The combination of R2R3-MYB gene AmRosea1 and hairy root culture is a useful tool for rapidly induction and production of anthocyanins in Antirrhinum majus L

Anthocyanins are the largest group of water-soluble pigments and beneficial for human health. Although most plants roots have the potential to express natural biosynthesis pathways required to produce specialized metabolites such as anthocyanins, the anthocyanin synthesis is specifically silenced in roots. To explore the molecular mechanism of absence and production ability of anthocyanin in the roots, investigated the effect of a bHLH gene AmDelila, and an R2R3-MYB gene AmRosea1, which are the master regulators of anthocyanin biosynthesis in Antirrhinum majus flowers, by expressing these genes in transformed hairy roots of A. majus. Co-ectopic expression of both AmDelila and AmRosea1 significantly upregulated the expression of the key target structural genes in the anthocyanin biosynthesis pathway. Furthermore, this resulted in strongly enhanced anthocyanin accumulation in transformed hairy roots. Ectopic expression of AmDelila alone did not gives rise to any significant anthocyanin accumulation, however, ectopic expression of AmRosea1 alone clearly upregulated expression of the main structural genes as well as greatly promoted anthocyanin accumulation in transformed hairy roots, where the contents reached 0.773–2.064 mg/g fresh weight. These results suggest that AmRosea1 plays a key role in the regulatory network in controlling the initiation of anthocyanin biosynthesis in roots, and the combination of AmRosea1 and hairy root culture is a powerful tool to study and production of anthocyanins in the roots of A. majus.

Heterologous Expression of Cyanobacterial PCS Confers Augmented Arsenic And Cadmium Stress Tolerance and Higher Artemisinin In Artemisia Annua Hairy Roots.

The present study provides the first report of heterologous expression of phytochelatin synthase from Anabaena PCC 7120 (anaPCS) into the hairy roots of Artemisia annua. Transformed hairy roots of A. annua expressing anaPCS gene showed better tolerance to heavy metals viz., arsenic (As) and cadmium (Cd) owing to 143 and 191 % more As and Cd accumulation respectively as compared to normal roots with a bioconcentration factor (BCF) of 9.7 and 21.1 for As and Cd respectively. Under As and Cd stresses, transformed hairy roots possessed significantly higher amounts of phytochelatins and thiols probably due to the presence of both AaPCS (Artemisia annua PCS) and anaPCS. In addition, artemisinin synthesis was also induced in transformed hairy roots under heavy metals stresses. In-silico analysis revealed the presence of conserved motifs in both AaPCS and anaPCS sequences as well as structural modelling of PCS functional domain was conducted. Interaction of AaPCS and anaPCS proteins with CdCl2 and sodium arsenate gene ontology analysis gave insights to anaPCS functioning in transformed hairy roots of A. annua. The study provides transformed hairy roots of A. annua as an efficient tool for effective phytoremediation with added advantages of artemisinin extraction from hairy roots used for phytoremediation.

A Stable Agrobacterium rhizogenes-Mediated Transformation of Cotton (Gossypium hirsutum L.) and Plant Regeneration From Transformed Hairy Root via Embryogenesis

Genetic transformation is a powerful tool to study gene function, secondary metabolism pathways, and molecular breeding in crops. Cotton (Gossypium hirsutum L.) is one of the most important economic crops in the world. Current cotton transformation methods take at least seven to culture and are labor-intensive and limited to some cultivars. In this study, we first time achieved plantlet regeneration of cotton via embryogenesis from transformed hairy roots. We inoculated the cotyledon explants of a commercial cultivar Zhongmian-24 with Agrobacterium rhizogenes strain AR1193, harboring a binary vector pBI-35S::GFP that contained the NPT II (neomycin phosphotransferase) gene and the GFP (green fluorescent protein) gene as a fluorescent marker in the T-DNA region. 82.6% explants produced adventitious roots, of which 53% showed GFP expression after transformation. 82% of transformed hairy roots produced embryonic calli, 12% of which regenerated into stable transformed cotton plants after 7 months of culture. The integration of GFP in the transformed cotton genomes were confirmed by PCR (Polymerase chain reaction) and Southern blot analysis as well as the stable expression of GFP were also detected by semi-quantitative RT-PCR analysis. The resultant transformed plantlets were phenotypically, thus avoiding Ri syndrome. Here we report a stable and reproducible method for A. rhizogenes-mediated transformation of cotton using cotyledon as explants, which provides a useful and reliable platform for gene function analysis of cotton.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Preliminary study on the preservation of Catharanthus roseus (L.) G. Don hairy root lines induced by Agrobacterium rhizogenes isolated in Vietnam

Agrobacterium rhizogenes-transformed hairy roots from Catharanthus roseus have been widely used in research and in life. Consequently, methods for preservation are essential to maintain valuable hairy root lines. Our results showed that Gamborg’B5 was the most suitable medium for hairy roots from VIN002 and VIN005 while solid White media was more comfortable for the hairy lines from VIN022 and VIN077. All hairy root lines must be preserved in the dark at 25–27 oC. Under suitable conditions, the rate of lines growing normally reached 69.3, 67.0, 57.3 and 60.7 % for VIN002, VIN005, VIN072, and VIN077, respectively. There were 90.0, 93.3, 80.0 and 93.3 % of lines could preserve rolB, a gene which has important effect on the growth of hairy roots.

Production of the Anticancer Compound Withaferin A from Genetically Transformed Hairy Root Cultures of Withania Somnifera

Withanolides (WTDs), well-known medicinally important compounds of Withania somnifera, including the anticancer compound withaferin A (WFA), are biosynthesized from their dedicated precursor squalene. Condensation of farnesyl pyrophosphate (FPP) molecules to produce squalene is catalyzed by squalene synthase (SQS). In the present study, the Arabidopsis thaliana squalene synthase gene ( AtSQS1) was transformed in W. somnifera by Agrobacterium tumefaciens C58C1 (pRiA4). The AtSQS1 gene was detected in 87.88 % of induced transformed hairy roots (THRs). The results showed that the growth index (GI) of THRs after five weeks of culture was 2-fold higher than that of adventitious hairy roots (AHRs). The biomass doubling time of THRs and AHRs was 18 and 30 days, respectively. Quantitative evaluation of WFA in the studied roots showed that THRs contain 1.51-fold more WFA (330±0.87μg g−1 dry weight (DW)) than AHRs (218±0.17μg g−1 DW). These findings can be used for the production of WFA as a valuable anticancer compound through controlled and scaled up cultures of W. somnifera THRs.