Abstract: The aim of the present investigation was to find the best Agrobacterium rhizogenes strain, which can induce hairy root initiation faster and the best inducer molecule which can potentiate hairy root induction in host plants. Four different wild strains of A. rhizogenes, namely A. rhizogenes 15834, A4, WS and WR were used for hairy root infection in four host plants (Ipomoea batatas, Solenostemon rotundifolius, Vigna vexillata and Canavalia sp). Agrobacterium rhizogenes strains initiated hairy roots from the host plants without inducer molecules (AS and sugars) to a lesser level. Phenolic compound acetosyringone (AS) and sugars (D-glucose, mannose and galactose) were used to activate the virulence genes of the root inducing (Ri) plasmid which initiates transcription of virulence genes for hairy root induction. AS and sugars activated the virulence genes of the root inducing (Ri) plasmid of A. rhizogenes strains, which further initiated the transfer of T-DNA region to the host plants and enhanced hairy root induction frequency. As enhanced hairy root induction percentage in the entire host plants ranges from 11 to 61.5% and sugars from 16.5 to 39.5%. A. rhizogenes 15834 was found to initiate hairy roots early when compared to other strains of A. rhizogenes.
INTRODUCTION
The genus, Agrobacterium can transfer DNA to a remarkably broad group of organisms including numerous dicot and monocot angiosperm species (Wordragen and Dons, 1992) and gymnosperms (Levee et al., 1999). In addition, Agrobacterium can transform fungi, including yeasts, ascomycetes and basidiomycetes (Piers et al., 1996).
Hairy roots are initiated by infecting the plant material with various strains of Agrobacterium rhizogenes (Shanks and Morgan, 1999). Hairy roots induced by Ri plasmid usually exhibited a vigorous growth and extensive lateral branching while growing in a media devoid of phytohormones. Hairy roots have several properties that which promoted their use for various plant biotechnological applications. Their fast growth and biosynthetic stability, low doubling time, ease of maintenance and their ability to synthesize a range of chemical compounds makes them a suitable system for in vitro production of secondary metabolites (Giri et al., 2001).
A. rhizogenes containing Ri (root inducing) plasmid respond more strongly to wound phenolic compounds, such as acetosyringone, which acts as a chemotactic agent at very low concentrations and it activates the vir (virulence) gene on the Ri plasmid, which initiate the infection process for the transfer of T-DNA (Rahimi et al., 2008) etosyringone (AS) acts as signal molecule for the vir gene induction and it is widely used in experiments aimed at increasing Ri transformation frequencies (Huang et al., 2001).
The virulence genes vir A/vir G two-component regulatory systems on the tumor-inducing plasmid of Agrobacterium tumefaciens enables this soil bacterium to cause tumors in dicotyledonous plants. When wounded, plant cells release specific phenolic signal molecules such as acetosyringone, which induce the virA/virG system and monosaccharides and acid pH in the surrounding environment further potentiate expressions by vir A and vir G (Gao and Lynn, 2005). The induction of vir A and vir G leads to transcription of vir genes and the resulting products generate and transfer a defined segment of DNA from the tumor-inducing plasmid to the plant nuclear genome, transforming the plant cell and leading to tumorous growth (Winans, 1992). Acetosyringone or related compounds (eg., α-hydroxy acetosyringone) have been reported to increase Agrobacterium tumifaciens mediated transformation frequency in a number of plant species- Salvia miltiorrhiza, Allium cepa, Glycine max, Nicotiana tabacum etc. Acetosyringone acts as a chemotactic agent in very low concentrations and it activates the vir gene on the root inducing (Ri) plasmid, which initiates the infection process for the transfer of T-DNA. Various sugars were also known to induce high levels of vir gene expression in Agrobacterium tumifaciens and are also used for enhancing transformation frequencies (Cangelosi et al., 1990). In the case of A. rhizogenes, integration and expression of T-DNA genes in host cell lead to the development of hairy roots, which can be excised and grown in vitro as hairy root cultures.
In the present investigation hairy roots were induced from four host plants, Ipomoea batatas, Solenostemon rotundifolius, Vigna vexillata and Canavalia sp. through the mediation of A. rhizogenes ATCC 15834 for co-cultivating vesicular arbuscular mycorrhizal fungi in these roots (Chandran and Potty, 2008). VAM fungi are symbiotic biotrophs and it requires living roots for their growth and multiplication. The present study reports the efficiency of different strains of A. rhizogenes in eliciting hairy root induction and the role of AS and sugars in enhancing the hairy root induction frequency in host plants. The use of AS and sugars as inducer molecules for A. rhizogenes 15834 in these host plants is the first report.
