Y-proteobacteria (Acinetobacter spp.,
Pseudomonas)
Ilex dumosa
Ñåãìåíòû óçëîâ ïîáåãà
Achromobacter, Stenotrophomonas
maltophilia
Echinacea
Ìèêðîðàñòåíèÿ
Acinetobacter, Bacillus, Pseudomonas, Stenotrophomonas, Wautersia (Ralstonia)
Carica papaya
Âåðõóøêè ïîáåãîâ
Agrobacterium (A. tumefaciens),
Bacillus (B. benzoevorans),
Brevundimonas (B. aurantiaca),
Enterobacter (E. cloacae),
Methylobacterium (M. rhodesianum),
Microbacterium (M. esteraromaticum),
Pantoea (P. ananatis) (70 %),
Sphingomonas, Wautersia (Ralstonia)
Potato
Ìèêðîðàñòåíèÿ
Bacillus pumilus
Carica papaya
Âåðõóøêè ïîáåãîâ
Lysinibacillus fusiformis, Paenibacillus sp.,
(1 ìåñ êóëüòèâèðîâàíèÿ)
Pantoea sp., Ralstonia mannitolilytica, Sphingomonas sp.
Limonium simuatum
Ìèêðîðàñòåíèÿ
Alcaligenes sp., Pasteurella multocida,
Stenotrophomonas maltophilia
Ananas comosus
Ìèêðîðàñòåíèÿ (5 ëåò êóëüòèâèðî- Actinobacteria, Alphaproteobacteria,
âàíèÿ)
Betaproteobacteria
Piper nigrum, Piper
Êàëëóñíàÿ êóëüòóðà (ïåðâè÷íûå
Aminobacter, Flavobacterium, Morococcus,
colubrinum, Taxus
ýêñïëàíòû)
Paracoccus Pseudomonas, Psychrobacter,
baccata subsp. wallichiRhizobacter
ana, Withania somnifera
(71)
(72)
(73)
(74)
(75)
(76)
(77)
(78)
9
è èñïîëüçîâàíèÿ ðàçíûõ ìåòîäîâ äëÿ èõ âûÿâëåíèÿ. Ýòî ïðåäñòàâëÿåò îïðåäåëåííóþ ñëîæíîñòü ïðè ðàáîòå ñ áîëüøèìè in vitro êîëëåêöèÿìè â ãåíåòè÷åñêèõ áàíêàõ ðàñòåíèé.
À í ò è á à ê ò å ð è à ë ü í à ÿ ò å ð à ï è ÿ. Äëÿ ýëèìèíàöèè áàêòåðèàëüíûõ ìèêðîîðãàíèçìîâ ïðèìåíÿþòñÿ àíòèáèîòèêè (79). Îïèñàíèå è
ñâîéñòâà íåêîòîðûõ àíòèáèîòèêîâ, ïðèìåíÿåìûõ äëÿ õèìèîòåðàïèè ðàñòåíèé, ïðèâåäåíû â îáçîðå G. Seckinger ñ ñîàâò. (80). Èäåàëüíûå àíòèáèîòèêè, ðåêîìåíäóåìûå äëÿ ýëèìèíàöèè áàêòåðèé â êóëüòóðå ðàñòèòåëüíûõ
òêàíåé, äîëæíû áûòü áàêòåðèöèäíûìè, íåäîðîãèìè, íåòîêñè÷íûìè äëÿ
÷åëîâåêà, ðàñòâîðèìûìè â ïèòàòåëüíûõ ñðåäàõ è íå èçìåíÿòü èõ pH (9,
61). Ðåçóëüòàòèâíîñòü âûáîðà íàèáîëåå ýôôåêòèâíûõ àíòèáèîòèêîâ (ñ øèðîêèì ñïåêòðîì äåéñòâèÿ èëè â ñïåöèôè÷åñêîé êîìáèíàöèè) ïîâûøàåòñÿ
ïðè èäåíòèôèêàöèè áàêòåðèé â êóëüòèâèðóåìûõ in vitro ðàñòèòåëüíûõ îáúåêòàõ.  ñëó÷àå èñïîëüçîâàíèÿ êîìáèíàöèè àíòèáèîòèêîâ (îñîáåííî ïðè
èõ ñèíåðãåòè÷åñêîì äåéñòâèè) ñíèæàåòñÿ ðèñê âîçíèêíîâåíèÿ óñòîé÷èâîñòè ê íèì ó áàêòåðèé, îäíàêî íåêîòîðûå àíòèáèîòèêè õèìè÷åñêè íåñîâìåñòèìû è â ñî÷åòàíèè ìîãóò íåéòðàëèçîâàòü äåéñòâèå äðóã äðóãà (71,
72). Ãðàìîòðèöàòåëüíûå áàêòåðèè (îíè ñîñòàâëÿþò áîëüøèíñòâî ñðåäè
èäåíòèôèöèðîâàííûõ â êóëüòóðå ðàñòèòåëüíûõ òêàíåé) îñîáåííî òðóäíî
ïîääàþòñÿ ýëèìèíàöèè èç-çà ôàêòè÷åñêè äâîéíîé êëåòî÷íîé ìåìáðàíû,
çàòðóäíÿþùåé ïðîíèêíîâåíèå ìíîãèõ àíòèáèîòèêîâ. Ïîñëå ïðèìåíåíèÿ
àíòèáàêòåðèàëüíîé òåðàïèè ðàñòèòåëüíûé ìàòåðèàë íåîáõîäèìî òåñòèðîâàòü íà ïðèñóòñòâèå áàêòåðèé íà ïðîòÿæåíèè 2-3 ïàññàæåé (71).
