Year : 2009 | Volume
: 5 | Issue : 20 | Page : 336--342
Agrobacterium rhizogenes Mediated Genetic transformation of Abrus precatorius L
Vijai Singh Karwasara, VK Dixit
Biotechnology Research Laboratory, Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar-470003 (M.P.), India
V K Dixit
Biotechnology Research Laboratory, Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar-470003 (M.P.)
Abrus precatorius L. known as Indian liquorice is a common deciduous vine containing sweet principle compound known as glycyrrhizin. Hence it can be used as a very good substitute for Liquorice. Genetic transformation has proved to be an effective way to enhance secondary metabolites in plant cell cultures. The transformation of Abrus precatorius L. mediated by Agrobacterium rhizogenes was studied using three bacterial strains i.e. MTCC 532, MTCCC 2364 and NCIM 5140. Putative hairy roots were obtained after the transformation. The effects of bacterial strains, bacterial concentration, acetosyringone and co-cultivation pH on transformation of Abrus were investigated. Co-cultivation with Strain MTCC 532 for 2 days with 100 µmol L -1 acetosyringone at pH 6.5 provided the optimal conditions under which transformation frequency approached 84%.
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Karwasara VS, Dixit V K. Agrobacterium rhizogenes Mediated Genetic transformation of Abrus precatorius L.Phcog Mag 2009;5:336-342
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Karwasara VS, Dixit V K. Agrobacterium rhizogenes Mediated Genetic transformation of Abrus precatorius L. Phcog Mag [serial online] 2009 [cited 2020 Jul 3 ];5:336-342
Available from: http://www.phcog.com/text.asp?2009/5/20/336/58563
Abrus precatorius L. is a slender shrubby woody vine that climbs shrubs and low trees and is ubiquitous in temperate and subtropical areas all over India. It is locally known as "Gumchi" and belongs to the Fabaceae family. Several triterpenoidsaponins,flavanolglycosides,isoflavaquinones, abrine, pentosans, anthocyanins, etc have been isolated and studied pharmacologically. Diverse activities have been reported in different parts of Abrus precatorius due to presence of a broad range of chemical constituents like abrine, abraline, abrussic-acid, abruquinone, anthocyanins, campesterol, cycloartenol, delphinidin, gallic-acid, glycyrrhizin, hypaphorine, trigonelline etc. ,,,,,, Preparations of Abrus have been used for centuries as folk remedies for cancer, contraceptive, convulsion, cough,diarrhea,diuretic,gonorrhea,jaundice,rheumatism, trachoma, and as vermifuge. It is also used as sweetener, aphrodisiac, abortifacient etc. ,,,
One of the most prominent secondary compounds of Abrus precatorius is glycyrrhizin which occurs in leaves and roots. Glycyrrhizin is the principal component of liquorice , which is used as natural sweetener, antiulcerative and anti-inflammatory agent , . Furthermore glycyrrhizin exhibit anti-hepatotoxic  , anti-HIV , , antitumor  and immunomodulatory activity  . Production of glycyrrhizin in cell culture of liquorice has not been successful and thus its production from alternative sources has assumed significant , . It is worthwhile to undertake the genetic transformation of such an important and varied activity bearing plant like Abrus precatorius. Hairy roots have been induced in many dicotyledonous plants by transformation with A. rhizogenes strains , . Hairy root cultures resulting from Agrobacterium rhizogenes transfection of plant material produce the same secondary metabolites as those usually synthesized in intact plant with similar or higher yields  . However, sometimes hairy root culture have been shown to accumulate the metabolites which are known to accumulate in the aerial parts of intact plant. e.g. Lawsonia inermis (lawsone) and Artimisia annua (artimisinin). This feature, together with genetic and biochemical stability and rapid growth with high lateral branching in simple media without phytohormones, makes them especially suitable for biochemical studies which is not achievable with intact plant roots  . However, the profiles of secondary metabolites depend on the genetic make up of the hairy roots  . The production of secondary metabolites by hairy root culture has been studied for many plants , . The transformed roots provide great potential as a production system of plant metabolites , .
