Erum Dilshad, Hammad Ismail, Waqas Khan Kayani and Bushra Mirza*
Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan
Received date: November 23, 2015; Accepted date: January 30, 2016; Published date: February 02, 2016
Citation: Dilshad E, Ismail H, Kayani WK, Mirza B (2016) Optimization of Conditions for Genetic Transformation and In Vitro Propagation of Artemisia carvifolia Buch. Curr Synthetic Sys Biol 4:129. doi:10.4172/2332-0737.1000129
Copyright: © 2016 Dilshad E, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The excessive attention in the use of plants as medicine is credited to the occurrence of active principles whose pharmacological activities have been investigated. Due to the limited production and very less quantity of these important metabolites present in the plant cells, their genetic engineering and increased in vitro production is the point of focus for many years and can be achieved by in vitro transformation and propagation of desired plant. In the current study, we report transformation protocol for Artemisia carvifolia Buch with Agrobacterium tumefaciens C58C1 harboring β-glucuronidase as reporter and neomycin phosphotransferase as selectable marker gene. We have optimized simple regeneration conditions after transformation involving two different types of explants (leaf and stem) on different media formulations for direct organogenesis and best regeneration response. MS media with 2.5 g/L benzylaminopurine (BAP), 0.25 g/L naphthalene acetic acid (NAA), giving maximum number of shoots was selected. Rooting was obtained on ½ MS medium supplemented with NAA (0.1 mg/L). Transient expression of gus reporter gene was observed in the leaf and stem explants after 2 days of bacterial infection. Artemisia carvifolia Buch can be successfully transformed with Agrobacterium tumefaciens strain C58C1 by using controlled in vitro regeneration conditions. Findings of the current study would be useful for micro propagation and genetic transformation of Artemisia carvifolia Buch in future.
Artemisia carvifolia; Agrobacterium tumefacien; Micropropagation; Gus expression
Gus: Glucuronidase; NPTII: Neomycin Phosphotransferase; MS: Murashige and Skoog; PGRs: Plant Growth Regulators; SRM: Shoot Regeneration Medium; BAP: Benzyl Amino Purine; NAA: Naphthalene Acetic Acid
Plants in vitro cultures are gaining substantial significance owing to their potential applications in the production of useful aromatics with broad range of pharmacological activities [1,2]. They offer a striking substitute to the whole plant for the production of vital plant secondary metabolites [3-5]. Well-built practices are presently accessible to help growers encounter the demand of the pharmaceutical industry. These practices are deliberated to make available prime levels of different nutritional (vitamins, mineral nutrients) and environmental factors (e.g. light, gaseous environment, temperature, and humidity). These techniques also provide the PGRs (Plant growth regulators essential to gain significant regeneration rates of many plant species in vitro), to assist commercially worthwhile micro propagation. Distinct tissue and cell culture approaches are also being practiced for the production of numerous significant secondary products .
Artemisia genus is an opulent source of terpenoids and other secondary plant products having uses in perfumery and pharmaceuticals . Among the secondary metabolites of genus Artemisia, sesquiterpene lactones and flavonoids are of high therapeutic importance. They show strong anti-inflammatory, antimalarial, antioxidant, antitumor activity, as well as they increase immunity and decrease the risk of atherosclerosis, arthritis and gastrointestinal disorders [8-10]. Due to the limited production and very little quantity of these important metabolites present in the plant cells, their increased in vitro production is the point of focus for many years and can be achieved by genetic engineering and in vitro propagation of desired plant. There are various reports regarding Agrobacterium mediated transformation of Artemisia species, although most of these describe transformation of A. annua [11-14]. Some reports are available regarding transformation of other Artemisia species such as Artemisia dubia [15-18] and A. absinthium . Micropropagation and organogenesis of different Artemisia species have also been formerly reported such as A. annua [20-23], A. scorpia , A. vulgaris L. , A. mutellina vill. . However, there is no report regarding genetic transformation and tissue culturing of A. carvifolia Buch.
In the current study, we have reported simple transformation and regeneration conditions involving two different types of explants for direct organogenesis and best regeneration response. MS media containing different combinations of growth hormones were tested and optimum combination giving maximum number of shoots was selected. Agrobacterium tumefaciens strain C58C1 was used for genetic transformation and gus reporter gene was used to find the transient β-glucoronidase expression in transformed leaves and stem. Stable integration was confirmed by performing the PCR of kanamycin resistant plants.
