Influence of acetosyringone concentration on induction of carrot hairy root by Agrobacterium rhizogenes

Carrot hairy root induction by two different strains of Agrobacterium rhizogenesMTCC532 and MTCC2364 grown in yeast extract mannitol agar medium (YEMA) was studied in carrot (Daucus carota). The maximum hairy root induction was observed when carrot was simmered with 48 h old culture of A. rhizogenes MTCC-532. With different concentrations of acetosyringone (AS) tested, addition of 150 μM acetosyringone was found to enhance the transformation frequency up to 75(±2.60) and 60 (±2.08) percentage by A. rhizogenes MTCC-532 and MTCC 2364 strains, respectively. Transformation efficiency was highly dependent on the acetosyringone concentrations, type of bacterial strains and carrot genotype. Transfer of Ri Ti-DNA was confirmed by polymerase chain reaction (PCR) analysis, the detection of ags gene in transformed carrot hairy root. A. rhizogenes transformed hairy roots had the ability to form copious lateral roots as well as a negative geotropic growth habit in a shorter period of time. The Murashige and Skoog (MS) medium was found to be the best medium for hairy root mass multiplication, which induced high root biomass production and rapid root tip elongation.


INTRODUCTION
Abundant studies have demonstrated that arbuscular mycorrhizal (AM) fungi are obligatory symbionts which colonize the roots of approximately 80% of terrestrial plants (Lekberg and Koids, 2005) and improve the nutrient mobilization from soil, plant growth and disease tolerance (Douds and Siedel, 2012;Elsen et al., 2008).The presently available open pot culture methods for mass production of arbuscular mycorrhizae is having limitations like high cross contamination, being time consuming process; besides, only small amount of inoculum production is acheived.To overcome this problem, in vitro cultivation of AM fungi by root organ culture seems to be promising.Mosse and Hepper (1975) first proposed the use of root organ cultures with excised roots as host partner in AM symbiosis.The Agrobacterium rhizogenes is a well known tumor inducting, Gram-negative soil bacterium, which is able to induce hairy roots rapidly at the infection site (Baranski, 2008).The agrobacterium mediated transformation frequency is based on vir gene expression (Mohiuddin et al., 2011).Transcription of high level vir gene expression is induced by acetosyringone released by wounded plant cells and it has been reported that the compound increases agrobacterium mediated transformation frequencies in a number of plant species (Kumar et al., 2006).Genetically modified carrot (Daucus carrota L.) roots, show profuse lateral branching and rapid root tip elongation within two to three weeks.Transformed hairy roots are genetically and biosynthetically stable for long periods (Sawsan et al., 2012).The negative geotropism of transformed roots facilitates contacts with hyphae of AM fungi.The success of in vitro cultivation on AM fungi depends on host partner growth.
Therefore, the objective of the study was to optimize the acetosyringone concentration, a wound response molecule known for enhanced hairy root formation for maximum transformation efficiency and also to select suitable A. rhizogenes strains for maximum hairy root induction in carrot.

Carrot discs preparation
The commonly cultivated carrot cultivar Ooty-1 obtained from Kavi farm, Santhur, Nilgiris, Tamil Nadu, India, was used as an experimental material.Freshly harvested carrots were washed 2 to 4 times thoroughly with tap water.Then they were surface sterilized with 0.1% HgCl 2 for 10 min with continuous stirring.They were further rinsed three times (each for 5 min) with sterile distilled water and dipped in 70% ethanol for 30 s and superficially flamed and peeled out.Each carrot was sliced into 0.5 cm thick discs and was placed on 0.5% MS (half strength) (Murashige and Skoog, 1962) plates with the basal sides facing upwards (Figure 1).The sterile needle was used to prick manually for wounding on carrot surface.For pre-cultivation, the plates were incubated at 28°C in dark for 24 h.

Co-cultivation and agrobacterium mediated transformation
After completion of pre-cultivation, the carrot discs were placed on sterilized Whatman No 1 filter paper in Petri dish to remove excess moisture present on the surface of the carrot discs.The carrot discs were simmered with a loopful of 48 h old bacterial suspension MTCC-532 and MTCC 2364, inoculated on the basal side of carrot discs for infection.Control carrot discs were simmered in an uninoculated YEMA broth.Then the discs were transferred to 1% MS (full strength) basal medium containing four different concentrations of acetosyringone (AS) (Sigma Aldrich) viz., 50, 100, 150 and 200 µM, which were added separately to the media before plating.The control treatment plate was kept without addition of acetosyringone compound.Then the plates were incubated at 28°C under darkness for two to three weeks.