Èñïîëüçîâàíèå àíòèáèîòèêîâ â êóëüòóðå ðàñòèòåëüíûõ òêàíåé îáúåêòèâíî îñëîæíÿåòñÿ ðàçëè÷íûìè ïðè÷èíàìè. Òàê, íåîáõîäèì ïîäáîð
ýôôåêòèâíîãî àíòèáèîòèêà â áàêòåðèöèäíîé äëÿ êîíêðåòíîãî áàêòåðèàëüíîãî îðãàíèçìà êîíöåíòðàöèè è îöåíêà âëèÿíèÿ àíòèáèîòèêà íà in vitro
êóëüòèâèðóåìûé ðàñòèòåëüíûé îáúåêò; íåèçáåæíî ïîÿâëåíèå áàêòåðèàëüíûõ øòàììîâ, ðåçèñòåíòíûõ ê ïðèìåíÿåìîìó àíòèáèîòèêó; àíòèáèîòèêè
âûçûâàþò ïîâðåæäåíèå õëîðîïëàñòîâ è ìèòîõîíäðèé, ÷òî ïðîÿâëÿåòñÿ â
õëîðîçå è ìîðôîëîãè÷åñêèõ èçìåíåíèÿõ ýêñïëàíòîâ (6, 9). Óñïåõè àíòèáàêòåðèàëüíîé òåðàïèè â êóëüòóðå òêàíåé ðàñòåíèé â çíà÷èòåëüíîé ñòåïåíè áóäóò îïðåäåëÿòüñÿ ðàçâèòèåì èññëåäîâàíèé â ýòîé îáëàñòè è ðàçðàáîòêîé íîâûõ êëàññîâ àíòèáèîòèêîâ.
Èòàê, òàêñîíîìè÷åñêîå ðàçíîîáðàçèå ñêðûòîé (ýíäîôèòíîé) áàêòåðèàëüíîé ìèêðîôëîðû â in vitro ðàñòèòåëüíûõ êóëüòóðàõ âåëèêî è ìîæåò
âêëþ÷àòü ôîðìû êàê ñ îòðèöàòåëüíûì, òàê è ñ ïîëîæèòåëüíûì âëèÿíèåì
íà êîëîíèçèðóåìûå èìè ðàñòèòåëüíûå îáúåêòû. Ïî ìåðå êóëüòèâèðîâàíèÿ
ðàñòèòåëüíîãî ìàòåðèàëà ìîæåò èçìåíÿòüñÿ òèòð, ñîñòàâ áàêòåðèàëüíûõ
àññîöèàöèé, ñïîñîáíîñòü ê êóëüòèâèðîâàíèþ. Ïðîáëåìà ïîëó÷åíèÿ ãíîòîáèîòè÷åñêèõ êóëüòóð (â ÷àñòíîñòè, â in vitro êîëëåêöèÿõ ãåíåòè÷åñêèõ áàíêîâ ðàñòåíèé) ñâÿçàíà ñî ñëîæíîñòüþ âûÿâëåíèÿ è ýëèìèíàöèè áàêòåðèàëüíîé ìèêðîôëîðû.
Àâòîðû âûðàæàþò ãëóáîêóþ áëàãîäàðíîñòü Ò.À. Ãàâðèëåíêî (Âñåðîññèéñêèé
ÍÈÈ ðàñòåíèåâîäñòâà èì. Í.È. Âàâèëîâà) è Â.È. Ñàôðîíîâîé (Âñåðîññèéñêèé ÍÈÈ
ñåëüñêîõîçÿéñòâåííîé ìèêðîáèîëîãèè) çà öåííûå çàìå÷àíèÿ ïðè îáñóæäåíèè ìàòåðèàëà, èçëîæåííîãî â ñòàòüå.
ËÈÒÅÐÀÒÓÐÀ
1.
2.
10
L e i f e r t C., W o o d w a r d S. Laboratory contamination management: the requirement for
microbiological quality assurance. Plant Cell Tiss. Cult., 1998, 52: 85-88 (doi: 1023/A:1005905604043).
L e i f e r t C., C a s s e l l s A.C. Microbial hazards in plant tissue and cell cultures. In Vitro
Cell. Dev. Biol. Plant., 2001, 37(2): 133-138 (doi: 10.1079/IVP2000129).
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
C a s s e l l s A.C. Contamination and its impact in tissue culture. Acta Hort., 2001, 560: 353-359.
L e i f e r t C., W a i t e s W.M., N i c o l a s J.R. Bacterial contamination of micropropagated
plant tissue cultures. J. Appl. Bact., 1989, 67: 353-361.
C a s s e l l s A.C. Problems in tissue culture: culture contamination. In: Micropropagation
technology and application /P.C. Debergh, R.H. Zimmerman (eds.). Kluwer Acad. Publishers,
1991: 31-44.
R e e d B.M., M e n t z e r J., T a n p r a s e r t P., Y u X. Internal bacterial contamination of
micropropagated hazelnut: identification and antibiotic treatment. Plant Cell Tiss. Cult., 1998,
52: 67-70.
T h o m a s P. A three-step screening procedure for the detection of covert and endophytic bacteria in plant tissue cultures. Current Sci., 2004, 87: 67-72.
P a n i c k e r B., T h o m a s P., J a n a k i r a m T., V e n u g o p a l a n R., N a r a y a n a p p a S.B. Influence of cytokinin levels on in vitro propagation of shy suckering chrysanthemum
«Arka Swarna» and activation of endophytic bacteria. In vitro Cell Dev. Biol. Plant, 2007,
43(6): 614-622 (doi: 10.1007/s11627-007-9061-6).
R e e d B.M., T a n p r a s e r t P. Detection and control of bacterial contaminants of plant tissue cultures. A review of recent literature. Plant Tissue Culture &. Biotechnology, 1995, 1(3):
137-142.