Tissue culture media and plant material
Murashige and Skoog (MS) medium  was used for micro propagation, organogenesis and transformation of A. carvifolia. MS media supplementation with different hormonal combination is given in Table 1. Seeds of Artemisia carvifolia were collected from Astore, in the Northern regions of Pakistan (35.3667°N, 74.8500°E; 8,500 ft elevation). No specific permissions were required for the mentioned location for collection of seeds. However, the project was approved by the institutional biosafety committee (IBC) Quaid-i-Azam University Islamabad, Pakistan. Surface sterilization of seeds was carried out with 70% ethanol for 30 sec followed by 0.1% (w/v) mercuric chloride (HgCl2) for different time durations including 20 sec, 40 sec, 60 sec and 2 min. After washing with sterile distilled water, seeds were sown on two types of media i.e. half strength MS medium and plane agar in petri dishes. They were given chill treatment at 4°C for 2 days in dark. Then petri dishes containing seeds were incubated in growth chamber at 25ºC 16 h of photoperiod, illumination of 45 μE m-2 s-1 and 60% relative humidity under aseptic conditions.
|Pre culturing/Co-cultivation medium||MS+BAP (2.5mg/l)+ NAA (0.25mg/l)+200µM acetosyringone|
|Shoot regeneration medium (SRM) 1||MS+BAP (0.1 mg/l)+NAA (0.1mg/l)|
|Shoot regeneration medium (SRM) 2||MS+BAP (0.5mg/l)+ NAA (0.1mg/l)|
|Shoot regeneration medium (SRM) 3||MS+BAP (1mg/l)+ NAA (0.1mg/l)|
|Shoot regeneration medium (SRM) 4||MS+BAP (2.5mg/l)+ NAA (0.25mg/l)|
|Rooting medium (RM)||MS+NAA (0.1mg/l)|
|Antibiotics used for selection||Kanamycin 50mg/l, Cefotaxime (300mg/l)|
Table 1: Media formulations used for organogenesis of A. carvifolia.
Bacterial strain and construct used for transformation
Agrobacterium tumefaciens strain C58C1 harboring binary vector p35SGUSINT was used for transformation . T-DNA of p35SGUSINT contained NPTII gene with NOS promoter and NOS terminator and β-glucuronidase (GUS) gene with CaMV35S promoter and NOS terminator (Figure 1). Prior to infection, bacterial strain was grown overnight in an incubator at 28°C with constant shaking at 120 rpm in Luria broth supplemented with 50 mg/L each of selective antibiotics ampicillin and kanamycin.
Figure 1: Schematic diagram of the T-DNA region of transformation vector p35SGUSINT: RB: Right border, NOS-PRO: nopaline synthase promoter; NPTII: neomycin phosphotransferase, NOS-TER: nopaline synthase terminator, CaMV35S-PRO: 35S promoter of cauliflower mosaic virus, GUS: gene for β-glucuronidase, LB: Left border.
Transformation of A. carvifolia was carried out following the reported protocol , with some modifications. Leaf and stem with nodal segments, excised from two month old seedlings of A. carvifolia were cut into 0.5-1 cm pieces under laminar flow hood and precultured for 4-5 days on pre-culturing media, after that they were dipped in 50 ml of the overnight grown bacterial culture with optical density 1.0 for varying time durations (5,10,15,20 and 25 minutes). The excess bacterial culture was removed by blotting the explants on autoclaved filter paper, after that they were shifted to co-cultivation medium (Table 1) for 24, 48 and 72 hours to find the best co-cultivation time period. After cocultivation and washing the explants with diluted antibiotic solution and sterile distilled water, they were cultured on selection media with different hormonal combinations for shooting (Table 1) supplemented with kanamycin 50 mg/l and cefotaxime 300 mg/l. Around 5-6 explants per petri plate were cultured and total 20 petri plates were used for each media formulation in completely randomized block design, the whole experiment was repeated at different time intervals.