MATERIALS AND METHODS
Host plants: Host plants, Ipomoea batatas (sweet potato), Solenostemon rotundifolius (chinese potato), Vigna vexillata and Canavalia sp. (sword bean) were used for hairy root induction. Cotyledons (cut into 0.5 cm2 blocks) and hypocotyls (cut into 1.0 cm long) were used as explants in all the host plants except S. rotundifolius. Stem cuttings and in vitro plants were used in S. rotundifolius for A. rhizogenes infection. Good quality seeds of all the above-mentioned plants were collected from Central Tuber Crops Research Institute (CTCRI), Sreekariyam, Thiruvananthapuram, Kerala State, India These experiments were done during January 2007.
Explant sterilization: The seeds of I. batatas, V. vexillata and Canavalia sp. were surface sterilized by treating the seeds with 0.1% HgCl2 for 10 min and washed well with sterile distilled water and poured 1% NaOCl3 and kept for 10 min and washed well with distilled water.
| Table 1: | YEB Media composition |
The surface sterilized seeds were placed in 1% agar and kept for incubation at 27°C in an incubator with continuous illumination under fluorescent lamps for 3 weeks.
Bacterial strains and growth media: Different wild strains of Agrobacterium rhizogenes used for the hairy root induction are A. rhizogenes ATCC 15834, A. rhizogenes A4, A. rhizogenes WR and WC (obtained from National Chemical Laboratory, Pune, India). Bacterial culture was maintained in Yeast Extract Broth (YEB) medium (Table 1).
Activation of bacterial culture: AS and sugars were incorporated with YEB culture medium in varying concentrations of 50, 100, 150, 200 and 250 μm L-1 were poured separately into sterilized Petri plates and allowed to solidify. Fresh bacterial colonies were streaked on the medium containing AS and sugars and incubated at 24°C for 24 to 48 h in the dark. The 24 to 48 h old activated cultures were used for infecting explants.
Agrobacterium transformation: Surface sterilized explants were wounded with a sterile scalpel, which was smeared with activated culture of A. rhizogenes. The infected explants were transferred to petri plates containing sterilized modified Murashige and Skoog medium (½ strength MS salts with 15 g L-1 sucrose, full strength B5 vitamins (myoinositol-100, nicotinic acid - 1.0, pyridoxine HCl-1.0, thiamine HCl- 10 mg L-1), 0.5 g L-1 cysteine HCl as antioxidant and pH 5.7). The plates were incubated at 24°C for two days in dark. After two days of incubation, the infected explants were transferred to fresh modified MS medium, containing 250 mg L-1 cefotaxime to eliminate bacterial growth (Danesh et al., 2006) for two days. Then the explants were transferred to fresh modified MS medium without antibiotics and kept for incubation at 24°C with 16 h photoperiod for thirty days for hairy root emergence. The hairy root initiation from the wounded explants was checked on a daily basis and the percentage of hairy root was also monitored in 100 explants each infected with AS and sugars activated A. rhizogenes. The percentage values were statistically analyzed.
Statistical analysis: One hundred explants were used in each case and the experiment was done in triplicates and all the data were processed using t-test as per (Hsu and Lee, 2010).
RESULTS AND DISCUSSION
After host plant infection, A. rhizogenes 15834 was found to be the most virulent strain because it initiated hairy root induction in all the four host plants early even in the presence and absence of inducer molecules. The early emergence of hairy roots indicated the enhanced virulence of A. rhizogenes. Similarly, David and Tempe (1988) reported that agropine type strains (A4 and 15834) were more virulent than manopine type strains of A. rhizogenes. I. batatas hairy roots were brittle, light yellow, branched with lot of root hairs and were negatively geotropic. V. vexillata hairy roots were thin, soft and were highly branched with lateral branching. The newly formed roots were pure white and become dark brown subsequently. Canavalia sp. roots were thick, highly branched and brittle. The growing root tips were white and the basal portions were brown. Rapid proliferation and tumor formation was also observed. S. rotundifolius hairy roots were white and very slender. Differences in pathogenecity among the four wild strains of A. rhizogenes were observed (Fig. 1) and similar observations were also made by Vanhala et al. (1995). The difference in virulence and morphology of hairy root could be explained by the plasmids harboured by bacterial strains (Nguyen et al., 1992). The genetic transformation mediated by Agrobacterium was affected by explant genotype (Fig. 1) and structure, chemical and physical factors, bacterial strains and signal molecules (Tao and Li, 2006). This could be due to variations in the concentration of phenolic compounds present in the respective host plants.