T a n p r a s e r t P., R e e d B.M. Detection and identification of bacterial contaminants from
strawberry runner explants. In vitro Cell Dev. Biol. Plant, 1997, 33: 221-226.
Á ó ð ã ó ò è í À.Á., Ô å î ê ò è ñ ò î â à Í.Â., Ï ó í è í à Í.Â., È ã í à ò î â À.Í. Îïðåäåëåíèå âèäîâîé ïðèíàäëåæíîñòè áàêòåðèé, êîíòàìèíèðóþùèõ êóëüòóðó äðåâåñíûõ ðàñòåíèé in vitro. Òåç. äîêë. IX Ìåæä. êîíô. «Áèîëîãèÿ êëåòîê ðàñòåíèé in vitro è áèîòåõíîëîãèÿ». Çâåíèãîðîä, 2008: 60-61.
L e i f e r t C., M o r r i s C., W a i t e s W.M. Ecology of microbial saprophytes and pathogens
in tissue cultured and field grown plants. CRC Crit. Rev. Plant Sci., 1994, 13: 139-183 (doi:
10.1080/713608058).
T h o m a s P., S w a r n a G.K., R o y P.K., P r a k a s h P. Identification of culturable and
originally non-culturable endophytic bacteria isolated from shoot tip cultures of banana cv.
Grand Naine. Plant Cell Tiss. Org. Cult., 2008, 93: 55-63 (doi: 10.1007/s11240-008-9341-9).
R a f f e r t y S.M., W i l l i a m s S., F a l k i n e r F.R., C a s s e l s A.C. Persistence in in vitro
cultures of cabbage (Brassica oleracea var. capitata L.) of human food poisoning pathogens: Esherichia
coli and Serratia marcescens. Proc. Int. Symp. on Meth. and Marks. for Qual. Assur. in Micropropagation /A.C. Cassels, B.M. Doyle, R.F. Curry (eds.). Acta Hort., 2000, 530(ISHS): 145-151.
P i r t t i l ä A.M., L a u k k a n e n H., P o s p i e c h H., M y l l y l a R., H o h t o l a A. Detection of intracellular bacteria in the buds of Scots pine (Pinus sylvestris L.) by in situ hybridization. Appl. Environ. Microbiol., 2000, 66: 3073-3077 (doi: 10.1128/AEM.66.7.3073-3077.2000).
P i r t t i l ä A.M. Endophytes in the buds of Scots pine (Pinus sylvestris L.). Doc. Thesis. Acta
Univ. Ouluensis, Ser. A Sci. Rerum Nat., 2001, 36: 1-55.
P i r t t i l ä A.M., J o e n s u u P., P o s p i e c h H., J a l o n e n J., H o h t o l a A. Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology
and mitigate browning of callus cultures. Physiologia Plantarum, 2004, 121: 305-312 (doi:
10.1111/j.0031-9317.2004.00330.x).
P i r t t i l ä A.M., P o d o l i c h O., K o s k i m ä k i J.J., H o h t o l a E., H o h t o l a A. Role
of origin and endophyte infection in browning of bud-derived tissue cultures of Scots pine (Pinus
sylvestris L.). Plant Cell Tiss. Org. Cult., 2008, 95: 47-55 (doi: 10.1007/s11240-008-9413-x).
K a m o u n R., L e p o i v r e P., B o x u s P. Evidence for the occurrence of endophytic prokaryotic contaminants in micropropagated plantlets of Prunus cerasus cv. Montmorency. In:
Pathogen and microbial management in micropropagation /A.C. Cassells (ed.). Kluwer Acad.
Publishers, Dordrecht, 1997: 145-148.
D e A l m e i d a C.V., A n d r e o t e F.D., Y a r a R., T a n a k a F.A.O., A z e v e d o J.L., De
A l m e i d a M. Bacteriosomes in axenic plants: endophytes as stable endosymbionts. Microbiol
Biotech., 2009, 25: 1757-1764 (doi: 10.1007/s11274-009-0073).
T h o m a s P., S w a r n a G.K., P a t i l P., R a w a l R.D. Ubiquitous presence of normally
non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to
quiescent cultivable form in tissue cultures. Plant Cell Tiss. Org. Cult., 2008, 93: 39-54 (doi:
10.1007/s112-40-008-9340-x).
H a b i b a U., R e z a S., S a h a M.L., K h a n M.R., H a d i u z z a m a n S. Endogenous
bacterial contamination during in vitro culture of Banana: identification and prevention. Plant
Tiss. Cult., 2002, 12: 117-124.
R y a n R.P., G e r m a i n e K., F r a n k s A., R y a n D., D o w l i n g D.N. Bacterial endophytes: recent developments and applications. FEMS Microbiol. Lett., 2008, 278(1): 1-9.
S a e n s E., B o r d a C., R e n a t a L., D a S i l v a M.G., P a d i l l a G. Searching for new
antibiotics in endophytic microorganisms. Proc. 2nd Int. Symp. on Biological Control of Bacterial Plant Diseases. Orlando, FL, USA, 2008: 71 (doi: 10.11 11/j.1574-6982.2007.00918.x).
Ì à ð ê î â à Þ.À., Ð î ì à í å í ê î À.Ñ., Ä ó õ à í è í à À.Â. Âûäåëåíèå áàêòåðèé ñåìåéñòâà Enterobacteriaceae èç ðàñòèòåëüíûõ òêàíåé. Ìèêðîáèîëîãèÿ, 2005, 74(5): 663-666.
11
26. L a c r o i x B., T z f i r a T., V a i n s t e i n A., C i t o v s k y V. A ñase of promiscuity:
Agrobacterium’s endless hunt for new partners. Trends Genet., 2006, 22: 29-37 (doi:
10.1016/j.tig.2005.10.004).