The explants were transferred to fresh medium after every two weeks for the first month, after which subcultures were made every 3rd week. After eight weeks, the concentration of cefotaxime was reduced to 150 mg/l. The cefotaxime was totally omitted after 12 weeks. Transgenic shoots of 2-3 cm in length were excised and placed on MS rooting medium (Table 1) containing 50 mg/l kanamycin for further selection. Two independent transformation experiments were conducted and 200 explants per transformation event were used. In each transformation experiment, 20-40 control explants were used without co-cultivation with Agrobacterium.
Histochemical GUS assay
Histochemical GUS assay was carried out essentially as reported . The leaf and stem explants after co-cultivation with bacteria were subjected to washing with sterile distilled water and incubated with GUS substrate solution overnight at 37°C. Untransformed explants were also incubated with the GUS substrate solution as control. The chlorophyll was removed by several washes with 70% ethanol after that transient GUS expression was observed.
In order to find out the stable integration of gus and neomycin phosphotransferase gene, kanamycin resistant plantlets were subjected to molecular analysis, which was performed after extraction of genomic DNA from aerial parts of 2 month old transformed and wild type plants by the CTAB method . The plasmid from C58C1 was isolated by the alkaline lysis method. PCR analysis was performed using a programmed DNA thermal cycler (Biometra, USA). The nptII gene forward 5’-AAGATGGATTGCACGCAGGTC-3’ and reverse primer 5´GAAGAACTCGTCAAGAAGGCG-3´, GUS gene forward 5´-AACGGCAAGAAAAAGCAGTC-3’ and reverse primer 5´-GAGCGTCGCAGAACATTACA-3 were used for PCR analysis. Conditions applied for PCR were as described previously . The annealing temperature for NPTII gene was 54°C and for that of GUS gene was 56°C.
The following research work was performed to optimize regeneration and transformation conditions for A. carvifolia Buch by using different explants, various media compositions and Agrobacterium tumefaciens strain C58C1 harboring p35SGUSINT with GUS reporter gene.
Effect of sterilization conditions and media on seed germination
Seeds were treated with mercuric chloride (HgCl2) 0.1% (w/v) for different durations of time. No contamination was observed in seeds of all treatments but germination efficiency was found to be different for each treatment (Figure 2a). Germination efficiency was found to be more on half MS medium which was 95% than plane agar on which 75% seeds germinated (Figure 2b). Seeds given chill treatment for 2-3 days were found to be more prone to germination than those without it. Seed germination took not more than one week without requiring any growth hormone for germination. These results are confirmed by the findings of another group who also found seeds germination within 6-7 days and observed that addition of growth hormones to the germination medium does not influence the percentage of seed germination . Later the germinated seedlings were maintained on MS medium with PGRs in order to obtain large no of plantlets for transformation experiment.
Effect of media formulation and explants on shoot organogenesis and transformation
In vitro regeneration of A. carvifolia after transformation was achieved successfully (Figures 3 and 4). The best shoot regeneration results were obtained on SRM4 (MS+2.5 mg/l BAP+0.25 mg/l NAA) followed by SRM2 (MS+0.5 mg/l BAP+0.1 mg/l NAA) (Figure 3), both were found to be optimal (P<0.05) for regeneration of maximum no. of transgenics. Whereas SRM1 was found to be best for shoot elongation. The regeneration response obtained on shoot induction medium (SRM4) was in accordance with the previous report of , who were able to regenerate transgenic plants of A. annua under similar growth hormone concentration. There is another report stating that nodal explants of Artemisia annua on 2.5 mg/l BAP gives highest regeneration response . Direct organogenesis takes less time for the rapid production of transformed plants  in contrast to callus tissue which needs to undergo few months of specialized culture before differentiating into shoot and root primordial . Rooting was obtained on half strength MS medium supplemented with NAA at concentration of 0.1 mg/l which is supported by the results of Nair et al.  and Jun-Li et al.  who were working with Agrobacterium tumefaciens mediated transformation of Artemisia annua and found rooting on 0.05-2.0 mg/l NAA and 0.1 mg/L NAA gave the best results.
Figure 3: Effect of different media compositions and explants on transformation efficiency: Values are means of two experiments performed independently ± S.E. Asterisk represents the significant difference (*=P<0.05, **=P<0.01) in data compared with the low regeneration efficiencies of explants on different shoot regeneration media.