| Fig. 1: | Hairy root induction by different strains of A. rhizogenes in the presence (150 μM) and absence of acetosyringone |
A. rhizogenes 15834 was further used in this study because of its high susceptibility to induce hairy roots faster in all the host plants. In the presence of 150 μm acetosyringone, the hairy root induction time was reduced by 2 days in I. batatas and S. rotundifolius and by 3 days in V. vexillata and by 4 days in Canavalia sp. Giri et al. (2001) observed that AS induced A. rhizogenes strains reduced the time of hairy root induction by one week in Artemisia annua when compared to non AS induced A. rhizogenes. Hairy root emerged from the host plants within 15 days of incubation and found that 24°C was optimum for vir gene expression. The present results also correlate with the observations made by Jin et al. (1993), that vir gene induction was maximal at approximately 25 to 27°C and indicated a temperature effect on transformation, because the pilus of Agrobacterium strains is most stable at 18 to 20°C and pilus may function as a hook to seize the recipient cells and bring the bacteria and plant into close proximity to effect molecular transfer (Gelvin, 2003).
A. rhizogenes infected explants incubated at 24°C showed hairy root emergence from the 8th day of infection and the percentage of hairy root induction enhanced considerably in the presence of inducer molecules. Table 2 shows that 150 μm of AS enhanced maximum hairy root induction frequency on the cotyledon explants of I. batatas (92.5%), followed by V. vexillata (82.5%). In Canavalia sp., 150, 200 and 250 μM AS provided a root initiation percentage of 71.5. The response of S. rotundifolius was low to AS and 150 μm provided a maximum of 32.5% hairy root induction in in vitro plants. Ming et al. (2007) observed the frequency of rose hybrid nodal segment showed GUS expression was higher at 50 μM acetosyringone and found that the inclusion of acetosyringone in co-cultivation medium increased the transformation frequency. Rahimi et al. (2008) observed that the transformation frequency of explants of V. sisymbriifolium by A. rhizogenes (15834) doubled when 100 μM acetosyringone AS incorporated into the bacterial culture medium.
Hairy root induction frequency of different explants in modified MS medium with and with out acetosyringone is given in Table 2 and the cotyledons were found to be the preferred explants in modified MS medium without acetosyringone because of its highest percentage of hairy root induction. On modified MS medium sharp variations were observed in the preferences of explants to Agrobacterium infection. In the case of I. batatas, cotyledon was the maximal explants but hypocotyls were the preferred explants in V. vexillata and Canavalia sp. From these results it was confirmed that the types of explants also influenced root induction efficiency.
V. vexillata responded very well to 150 μm acetosyringone and showed a hairy root initiation frequency of 82.5% in cotyledon explants. The corresponding values of other host plants are also given in Table 3. Kumar et al. (2006) also reported 86% transformation frequency in Nicotiana tabacum by sonication of A. rhizogenes with 100 μM/L AS. Giri et al. (2001) reported that the incorporation of 50 μm acetosyringone in bacterial culture medium and the same concentration in the co-cultivation medium resulted in the hairy root induction frequency ranging from 75 to 100% in Artemisia annua by different strains of A. rhizogenes. In the present investigation AS was incorporated with the bacterial culture medium and a similar method was also adopted by Wang et al. (2001). They observed a higher transformation frequency of 59.2 to 84.7% in the presence of AS induced A. rhizogenes 15834 and a low of 10.6 to 22.4% in cotyledons and hypocotyls of Alhagi pseudoalhagi in the absence of AS (Table 2). In the present study AS activated A. rhizogenes 15834 showed hairy root induction frequency from 31-92.5% in I. batatas, 28-82.5 % in V. vexillata, Canavalia sp. 27-71.5%, S. rotuntifolius 20-32.5%. High concentration of acetosyringone, (200 μM) in the A. rhizogenes culture medium was used for transformation in three days old cotyledonary nodes of Vigna unguiculata and found that acetosyringone was indispensable for successful transformation (Raveendar and Ignacimuthu, 2010). The interaction among the host plants analyzed revealed that Canavalia sp. with S. rotundifolius is highly significant and the least significance was found with V. vexillata with Canavalia sp. The interaction of all other host plants was found to be statistically significant at 0.5% level (Table 2).