27. B u l g a k o v V.P., K i s e l e v K.V., Y a k o v l e v K.V., Z h u r a v l e v Y.N., G o n t c h a r o v A.A., O d i n t s o v a N.A. Agrobacterium-mediated transformation of sea urchin
embryos. Biotechnol. J., 2006, 1: 454-461 (doi: 10.1002/biot.200500045).
28. Ò è õ î í î â è ÷ È.À., Ï ð î â î ð î â Í.À. Ñåëüñêîõîçÿéñòâåííàÿ ìèêðîáèîëîãèÿ êàê îñíîâà ýêîëîãè÷åñêè óñòîé÷èâîãî àãðîïðîèçâîäñòâà: ôóíäàìåíòàëüíûå è ïðèêëàäíûå àñïåêòû. Ñåëüñêîõîçÿéñòâåííàÿ áèîëîãèÿ, 2011, 3: 3-9.
29. T h o m a s P. In vitro decline in plant cultures: detection of a legion of covert bacteria as the
cause for degeneration of long-term micropropagated triploid watermelon cultures. Plant Cell
Tiss. Org. Cult., 2004, 77: 173-179 (doi: 10.1023/B:TICU.0000016824.09108.c8.23).
30. H o l l a n d M.A., P o l a c c o J.C. PPFMs and other covert contaminants: is there more
to plant physiology than just plant? Annu. Rev. Plant Physiol., 1994, 45: 197-209 (doi:
10.1146/annurev.pp.45.060194.001213).
31. H o r s c h R.B., K i n g J. A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium sp. from cultures of Datura innoxia (Mill.). Plant Cell Tiss. Org. Cult., 1983, 2: 21-28.
32. H a l l m a n n J., Q u a d t - H a l l m a n n A., M a h a f t e e W.F. Endophytic bacteria in agricultural crops. Can. J. Microbiol., 1997, 43: 895-914.
33. B a l d a n i J.I., C a r u s o L., B a l d a n i V.L.D., G o i S.R., D o b e r e i n e r J. Recent advances in BNF with non-legume plants. Soil Biol. Biochem., 1997, 29: 911-922.
34. U l r i c h K., U l r i c h F., E w a l d D. Diversity of endophytic bacterial communities in poplar grown under field conditions. FEMS Microbiol. Ecol., 2008, 63: 169-180 (doi: 1111/15746941.2007.00419.x).
35. V a n D o o r n W.G., D e S t i g t e r H.C.M., D e W i t t e Y., B o e k e s t e i n A. Microorganisms at the cut surface and in xylem vessels of rose stems: a scanning electron microscope
study. J. Appl. Bact., 1991, 70: 34-39.
36. T h o m a s P. Reemergence of covert bacteria Bacillus pumilus and Brevibacillus sp. in microbe-freed grape and watermelon stocks attributable to occasional autoclaving defying residual spores from previous cycles. Plant Cell Tiss. Org. Cult., 2006, 87: 155-165 (doi:
10.1007/s11240-006-9150-y).
37. T h o m a s P. Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus
sp. as a covert contaminant in grape tissue cultures. J. Appl. Microbiol., 2006, 101: 764-774
(doi: 10.1111/j.1365-2672.2006.02993.x).
38. L a u k k a n e n H., S o i n i H., K o n t u n e n - S o p p e l a S., H o h t o l a A., V i l j a n e n M.
A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth
of Scots pine seedlings. Tree Physiol., 2000, 20(13): 915-920.
39. P e n c e V.C., S a n d o v a l J.A. Controlling contamination during in vitro collection. In: In vitro
collection techniques for germplasm conservation /V.C. Penke, J.A. Sandoval, V.M. Villalobos,
F. Engelmann (eds.). IPGRI Technical Bulletin (Rome, Italy), 2002, 7: 30-40.
40. R o y A., S a h a P.K. Factors involved during in vitro culture of Calamus rotang. J. Trop. For.
Sci., 1997, 10: 225-232.
41. S m i t h R.H., B u r r o w s J., K u r t e n K. Challenges associated with micropropagation of Zephyranthes and Hippeastrum sp. (Amaryllidaceae). In vitro Cell Dev. Biol. Plant., 1999, 35: 281-282.
42. B u c k l e y P.M., D e W i l d e Å.N., R e e d B.M. Characterization and identification of bacteria isolated from micropropagated mint plants. In vitro Cell Dev. Biol. Plant, 1995, 31: 58-64.
43. R e e d B.M., B u c k l e y P.M. Tissue Culture Contaminants Handbook. USDA-ARS, Corvallis, OR. 1999 (Lab manual).
44. S i n g h a S., B i s s o n n e t t e G.K., D o u b l e M.L. Methods for sterilizing instruments contaminated with Bacillus sp. from plant tissue cultures. Hort. Sci., 1987, 22: 659.
45. S t e a d D.E., E l p h i n s t o n e J.G., W e l l e r S., S m i t h N., H e n n e s s y J. Modern
methods for characterizing, identification and detecting bacteria associated with plants. Acta Hort.,
2000, 530: 45-57.
46. Îïðåäåëèòåëü áàêòåðèé Áåðäæè (ïåð. ñ àíãë.) /Ïîä ðåä. Äæ. Õîóëòà, Í. Êðèãà, Ï. Ñíèòà,
Äæ. Ñòåéëè, Ñ. Óèëüÿìñà. Ò. 1. Ì., 1997.
47. http://www.arb-silva.de
48. G r e i z e n K., L o e f f e l h o l z M., P u r o h i t A., L e o n g D. PCR primers and probes for
the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol., 1994, 32: 335-351.