Agrobacterium rhizogenes strains contain a single copy of a large Ri T-DNA which is transferred to wounded plant cells and becomes stably integrated into the host genome  . Hairy roots are able to regenerate whole viable plants with high genetic stability. Like most members of the Leguminosae (Fabaceae) family, A. precatorius is very difficult to transform with Agrobacterium rhizogenes. The transformation of Abrus mediated by A. rhizogenes has not yet been reported. In this study, we describe the Ri plasmid-mediated transformation of Abrus and optimized the effects of several major physical and chemical factors, on Abrus transformation viz. effect of Agrobacterial strain and concentration, infection time, acetosyringone, cocultivation days, effect of pH of pre and co-cultivation medium.
Materials and Methods
Seeds of Abrus precatorius were collected from medicinal plant garden, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, M.P. The seeds were surface sterilized by 70% ethanol for 1-2 min, then with sodium hypochlorite (4% v/v) for 10-12 min and then rinsed 7-8 times with sterilized double distilled water to remove all traces of chemical sterilant. Seed germination was tested by putting few seeds on absorbent cotton moistened on half-strength Murashige and Skoog medium (1/2 MS)  and few seeds were placed on absorbent cotton dampened with sterile distilled water. Cultures were maintained at 252 C under a 12-h photoperiod regime with white fluorescent light. For transformation experiments, petiole, leaves and epicotyl were obtained from aseptically germinated young Abrus plantlet.
Agrobacterial strains MTCC 532 and MTCC 2364 were obtained from Institute of Microbial Technology (IMTECH), Chandigarh. Bacterial strain NCIM 5140 was obtained from National Collection of Industrial Microorganisms, Pune. A. rhizogenes strains MTCC 532 and MTCC 2364 belong to the agropine type and harbor the Ri plasmid with two parts of T-DNA (left T-DNA and right T-DNA). For the transformation experiments, MTCC 532 strain was inoculated into liquid YEB medium (sucrose 5g L -1 , beef extract 1 g L -1 , yeast extract 1 g L -1 , peptone 5 g L -1 ) and the MTCC 2364 strain was inoculated into liquid xanthomonas medium (galactose 20 g L -1 , yeast extract10 g L -1 , calcium carbonate 20 g L -1 ) and shaken for 48 h at 28C. NCIM 5140 strain was inoculated on nutrient agar plates having following composition: beef extract 1 g L -1 , yeast extract 2 g L -1 , peptone 5 g L -1 sodium chloride 5 g L -1 and shaken for 48 h at 26 C. The cultures were then centrifuged at 1500Χg and resuspended in liquid MS medium containing 100 ΅mol L -1 acetosyringone.
Abrus explants were pre-cultivated on MS medium solidified with 0.8% (w/v) agar at a varying pH range (4.5, 5.5, 6.5, 7.5) and concentrations of acetosyringone (0, 50, 100, 150, 200 ΅mol L -1 ) for 2 days. The explants were then transferred to the Agrobacterium suspensions containing 100 ΅mol L -1 acetosyringone and incubated for definite time and then blotted dry on sterilized filter paper. The explants were returned to the same precultivation medium for co-cultivation. The explants were transferred to media (MS medium containing 500 mg L -1 cefotaxime) after every four days until hairy root formation observed. The numbers of explants with roots (a minimum 22 days after infection) were recorded. The standard condition maintained were 45 min infection and 2 days pre- and co-cultivation at pH 6.5 using a bacterial concentration of 1.0 (OD600). To determine the optimum transformation conditions, one factor of the standard conditions was changed each time and the effects on hairy root formation observed. The Agrobacterium rhizogenes MTCC532, MTCC2364 and NCIM 5140 strain were streaked and subcultured for 48 hr in the dark at 261C on recommended solidified agar medium.
