Espand (Peganum harmala) is a valuable medicinal plant with antioxidant, antimutagenic and anticancer properties. To increase certain substances in cells, metabolic engineering is a powerful tool which needs an optimized regeneration and transformation protocol. The aim of current study was to setting up a regeneration and transformation protocol for Espand. Hypocotyls of three Iranian Espand cultivars (Yazd, Amol and Uremia) were cultured on MS medium supplemented with different concentration of BA (0, 1, 2 and 3 mg/l) and NAA (0, 0.1 mg/l). The highest frequency of regeneration achieved by Amol ecotype with 93% shoot regeneration on 0.01 mg/l NAA and 2 mg/l BAP medium. For transformation, Amol hypocotyls inoculated with Agrobacterium tumefaciens strain LBA4404 (pBI121) which contained neomycin phosphotransferase (nptII) and an intron-containing β-glucuronidase (GUS) genes. Kanamycin-resistant shoots were successfully regenerated on selection medium. An overall transformation frequency of 37% was achieved and confirmed by polymerase chain reaction. T1 segregation confirmed the genotypic ratio of 1:2:1 based on NPTII selectable marker gene. Also GUS expression of randomly green selected T1 transgenic plants, confirmed stability of transgene.
CTAB Cetyltrimethylammonium bromide
nptII neomycin phosphotransferase
CaMV cauliflower mosaic viru
Peganum harmala in family of Nitrariaceae is a medicinal aromatic perennial herb commonly known as Espand. It grows in semi-arid conditions and is extremely drought tolerant (Zhang, Chen and Hu 1992). It is wildly distributed in North Africa, the Middle East, Turkey, Pakistan, India and Iran. P. harmala is valued for its multipurpose uses in the field of pharmaceuticals (Moloudizargari et al. 2013). The active β-carboline alkaloids, harmine and harmaline, found in seeds and aerial parts of Espand have anti-tumor effects (Lamchouri et al. 1999; Wink 2001; Chen et al. 2005; Hamsa and Kuttan 2010; Zhao and Wink 2013; Seyed Tehrani et al. 2014). Espand dried seeds and extracts are sold as antispasmodicum, anthelminticum, antiasthmaoticum or antiheumaticum (Bajaj 1999). Its quinoline alkaloids, peganine and vasicinone, act as a respiratory stimulant (Baxter, Harborne and Moss 1993; Zayed and Wink 2005). P. harmala propagation is done by seeds which have a short span of viability (Singh and Ratnam 1983). In vitro strategies not only make rapid multiplication of favorite clones but also provide a proper source of materials for genetic manipulation. There are only a few reports on Espand tissue culture (Saini and Jaiwal 2000; Ehsanpour and Saadat 2002; Khawar et al. 2005; Goel, Singh and Saini 2009) which show explant type, plant hormone type and its concentration are important factors affecting on regeneration frequency of P. harmala. Different type of explant including cotyledonary node (Saini and Jaiwal 2000; Goel, Singh and Saini 2009), hypocotyl segments (Ehsanpour and Saadat 2002) and shoot apices (Khawar et al. 2005; Goel, Singh, and Saini 2009) have been reported as proper explant for shoot regeneration. Espand has been proposed as a useful system to study secondary pathways. Its serotonin content was overproduced when a tryptophan decarboxylase gene from Catharanthus roseus was overexpressed in Espand suspension culture (Berlin et al. 1993, 1994). In this study we compare the regeneration frequency of three Iranian Espand ecotypes. GUS gene transformation and expression in T0 and T1 is reported here.
Seeds of three ecotypes of Espand including Amol, Yazd and Uremia prepared from Iranian Forests, Range and Watershed management organization. Seeds surface sterilized by 70% (v/v) ethanol and sodium hypochlorite 2% for 30s and 10 min respectively. They rinsed three times with sterile-distilled water for 3 min per rinse and were cultured on MS medium containing 3% (w/v) sucrose, solidified with 0.7 (w/v) agar [Duchefa, Haarlem, The Netherlands]. The pH of medium was adjusted to 5.8 with 1N NaOH prior to autoclaving (121 °C for 20 min) and were incubated at 25 ± 2 °C with 16/8 hour photoperiod under cool-white fluorescent light at 40 µmol m−2s−1. Hypocotyl explants prepared from four-days-old in vitro seedlings. Explants were cultured on MS medium with different concentrations of BA (0, 1, 2 and 3 mg/l) and NAA (0 and 0.01 mg/l) [Sigma–Aldrich, Steinheim, Germany]. The optimum pH of all culture media were adjusted to 5.8 with 1N NaOH before sterilization. Cultures maintained in a growth chamber at 25 °C under 16/8h light/dark photoperiod and after four weeks, regeneration frequency was recorded.