49. W i l s o n K.H. Detection of culture-resistant bacterial pathogens by amplification and sequencing of ribosomal DNA. Clin. Infect. Dis., 1994, 18: 958-962 (doi: 10.1093/clinids/18.6.958).
50. G a r c i a - M a r t i n e z J., A c i n a s S.G., R o d r i g u e s - V a l e r e F. Use of the 16S-23S ribosomal genes spacer region in studies of prokaryotic diversity. J. Microbiol. Meth., 1999, 36: 55-64.
51. T h o m a s P., S o l y T.A. Endophytic bacteria associated with growing shoot tips of banana
(Musa sp.) cv. Grand Naine and the affinity of endophytes to the host. Microb. Ecol., 2009,
58(4): 952-964 (doi: 10.1007/s00248-009-9559-z).
52. Ñ à ô ð î í î â à Â.È., × è æ å â ñ ê à ÿ Å.Ï., Á å ë è ì î â À.À., Ï à â ë î â à Å.À. Îïðåäåëåíèå òàêñîíîìè÷åñêîãî ïîëîæåíèÿ ìèêðîñèìáèîíòîâ êîïåå÷íèêà (Hedysarum) è àñòðà-
12
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
ãàëà (Astragalus) íà îñíîâå àíàëèçà ãåíîâ ðèáîñîìàëüíûõ ÐÍÊ. Ñåëüñêîõîçÿéñòâåííàÿ
áèîëîãèÿ, 2011, 3: 61-64.
http://www.ncbi.nlm.nih.gov/
Ï è í å â è ÷ À.Â. Ìèêðîáèîëîãèÿ. Áèîëîãèÿ ïðîêàðèîòîâ. Ò. 1. Ñïá, 2007.
L e i f e r t C. Quality assurance systems for plant cell and tissue culture: The problem of latent
persistence of bacterial pathogens and Agrobacterium-based transformation vector systems. Acta
Hort., 2000, 530: 87-91.
E w a l d D., Z a s p e l I., N a u j o k s G., B e h r e n d t U. Endogenous bacteria in tissue cultures of conifers — appearance and action. Acta Hort., 2000, 530: 137-143.
T h o m a s P., P a n i c k e r B., J a n a k i r a m T., S a t h y a n a r a y a n a B.N. In vitro
propagation of «Arka Ravi» chrysanthemum on growth regulator-free medium harboring endophytic bacteria. J. Hortic. Sci. Biotech., 2009, 84(6): 653-659.
Î ñ ë å ä ê è í Þ.Ñ., Ë å â ÷ ó ê Ñ.Ñ., Î ã î ð î ä í è ê î â à Â.Ô., Ä ó í à å â à Ñ.Å., Ë ó ï û ø å â à Þ.Â., Î ð ë î â à Ñ.Þ., Ï à ç î â à Ç.Õ., Ò ð ó ñ ê è í î â Ý.Â., Ã à â ð è ë å í ê î Ò.À.
Áàêòåðèàëüíûå èíôåêöèè â êóëüòóðå in vitro ðàñòåíèé. Ìåæä. íàó÷. êîíô. «Ìîëåêóëÿðíàÿ ãåíåòèêà, ãåíîìèêà è áèîòåõíîëîãèÿ». Ìèíñê, 2004: 173-174.
Ä ó í à å â à Ñ.Å., Ï å í ä è í å í Ã.È., À í ò î í î â à Î.Þ., Ø â à ÷ ê î Í.À., Â î ë ê î â à Í.Í., Ã à â ð è ë å í ê î Ò.À. Ñîõðàíåíèå âåãåòàòèâíî ðàçìíîæàåìûõ êóëüòóð â in vitro
è êðèîêîëëåêöèÿõ. Ìåòîä. óêàç. /Ïîä ðåä. Ò.À. Ãàâðèëåíêî. ÑÏá, 2011.
C o o k e D.L., W a i t e s W.M., L e i f e r t C. Effects of Agrobacterium tumefaciens, Erwinia
carotovora, Pseudomonas syringae and Xanthomonas campestris on plant tissue culture of Aster,
Cheiranthus, Delphinium, Iris and Rosa: disease development in vivo as results of latent infection
in vitro. J. Plant Dis. Protect., 1992, 99: 469-481.
T h o m a s P., P r a b h a k a r a B.S., P i t c h a i m u t h u M. Cleansing the long-term micropropagated triploid watermelon cultures from covert bacteria and field testing the plants for
clonal fidelity and fertility during the 7-10 year period in vitro. Plant Cell Tiss. Org. Cult., 2006,
85: 317-329 (doi: 10.1007/s11240-006-9083-5).
M i s r a P., G u p t a N., T o p p o D.D., P a n d e y V., M i s h r a M.K., T u l i R. Establishment of long-term proliferating shoot cultures of elite Jatropha curcas L. by controlling
endophytic bacterial contamination. Plant Cell Tiss. Org. Cult., 2010, 100: 189-197 (doi:
10.1007/s11240-009-9636-5).
L e i f e r t C., W a i t e s W.M., C a m o t t a H. Lactobacillus plantarum: a deleterious contaminant of plant tissue cultures. J. Appl. Bact., 1989, 67: 363-370.
D i a s A.C.F., C o s t a F.E.C., A n d r e o t e F.D., L a c a v a P.T., T e i x e i r a M.A., A s s u m p ç ã o L.C., A r a ú j o W.L., A z e v e d o J.L., M e l o I.S. Isolation of micro propagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion. World J. Microbiol. Biotechnol., 2009, 25: 189-195 (doi: 10.1007/s11274-008-9878-0).
