Allelopathic effects of Chenopodium murale L. hairy roots in vitro

Korenov sistem C. murale produkuje i luči hemijske materije koje imaju alelopatsku aktivnost. Proučavanje efekata na druge organizme, kao i izolovanje i karakterizacija metabolita koji su izlučeni iz korenova u zemljište je veoma kompleksan proces. Zbog toga je cilj ovog istraživanja bio uspostavljanje kulture transformisanih korenova C. murale in vitro, kao novog sistema pogodnog za izučavanje alelopatskog efekta. Transgeni korenovi su indukovani pomoću Agrobacterium rhizogenes A4M70GUS na eksplantatima korenova, kotiledona, listova i internodija klijanaca C. murale. Eksplantati korenova su imali najveću efikasnost transformacije (11,1%). Uspostavljeni su klonovi transformisanih korenova različite morfologije i potencijala rasta koji se gajenjem (subkultivisanjem) u tečnom MS medijumu održavaju više od dve godine. Potvrda transformacije ovih klonova je urađena pomoću PCR, RT–PCR i histohemijske GUS analize. Nije ustanovljena razlika u prisustvu rol gena među klonovima. Ispitivan je alelopatski efekat tečnih medijuma u kojima su rasli transformisani i netransformisani korenovi C. murale (fitotoksični medijum) na klijanje i rast klijanaca test biljaka arabidopsisa (Arabidopsis thaliana (L.) Heynh), pšenice (Triticum aestivum L.) i salate (Lactuca sativa L.). Kod sve tri test biljke ustanovljen je inhibitorni efekat na klijanje semena i rast klijanaca. Klonovi transformisanih klonova: R1 i R8, imali su najveći alelopatski potencijal. Kod test biljaka arabidopsisa i pšenice, koje su bile tretirane fitotoksičnim medijumima, praćena je ukupna aktivnost antioksidativnih enzima – katalaza, peroksidaza i superoksid dismutaza (spektrofotometrijski), kao i zastupljenost i aktivnost pojedinih izoformi navedenih enzima (elektroforetski). Aktivnost ovih enzima (izuzev katalaza kod arabidopsisa) bila je povećana kod klijanaca isklijavanih i gajenih na fitotoksičnim medijumima, što je u skladu sa aktiviranjem odbrambenih mehanizama u biljkama kada se one bile izlažu stresu (fitotoksinima, tj. alelohemikalijama). Fitotoksični medijumi različitih klonova transformisanih korenova dovodili su do različitog nivoa povećanja aktivnosti katalaza, peroksidaza i superoksid dismutaza kod različitih test biljaka...Root system produces and releases chemical compounds with allelopathic activity. Isolation and characterization of these metabolites, as well as investigation of their effects on other organisms, are very complicated processes. The aim of the present study was to initiate and establish Chenopodium murale L. transgenic hairy root in vitro culture system, as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established 13 hairy root clones differed in their morphology and growth potential. The liquid culture system of characterized hairy root clones was maintained for over two years. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene presence were observed. We investigated allelopathic effect of wild-type and hairy root growth media (phytotoxic media) against germination and seedling development of Arabidopsis thaliana (L.) Heynh, wheat (Triticum aestivum L.) and lettuce (Lactuca sativa L.) test plants. The inhibitory potential varied depending on the test plant and hairy root clone. R1 and R8 clones had strong allelopathic effects. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. In test plants A. thaliana and wheat, after treatment with phytotoxic media, activity of different antioxidative enzymes (catalase, peroxidase and superoxide dismutase) were analyzed spectrophotometrically and electrophoretically. Activities of these antioxidative enzymes were higher in comparison to the control, except the catalase activity in A. thaliana, which was the same or lower. Phytotoxic media from different hairy root clones cause different changes in activities of catalases, peroxidases and superoxide dismutases in test plants..

Physiological and cell ultrastructure disturbances in wheat seedlings generated by Chenopodium murale hairy root exudate.

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Nataša seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H2O2 and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.This is a post-peer-review, pre-copyedit version of an article published in Protoplasma. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s00709-018-1250-0