Figure 4: Seeds germination, regeneration of explants and gus expression: seeds germination (a), giving rise to plantlets used for transformation (b). Regeneration of leaf and stem explants on selection media (c), kanamycin resistant plants on shoot regeneration media SRM4 (d) and rooting media (e). Expression of gus gene in stem and leaf explants after bacterial infection.
For the selection of transformed plants 50 mg/l kanamycin and 300 mg/l cefotaxime were used in the selection media. Various reports have shown that 50 mg/l kanamycin is sufficient for the selection of transformed plants [33-35]. The control of Agrobacterium growth at 300 mg/l cefotaxime has been shown by some other groups as well [36-38].
The highest transformation efficiency was shown by stem explants with nodal regions (50%) on all types of media as compared to leaf explants (25%). The type of explant contributes significantly towards efficient transformation system . The increased transformation efficiency of stem explants as compared to leaf explants has also been reported [40-44]. Analysis of variance showed highly significant interaction (P<0.01) between different types of media and explants used for transformation (Table 2).
|Source||Degree of Freedom||Sum of Squares||Mean Square||F Value||Prob|
|Factor A (explants)||1||9009.375||9009.375||1221.6102||0.0000|
|Factor B (media)||3||1741.125||580.375||78.6949||0.0000|
|Coefficient of Variation: 10.77%.|
Table 2: ANOVA for the effect of shoot regeneration media (SRM) on different types of explants on transformation efficiency.
The age of explant is also considered to be critical factor for transformation. In our report one week old explants were found capable of being transformed sufficiently. These findings are also supported by previous findings [45,46] showing that transformation efficiency of Agrobacterium decreases as age of explants increases.
Effect of infection time and co-cultivation period
The bacterial suspension used for transformation at the optical density of 1.0 was found to be optimum for efficient transformation of A. carvifolia. Reports are available showing that bacterial solution with 1.0 OD gave maximum number of transformants of A. absanthium  and Vigna radiata [47,48]. Bacterial density higher than this does not increase transformation efficiency but affects regeneration potential of explants due to stress encountered .
Infection and co-cultivation time greatly affect transformation efficiency. Infection time of 10-15 minutes was sufficient for getting maximum regeneration response on selection media, longer infection time results in the death of the explants (Figure 5a). The time duration of co-cultivation also positively influences the transformation efficiency, probably it allows the T-DNA transfer, integration, transcription and sufficient enzyme production leading to the expression of kanamycin resistant and GUS phenotypes. It is reported that co-cultivation of explants with bacteria in the presence of acetosyringone increases transformation efficiency up to 20% . However, we observed that co-cultivation period of 48 hours was found optimum for maximum transformation efficiency (Figure 5b), as previously reported that longer co-cultivation time only results in overgrowth of bacteria on explants and does not increase the transformation efficiency .
Histochemical gus assay and molecular analysis
Transformed explants after staining with GUS solution showed positive GUS expression in the form of blue dots scattered over the stem and leaf tissue (Figure 4f). On the contrary, the untransformed control explants were unable to show the GUS expression. The GUS expression was found to be maximum for co-cultivation of 48-72 hours after that it started to decline. Similar findings were obtained by Mannan et al.  who got the GUS+expression in the leaf, root and hypocotyl explants of A. absinthium L. after infection and co-cultivation of these explants with Agrobacterium tumefaciens C58C1 harboring the harboring p35SGUSINT vector.
PCR reaction performed to confirm the stable transgene integration showed the amplified products of 895bp for gus and 781 bp for nptII gene. Plasmid DNA also showed the similar amplified products, on the contrary wild type plant was unable to show these amplified PCR products (Figure 6).
Above results allow us to conclude that Artemisia carvifolia can be transformed with Agrobacterium tumefaciens strain C58C1 at optical density 1.0 with infection time of 15 minutes and co-cultivation period of 48 hours. The best regeneration response and transformation efficiency can be obtained by using stem explants with nodes at shoot regeneration medium containing 2.5 mg/l BAP, 0.25 mg/l NAA. Rooting can be obtained at NAA concentration of 0.1mg/l in half MS. These conditions would be helpful in conducting the experiments of in vitro propagation and transformation of A. carvifolia Buch in future.
We are thankful to the Higher Education Commission of Pakistan for providing the scholarship to author Erum Dilshad during her PhD studies.