Ï î ë ÿ ê î â À.Â., × è ê ð è ç î â à À.Ô., Ê à ë ÿ å â à Ì.À., Ç à õ à ð ÷ å í ê î Í.Ñ., Á à ë î õ è í à Í.Â., Á ó ð ü ÿ í î â ß.È. Òðàíñôîðìàöèÿ ðàñòåíèé ëüíà-äîëãóíöà. Ôèçèîëîãèÿ ðàñòåíèé, 1998, 45(4): 882-887.
Ê à ë ÿ å â à Ì.À., Ç à õ à ð ÷ å í ê î Í.Ñ., Ä î ð î í è í à Í.Á., Ð ó ê à â ö î â à Å.Á., È â à í î â à Å.Ã., À ë å ê ñ å å â à Å.Å., Ò ð î ö å í ê î Þ.À., Á ó ð ü ÿ í î â ß.È. Ñòèìóëÿöèÿ
ðîñòà è ìîðôîãåíåçà ðàñòåíèé in vitro àññîöèàòèâíûìè ìåòèëîòðîôíûìè áàêòåðèÿìè.
Ôèçèîëîãèÿ ðàñòåíèé, 2001, 48(4): 596-599 (doi: 10.1023/A:1016715800238).
Ê à ë ÿ å â à Ì.À., Ä î ð î í è í à Í.Á., È â à í î â à Å.Ã., Ò ð î ö å í ê î Þ.À., Á ó ð ü ÿ í î â ß.È. Ïðèìåíåíèå àýðîáíûõ ìåòèëîáàêòåðèé è ìåòàíîòðîôîâ äëÿ èíäóêöèè ìîðôîãåíåçà ïøåíèöû ìÿãêîé (Triticum aestivum L.) in vitro. Áèîòåõíîëîãèÿ, 2003, 2: 38-44.
Ø è ð î ê è õ È.Ã., Ø ó ï ë å ö î â à Î.Í., Ø è ð î ê è õ À.À. Îöåíêà âëèÿíèÿ ìåòèëîòðîôíûõ áàêòåðèé íà ðàñòåíèÿ in vitro. Äîêëàäû Ðîññèéñêîé àêàäåìèè ñåëüñêîõîçÿéñòâåííûõ íàóê, 2007, 5: 23-25.
M c F a l l - N g a i M. Unseen forces: the influence of bacteria on animal development. Devel.
Biol., 2002, 242: 1-14 (doi: 10.1006/dbio.2001.0522).
M u r t h y B.N.S., V e t t a k k o r u m a k a n k a v N.N., K r i s h n a R a j S., O d u m e r u J.,
S a x e n a P. Characterization of somatic embryogenesis in Pelargonium ½ hortorum mediated
by a bacterium. Plant Cell Rep., 1999, 18: 607-613.
L u n a C., C o l l a v i n o M., M r o g i n s k i L., S a n s b e r r o P. Identification and control
of bacterial contaminants from Ilex dumosa nodal segments culture in a temporal immersion
bioreactor system using 16S rDNA analysis. Plant Cell Tiss. Org. Cult., 2008, 95(1): 13-19.
L a t a A.H., L i X.C., S i l v a B., M o r a e s R.M., H a l d a - A l i j a L. Identification of
IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA
sequencing. Plant Cell Tiss. Org. Cult., 2006, 85(3): 353-359 (doi: 10.1007/s11240-006-9087-1).
T h o m a s P., K u m a r i S., S w a r n a G.K., G o w d a T.K.S. Papaya shoot tip associated
endophytic bacteria isolated from in vitro cultures and host-endophyte interaction in vitro and
in vivo. Can. J. Microbiol., 2007, 3(3): 380-390 (doi: 10.1139/WO6-141).
I s e n e g e r D.A., T a y l o r P.W.J., M u l l i n s K., M c G r e g o r G.R., B a r l u s M.,
H o l c h i n s o o J.F. Molecular detection of a bacterial contaminant Bacillus pumilus
in symptomless potato plant tissue cultures. Plant Cell Rep., 2003, 21(8): 814-820 (doi:
13
10.1007/s00299-003-0583-z ) .
75. T h o m a s P., K u m a r i S. Inconspicuous endophytic bacteria mimicking latex exudates in shoottip cultures of papaya. Sci. Hort., 2010, 124(4-1): 469-474 (doi: 10.1016/j.scienta.2010.02.013).
76. L i u T.-H., H s u N.-W., W u R.-Y. Control of leaf-tip necrosis of micropropagated ornamental
statice by elimination of endophytic bacteria. In vitro Cell. Devel. Biol. Plant, 2005, 41(4): 546549 (doi: 10.1079/IVP2005673).
77. A b r e u - T a r a z i M.F., N a v a r r e t e A.A., A n d r e o t e F.D., A l m e i d a C.V., T s a i S.M.,
A l m e i d a M. Endophytic bacteria in long-term in vitro cultivated «axenic» pineapple
microplants revealed by PCR–DGGE. J. Microbiol. Biotechnol., 2010, 26(3): 555-560
(doi: 10.1007/s11274-009-0191-3).
78. K u l k a r n i A.A., K e l k a r S.M., W a t v e M.G., K r i s h n a m u r t h y K.V. Characterization and control of endophytic bacterial contaminants in in vitro cultures of Piper spp., Taxus
baccata subsp. wallichiana and Withania somnifera. Can. J. Microbiol., 2007, 53: 63-74 (doi:
10.1139/WO6-106).
79. F a l k i n e r F.R. Antibiotics and antibiotic resistance associated witch plants, fruits and vegetables. Acta Hort., 2000, 530: 83-86.
80. S e c k i n g e r G.R., T o r r e s K.C. Physical and chemical means of controlling contamination in
plant tissue culture. Proc. World Congress on in vitro Biol. S. Francisco, California, USA, 2004
(http://www.phytotechlab.com/pdf/SIVBMay2004.pdf).