Conformation of transformed hairy root by PCR analysis
Carrot hairy root were cut into the small pieces and the genomic DNA was isolated from transformed carrot hairy root line by CTAB methods (Doyle and Doyle, 1990).The PCR was performed to amplify T-DNA agropine synthase (ags) transformed hairy root.The specific primer used to amplify ags genes were forward primer (5-GCGCATCCCGAGGCGAT-3) and reverse primer (5-AGGTCTGGCGATCGCAGGA-3).
PCR amplification was performed with a program of initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 1 min.annealing at 55°C for 1 min, extension at 72°C for 1 min.and a final extension at 72°C for 7 min and storage at 4°C.The amplification was analyzed by agarose gel electrophoresis.Plasmid DNA from A. rhizogenes strains MTCC-532 and 2364 was used as a positive control.

Mass production of hairy roots
After two to three weeks of incubation, carrot discs showed profuse white, turgescent and non-ramified apexes hairy roots.Fresh hairy roots were cut into minimum 3 cm long and then transferred to MS medium containing plates and incubated in inverted position under dark at 27°C.Bacteria free hairy root were obtained by subsequent subculture three times in fresh MS medium containing, cefotaxime at 250 mg/l (HiMedia, Mumbai, India) to make it free of A. rhizogenes.The bacterial free hairy roots could be used for in vitro mass production of AM fungi.

Statistical analyses
All the data were subjected to statistical analysis with software, Microsoft Excel for Windows 2007 add-in with XLSTAT Version 2010.5.05 (XLSTAT, 2010).Statistically significant differences between the treatments were analyzed using analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) at a 5 % significance level.

Transformation
After 5 to 10 days of co-culture with A. rhizogenes, callus initiation (Figure 2.) was observed on the surface of carrot discs, followed by appearance of the transformed roots on the side wall of discs (Figure 3).Hairy root initiation continued to occur from 10 days to two to three weeks.A typical hairy root was formed quickly from    negative geotropic growth habit (Figure 5).Some carrot discs were observed without any hairy root induction.This may be due to instability of the new genome or due to non-expression of genes involved in root induction.The carrot discs produced hairy roots up to three weeks and later on rotting was noticed.Among the four different concentrations of acetosyringone tested, 150 µM was the most effective in enhancing hairy root induction from both strains, as compared to the control.The maximum hairy root induction percentage (75±2.60a ), number of lateral roots (12±0.42 a ) and number of negative geotropic roots (9±0.31 a ) was observed from carrot discs simmered with 48 h old A. rhizogenes MTCC-532 on MS plates supplemented with 150 µM acetosyringone (Table 1).In the A. rhizogenes MTCC 2364 strain used for same condition only (60±2.08 a )) percentage of hairy root initiation, (9 (0.31 a )) number of lateral roots and (5 (0.17 a )), number of negative geotropic roots was observed.The transformation frequency declined at both lower (<150 µM) and higher concentrations of acetosyringone (Table 2).These transformed hairy roots were cut around 3 cm long roots and transferred to a sterile hormone-free MS medium (Figure 6).The transformed carrot hairy root was multiplied as per th e above p r o c e d u r e described in Materials and Methods (Figures 7 and 8).Hairy roots were maintained with regular sub culturing at three weeks interval.

Detection of agropine synthase (ags) gene in transformed carrot hairy root
PCR analysis was done using a pair of gene specific primer (forward and reverse) which amplifies the T-DNA agropine synthase gene.The total genomic a b      DNA was extracted from the transformed carrot hairy roots to observe the presence of Ri Ti-DNA.Also, for positive control of PCR analysis, the total genomic DNA was extracted from A. rhizogenes and resolved in agarose gel electrophoresis.The primer showed amplification, which confirmed the successful transformation of T-DNA agropine synthase gene, which produced the band of size approximately 341 bp (Figure 9).

Conclusion
This study demonstrates the ability of A. rhizogenes (MTCC-532) strain and 150 µM concentration of acetosyringone combination showed maximum hairy root induction and growth under in vitro conditions.The potential role of Agrobacterium strain and host explant genotype in hairy root induction is of great scientific interest, which may allow the rational manipulation of hairy root biomass production on large scale to develop monoxenic culture of AM Fungi.

Figure 3 .
Figure 3. Initiation of hairy root on carrot discs.

Figure 4 .
Figure 4. Growth of hairy root on carrot discs.
(±SE) (N=20) and values followed by the same letter in each column are not significantly different from each other as determined by DMRT (p0.05).

Figure 6 .
Figure 6.Transformed hairy root on MS medium.

Figure 7 .
Figure 7. Lateral branches formation after 3 days of incubation.

Table 1 .
strain with their ability to induce carrot hairy roots on various concentrations of acetosyringone.

Table 2 .
Effect of A. rhizogenes (MTCC-2364)strain with their ability to induce carrot hairy roots on various concentrations of acetosyringone.