Total genomic DNA was isolated using the AuPrep Kit (Life Technologies Pvt. Ld., India). Integration of the TL DNA (rol gene) and TR DNA (ags gene) regions of the pRi plasmidwasconfirmedbyPCRanalysisof thegeneslocated in this region. All primers were obtained from Microsynth (Switzerland). Presence of ags gene was confirmed by using 5'--CGGAAATTGTGGCTCGTTGTGGAC-3' and 5'--AATCGTTCAGAGAGCGTCC GAAGTT-3' as primers. Amplification by PCR was performed under following conditions: initial denaturation at 94 C for 4 min, annealing at 58 C for 1 min and extension at 72 C for 1 min for 35 cycles, with a final extension at 72 C for 5 min. The amplicons were analyzed by electrophoresis on 1% agarose gel (w/v).
The data on hairy root formation are the mean of three independent experiments with a minimum of 20 explants per treatment. All roots that survived in the selection medium for 20 days were designated as hairy roots. The data among different treatment were tested by Graph Pad InStat 3 software run on XP professional Intel Pentium IV processor.
Abrus seed germination
Seeds germinated on both 1/2 MS agar medium as well as on damp absorbent cotton with or without fluorescent light after 4 days. The percentage of germinated seeds on the half strength MS medium after 10 days was much higher than that of sterile distilled water damp absorbent cotton [Table 1].
Agrobacterium and Abrus transformation optimization protocol
Three strains of A. rhizogenes, MTCC 532, MTCC 2364 and NCIM 5140 were tested for their transformation efficiency [Figure 1]A. MTCC 532 exhibited maximum transformation efficiency (65%) and was the best amongst the tested agrobacterial strains. Transformation efficiency of 29.5% and 20% was recorded with MTCC 2364 and NCIM5140 respectively suggesting low transformation by these strains. Among the various explants taken from the aseptically germinated Abrus plantlet [Figure 2]A, the leaf explant shows maximum transformation efficiency while it was least with petiole [Figure 1]B. A. rhizogenes MTCC 532 induced hairy root has been shown in [Figure 2]B . [Figure 3]A shows the effect of infection time on the frequency of hairy root formation. The transformation frequency markedly increased with increase in infection time from 15 to 45 min. At 15 min. the transformation frequency was 26.5 % which was increased to nearly three fold i.e. 67.5% at 45 min. The hairy root formation frequency increased two-folds when the bacterial concentration was raised from 0.5 to 1.0 (OD600). It however, slowly decreased with further increase in agrobacterial concentrations [Figure 3]B.The hairy root frequency was increased progressively with increasing co-cultivation time from 2 to 4 days [Figure 3]C. At longer co-cultivation period, problem of increased bacterial cell densities in the selection medium was encountered. Therefore, co-cultivation was restricted to 2 or 3 days and this was found to be sufficient for transformation. The concentration of acetosyringone has profound effect on hairy root frequency, which was most favorable at about 100 ΅ mol L -1 [Figure 3]D. The pH of co-cultivation medium had a strong influence on hairy root induction and the most favorable pH was 6.5, higher pH inhibited growth [Figure 3]E. Effect of mode of explant wounding was also studied which showed a better transformation with manual wounding as compared to the high speed vortexing. Hairy root production under the optimized conditions determined as above was performed: Bacterial strain MTCC 532, co-cultivation for 2 days, 100 ΅molL -1 acetosyringone, and a pH 6.5 in the co-cultivation medium yielded the hairy root production with 84.4% frequency.
Confirmation of transformation
Root clones each from MTCC 532 and MTCC 2364 were randomly selected for opine assay. All roots analysed show the presence of opine as a dark spot on paper electrophoretogram, which indicate that the roots were transformed. For PCR amplification of the ags gene, roots were excised from rooting explants that had been infected for one month. All roots were found to harbor the ags gene which shows their transformed nature [Figure 4].