1ÃÍÓ
Âñåðîññèéñêèé ÍÈÈ ðàñòåíèåâîäñòâà
èì. Í.È. Âàâèëîâà Ðîññåëüõîçàêàäåìèè,
Ïîñòóïèëà â ðåäàêöèþ
8 îêòÿáðÿ 2013 ãîäà
190000 Ðîññèÿ, ã. Ñàíêò-Ïåòåðáóðã, óë. Áîëüøàÿ Ìîðñêàÿ, 42,
e-mail: dunaevase@mail.ru;
2ÃÍÓ Âñåðîññèéñêèé ÍÈÈ ñåëüñêîõîçÿéñòâåííîé
ìèêðîáèîëîãèè Ðîññåëüõîçàêàäåìèè,
196608 Ðîññèÿ, ã. Ñàíêò-Ïåòåðáóðã—Ïóøêèí, ø. Ïîäáåëüñêîãî, 3
Sel’skokhozyaistvennaya biologiya [Agricultural Biology], 2015, V. 50, ¹ 1, pp. 3-15
BACTERIAL MICROORGANISMS ASSOCIATED WITH THE PLANT
TISSUE CULTURE: IDENTIFICATION AND POSSIBLE ROLE
(review)
S.E. Dunaeva1, Yu.S. Osledkin2
1N.I.
Vavilov All-Russian Institute of Plant Industry, Russian Academy of Agricultural Sciences, 42, ul. Bol’shaya
Morskaya, St. Petersburg, 190000 Russia, e-mail dunaevase@mail.ru;
2All-Russian Research Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, 3, sh.
Podbel’skogo, St. Petersburg, 196608 Russia
Received October, 8, 2013
doi: 10.15389/agrobiology.2015.1.3eng
Abstract
Effective sterilization of plant explants and antiseptics rules compliance do not exclude the
presence of so-called covert (endophytic) bacteria in in vitro cultures. But the role of these bacteria
in tissues cultures has been not enough studied whereas it was related to the explants regeneration
capacity and the possibility of animal and human cells transformation under in vitro cultivation.
Bacterial strains pathogenic to humans can be stably maintained in cultivated tissues and ex vitro
plants. The broadening of bacterial environments creates ecological and genetic risks leading to necessity of careful monitoring of endophytic communities in plants used as raw food and at use of in
vitro technologies in practical plant growing and food production. Identification of bacterial microorganisms colonizing in vitro plant cultures allows studying the bacteria effect on the host, realizing
special chemotherapy and developing the microorganisms’ databases. Two methods of identification
are the most widespread: more available traditional one that does not allow detecting non-cultured
forms (its base is the use of cultural and morphological characteristics as well as chemical and biochemical reactions) and molecular-genetic one. At the second approach different 16S-rRNA sequences are studied using metagenomic DNA and appropriate specific primers; these sequences have
conserved sites identical for all prokaryotes and variable ones suitable for species specific regions
identification. Internal transcribed spacers (ITS) are being mainly used to distinguish the microorganisms at the species level and even at strains one. Taxonomy of in vitro cultures’ bacterial endophytes
indicates to their diversity and absence of specific composition as for cultures of plants belonging to
different taxa as for different plant organs explants. Among identified endophytic bacteria potentially
useful for intact plants Streptomycete, Pantoea agglomerans and others were found as well as those
pathogenic for humans, e.g. Ralstonia mannitolytica, Staphylococcus epidermidis, Corynebacterium
amycolatum, Bacillus neonatiensis, Salmonella and Nocaridia spp. At in vitro plant cultivation durable
symptomless bacterial presence is caused on the one hand by bacterial growth repression with factors
14
accompanying plant explants cultivation (PH, temperature below bacterial optimum, activation of the
defense mechanisms), and on the other hand by simultaneous bacteria support due to exudates secreted
by plant explants. The rapid bacterial cells proliferation can begin even at small changes in initial
conditions, at increase in plant exudates concentrations and per se in consequence of in vitro cultivation as a stress at the absence of whole organism regulatory role. As the number of subcultivations
increases a portion of plant cultures with latent bacterial contamination increases too; no-cultured
endophytes have been reported to acquire the status of cultured ones. Covert bacterial contamination
could depress regeneration, micropropagation, cause death of in vitro cultivated objects, restrict the
protocols repeatability and concern induction of epigenetic somaclonal variability. For instance
Acinetobacter and Lactobacillus plantarum filtrates extracted from degrading calluses strongly reduced
shoot regeneration at inoculation in explants or addition into a medium; bacteria Mycobacterium
obuense and M. aichiense repressed seeds development in in vitro cultures. The article accents the
problem of gnotobiological plant cultures (specifically in in vitro collections of plants genetic banks) development caused by difficulties in identification and elimination of bacterial microorganisms.
Êeywords: plant tissue culture, bacterial microorganisms, antibacterial therapy.