The explants of Canavalia sp. showed the higher hairy root induction frequency of 71.5% at 150 μM of AS and this concentration showed the highest hairy root initiation frequency in all the other host plants (Table 2).
| Table 2: | The effect of AS concentrations on hairy root induction frequency using A. rhizogenes 15834 |
| *Significatant at p = 0.05 | |
| Table 3: | Effect of sugars on hairy root induction frequency using A. rhizogenes 15834 |
| *Significatant at p = 0.05 | |
Further increase in AS concentration did not increase hairy root induction frequency and this may be due to the inhibitory effect of AS on transformation and it can radically affect the relative virulence of different strains and these effects are not consistent across species (Vanhala et al., 1995). Bond and Roose (1998) found that 250 μM of AS was the adequate concentration supplemented to the co-culture medium to induce hairy roots in citrus Washington navel orange.
Glucose, mannose and galactose also induced vir genes expression strongly, but to a lesser level than that of AS at concentrations ranging from 150 to 250 μm. Among the promising host plants, I. batatas was observed to be the most susceptible plant, both to acetosyringone and glucose with a 37.5% increase in hairy root induction frequency (Table 3). According to (Cangelosi et al., 1990) certain sugars induce vir genes synergistically with acetosyringone, even in low concentration of the latter they strongly induce vir gene expression in wild type cells of A. tumifaciens. The increase in hairy root initiation frequency attained due to the sugars activation of A. rhizogenes is in full agreement with the observation made by Shimoda et al. (1990). Besides increasing hairy root induction, sugars also promote rapid growth of hairy roots.
Glucose activated A. rhizogenes 15834 strain showed a high hairy root initiation frequency of 68.5% at concentration ranging from 150 to 250 μM in I. batatas and in V. vexillata explants, a maximum hairy root induction frequency of 50.5% in 250 μM of glucose. Canavalia sp. 200 to 250 μM glucose showed a similar hairy root initiation frequency of 62.5% and further increase in concentration of glucose did not elevate hairy root initiation frequency (Table 3).
In most of the cases 200 μM of mannose showed a maximum hairy root initiation frequency in I. batatas (66.5%) and V. vexillata (54%) explants and in Canavalia sp. explants a maximum of 66.5% was observed in 150 μM of mannose and further increase in concentration showed a negative trend in hairy root initiation frequency (Table 3).
In explants of I. batatas, galactose activated A. rhizogenes 15834 showed a higher hairy root induction frequency of 64.5% in both 150 μM and 200 μM galoctose. In V. vexillata and Canavalia sp. 250 μM galoctose showed a higher hairy root initiation frequency of 50.5 and 70.5% in the respective explants. The interaction of various sugars with host plants is given in Table 3 and all the sugars showed good increase in hairy root initiation and are statistically significant at 0.5% level.
From these results it was very clear that D-glucose, galactose and mannose activated the virulence genes of A. rhizogenes strain 15834 at different concentration and showed that the sugars can also contribute substantially to hairy root induction frequency. Citovsky et al. (1992) also reported that when AS concentration is low or not detectable, vir gene expression was significantly increased by monosaccharides (glucose or galactose) and various sugars can also act synergistically with AS to induce high levels of vir gene expression (Delmotte et al., 1991). Acetosyringone and sugar treatment of A. rhizogenes might be useful for eliciting hairy roots and also to enhance transformation frequencies of other host plants and this has immense potential in biotechnology. The inducer molecules were used to initiate hairy root from recalcitrant plant species, which are very difficult to propagate by known methods (Hansen et al., 1989) and after hairy root initiation they were planted as new plants. The inducer molecules are also used for genetic engineering experiments, to reduce the time of hairy root initiation and this has commercial significance also, hairy roots from medicinally important plants are used for the production of secondary metabolites (Shanks and Morgan, 1999). Hairy roots can also produce recombinant proteins from transgenic roots and they hold immense potential for the pharmaceutical industry (Guillon et al., 2006).
CONCLUSION
Because of the tremendous commercial potential of hairy roots in research and industry, the role of inducer molecule in enhancing hairy root initiation in host plants is vital in achieving success in transformation. The results obtained in this study highlighted the role played by inducer molecule AS and sugars in Agrobacterium mediated transformation.