The germination of Abrus precatorius seeds in MS medium was similar to MS medium solidified by agar and damp absorbent cotton, and 12/12 light/dark cycle strongly promoted this process. The maximum seed germination was found on absorbent cotton soaked with half strength MS medium while the seed germination frequency was lower on the distilled water damp absorbed cotton due to the nutrient constraint. Unexpectedly seed germination percentage was lowest on the MS solidified medium due to frequent seed callus development instead of seedling germination. The genetic transformation mediated by Agrobacterium is affected by various factors like explant genotype and structure, chemical and physical factors, bacterial strains and signal molecules etc. Different strains of A. rhizogenes vary in their transforming ability , . This was confirmed in our study that the MTCC 532 strain had greater hairy root transformation capacity than the MTCC 2364 in Abrus transformation. The plasmids contained by these strains carry genes of Ri T L -DNA which direct the synthesis of substances that induce the cell to differentiate the root formation under the influence of endogenous auxins , . The tms loci in the Ri T R-DNA also synthesis auxin which induce root formation. Variations in plasmid of strains may be responsible for the variability of transformation efficiency  . Bacterial concentration plays an important role in the production of transformed roots. Suboptimal agrobacterial concentrations result in low availability of bacteria for transforming the plant cells while high concentrations decreased their potential by competitive. The results show the optimum bacterial concentration was 1.0 (OD 600 ) , which was similar to the effect of bacterial concentration on hairy root formation in soybean transformation described by Kumar et al. 1991. Therefore it may have the same mechanism for the transformation.
Co-cultivation is very important step in the transformation process. Bacteria attachment, T-DNA transfer and integration occurs during this stage , . These processes can be accelerated by supplementing some ingredients in the co-cultivation medium or prolonging co-cultivation time so that these processes can be completed effectively. Our results indicated that, when the co-cultivation time was too short then these transformation processes could not be completed, while long co-cultivation time could negatively affect transformation by reducing bacterial affinity to the plant cell or by competitive inhibition. Some recalcitrant plant species can be transformed by inducing the vir genes of the bacteria by signal molecules or it can be achieved in vitro by co-cultivating agrobacterium with wounded tissue  . Acetosyringone or related compounds functions as signal molecules, which have been reported to improve the agrobacterium-mediated transformation in several plant species  . In our work, exogenous acetosyringone at 50 to 100 ΅mol L -1 was must for the hairy root formation, which according to the finding of Cervera et al. 1998  . The transformation frequency was not significantly increased with application of higher concentration of (>100΅mol L -1 ) acetosyringone. We suggest that, in Abrus transformation, presence of acetosyringone in relatively lesser concentration is obligatory for signaling but at high concentration the molecule function is not increased suggesting optimization of response at lower concentration.
Studies indicate that low medium (e.g. Nutrient broth medium and Xanthomonas medium) pH for activating bacteria promotes activation of some vir genes. Like VirD2, which are effectively activated under acidic pH, optimum being pH 5.1-5.2  . When pH of medium is decreased from 7.2 to 5.8, the hairy root formation frequency markedly increased which is in agreement with the finding of Yu et al. 2001  . Hairy root formation frequency increased when pre- and co-cultivation medium pH was increased from 4.5 to 6.5, but decreased when pH was higher than 6.5. This is in accordance with the findings of Aida and Shibata 1995  the underlying mechanism of which is not clear. The transformation of roots induced from Abrus leaves by A. rhizogenes was obtained with relative ease and it was demonstrated that the transformation frequency could approach 84.4% under optimal conditions. Abrus could become an important research plant with special reference to genetic transformation and the secondary metabolite production by virtue of its wide-ranging list of chemical constituents present in its different parts.
One of the authors wishes (Vijai Singh Karwasara) to acknowledge All India Council for Technical Education, New Delhi for providing the scholarship for the research work.
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