Íàó÷íûå ñîáðàíèÿ
VIII ÌÎÑÊÎÂÑÊÈÉ ÌÅÆÄÓÍÀÐÎÄÍÛÉ ÊÎÍÃÐÅÑÑ
«ÁÈÎÒÅÕÍÎËÎÃÈß: ÑÎÑÒÎßÍÈÅ È ÏÅÐÑÏÅÊÒÈÂÛ
ÐÀÇÂÈÒÈß»
XIII ÌÅÆÄÓÍÀÐÎÄÍÀß ÑÏÅÖÈÀËÈÇÈÐÎÂÀÍÍÀß
ÂÛÑÒÀÂÊÀ «ÌÈÐ ÁÈÎÒÅÕÍÎËÎÃÈÈ-2015»
(17-20 ìàðòà 2015 ãîä, ã. Ìîñêâà, Íîâûé Àðáàò, 36/9)
Îðãàíèçàòîðû êîíãðåññà è âûñòàâêè:
Ìèíèñòåðñòâî îáðàçîâàíèÿ è íàóêè Ðîññèéñêîé Ôåäåðàöèè, Ìèíèñòåðñòâî ïðîìûøëåííîñòè è òîðãîâëè Ðîññèéñêîé Ôåäåðàöèè, Ôåäåðàëüíîå àãåíòñòâî íàó÷íûõ îðãàíèçàöèé,
Ðîññèéñêàÿ àêàäåìèÿ íàóê, Ðîññèéñêèé ôîíä ôóíäàìåíòàëüíûõ èññëåäîâàíèé, Ðîññèéñêèé ñîþç õèìèêîâ, ÇÀÎ «Ýêñïî-áèîõèì-òåõíîëîãèè».
Îñíîâíûå òåìàòè÷åñêèå íàïðàâëåíèÿ:
Ôóíäàìåíòàëüíûå èññëåäîâàíèÿ è áèîòåõíîëîãèÿ
Áèîòåõíîëîãèÿ è ìåäèöèíà
Áèîìåäèöèíñêèå ñåíñîðû è òåõíîëîãèè
Èììóííàÿ áèîòåõíîëîãèÿ
Ñåëüñêîõîçÿéñòâåííàÿ áèîòåõíîëîãèÿ:
•
Áèîòåõíîëîãèÿ â ñåëåêöèè è ðàñòåíèåâîäñòâå
•
Ðåãóëÿöèÿ ýêñïðåññèè ãåíîâ è ïðîäóêòèâíîñòü ñåëüñêîõîçÿéñòâåííûõ îðãàíèçìîâ
•
Áèîòåõíîëîãèÿ ãåíåòè÷åñêè èíòåãðèðîâàííûõ íàäîðãàíèçìåííûõ ñèñòåì
•
Ìèêðîáíî-ðàñòèòåëüíûå ñèñòåìû è èõ áèîòåõíîëîãèÿ
•
Ìîëåêóëÿðíàÿ äèàãíîñòèêà áîëåçíåé ðàñòåíèé è æèâîòíûõ
•
Áèîòåõíîëîãèÿ â âîñïðîèçâîäñòâå öåííûõ ïîðîä ðûá
Ñîâðåìåííûå ïðîáëåìû æèâîòíîâîäñòâà è áèîòåõíîëîãèè èõ ðåøåíèÿ (êðóãëûé ñòîë)
Ëåñíàÿ áèîòåõíîëîãèÿ: îò èññëåäîâàíèé ê èííîâàöèÿì (êðóãëûé ñòîë)
Áèîòåõíîëîãèÿ è ïðîìûøëåííîñòü
Áèîìàòåðèàëû è èõ ðîëü â ñîâðåìåííûõ áèîòåõíîëîãèè è ìåäèöèíå
Áèîòåõíîëîãèÿ ïèùè. Ïðîäóêòû çäîðîâîãî ïèòàíèÿ
Áèîêàòàëèç è áèîêàòàëèòè÷åñêèå òåõíîëîãèè
Áèîòåõíîëîãèÿ â ðåøåíèè ïðîáëåì îõðàíû îêðóæàþùåé ñðåäû
Áèîãåîòåõíîëîãèÿ
Áèîýíåðãåòèêà — âàæíûé ôàêòîð â ðåøåíèè ýêîëîãè÷åñêèõ è ýêîíîìè÷åñêèõ ïðîáëåì
Áèîèíôîðìàòèêà
Òåìàòèêà âûñòàâêè: Ïðîöåññû, àïïàðàòû, ëàáîðàòîðíî-àíàëèòè÷åñêîå îáîðóäîâàíèå, äèàãíîñòè÷åñêèå íàáîðû, áèî÷èïû è áèîñåíñîðû äëÿ áèîòåõíîëîãè÷åñêèõ ïðîèçâîäñòâ è ëàáîðàòîðíûõ èññëåäîâàíèé. Êîìïüþòåðíûå òåõíîëîãèè. Âåñü ñïåêòð áèîïðîäóêòîâ äëÿ ôàðìàöåâòè÷åñêîé è ïèùåâîé ïðîìûøëåííîñòåé, àãðîïðîìûøëåííîãî êîìïëåêñà, âåòåðèíàðèè, ãåîëîãèè, ïðîìûøëåííûõ ïðîèçâîäñòâ, à òàêæå áèîàãåíòû äëÿ îõðàíû è âîññòàíîâëåíèÿ îêðóæàþùåé ñðåäû. Áèîëîãè÷åñêè àêòèâíûå äîáàâêè. Òåñò-ñèñòåìû äëÿ îïðåäåëåíèÿ àëêîãîëÿ è íàðêîòè÷åñêèõ âåùåñòâ. Áèîêàòàëèç è áèîêàòàëèòè÷åñêèå òåõíîëîãèè. Ïèòàòåëüíûå ñðåäû. Áèîïðåïàðàòû äëÿ ìåäèöèíû è êîñìåòîëîãèè, à òàêæå ãîòîâûå ïðîäóêòû
íà èõ îñíîâå. Àëüòåðíàòèâíûå èñòî÷íèêè ýíåðãèè, íàíîìîëåêóëÿðíûå ïðåîáðàçîâàòåëè
ýíåðãèè. Ïðîìûøëåííàÿ è ëàáîðàòîðíàÿ áåçîïàñíîñòü.
Êîíòàêòû è èíôîðìàöèÿ: http://www.mosbiotechworld.ru,
e-mail: aleshnikova@mosbiotechworld.ru, atv@biomos.ru, ser@biomos.ru
15