In order to determine the optimal concentration of kanamycin, hypocotyl explants of Amol cultivar were tested at MS medium supplemented with different concentrations of kanamycin (0, 10, 25, 50, 75 and 100 mg/l) and 0.01 mg/l NAA+ 2 mg/l BA. This experiment was performed with three replications and 4 explants in each replication. Survival rate was recorded after 4 weeks. Amol ecotype with the highest regeneration frequency was selected for transformation experiment. Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121 carrying the NPTII and GUS-Intron was used for inoculation. A. tumefaciens was grown overnight in a reciprocal shaker (130–140 cycles min-1) at 28 ◦C in 50 ml LB (liquid Luria-Bertani) medium (10 g/l tryptone, 10 g/l NaCl, 5 g/l yeast extract, pH 7) [Duchefa, Haarlem, The Netherlands] with 50 mg/l kanamycin [Sigma–Aldrich, Steinheim, Germany] and rifampicin [Sigma–Aldrich, Steinheim, Germany]. The bacterial suspension was centrifuged for 10 min (4000 g), then re-suspended in inoculation medium and diluted to OD600=0.8. Sixty hypocotyl explants were inoculated with Agrobacterium for 15 min, and then co-cultivated on 0.7 (w/v) agar-solidified MS medium including 3% (w/v) sucrose, pH 5.5 for 2 days under dark condition at 25 ◦C. After co-cultivation, explants transferred to regeneration medium. The MS medium including 25 mg/l kanamycin, 0.01 mg/l NAA + 2 mg/l BA [Sigma–Aldrich, Steinheim, Germany] was used for transformation experiment as it showed the highest frequency of regeneration. To prevent Agrobacterium overgrowth 30 mg/l cefotaxime [Sigma–Aldrich, Steinheim, Germany] was used. Regenerated shoots were transferred to MS medium supplemented with 3% (w/v) sucrose, 0.7 (w/v) agar and supplemented with 10 mg/l kanamycin without any plant growth regulator. The rooted plantlets were rinsed under running tap water to remove agar and transferred to the plastic pots containing a ratio of sterile peat moss and perlite (1:1) mixture. They successfully acclimatized in a transparent plastic box in a growth chamber condition 16/8h (light/dark) photoperiod, fluorescent illuminations (40 µmol m-2 s-1) and 22◦C ± 1◦C for 2 weeks.
All seeds (128) of a random transgenic plant (T0) surface sterilized on 2% sodium hypochlorite for 10 min and were cultured on selection medium (MS medium+25 mg/l kanamycin). Number of green and completely yellow seedlings were recorded. Non-transformed seedlings turned yellow and died while NPTII transformed seedlings grew healthy. For segregation analysis to compare observed values against theoretical values the χ2 test was done.
DNA extraction and PCR
Genomic DNA was isolated from the young leaves of regenerated plants according to the modified CTAB method of Doyle and Doyle (1987). The PCR amplification was performed in 25 µl reaction mixture containing 1µl of DNA template, 1µl of each primer and 12.5 µl of master mix [i-Taq from iNtRON Biotechnology]. GUS specific primers of 5’-GGTGGTCAGTCCCTTATGTTACG- 3’ (Forward primer) and 5’-CCGGCATAGTTAAAGAAATCATG-3’ (reverse primer) were used to amplify the GUS-specific product. Thermal cycling (BIO-RAD T100) started with 5 min at 94 ◦C, and 35 cycles of 1 min at 94 ◦C, 1 min at annealing temperature (58◦C) and 45 sec at 72 ◦C ended by an extension for 5 min at 72 ◦C. The PCR products were separated on 1% agarose gel, stained with ethidium bromide and documented using a UV transilluminator system.
To detect β-glucuronidase activity, putative plants disks were fully immersed in the GUS reagent for 16 h at 25 ◦C. The tissue was destined in 70% ethanol for several hours (Hamilton et al. 2000). Controls were treated identically. GUS assay also was applied on randomly selected green plants of T1 transgenic plants.
Factorial experiment in a completely randomized design (CRD) with three replications and 5 explants per replication was performed. Percentage data of shoot regeneration subjected to arc sine (√x) through SPSS analysis. The normalized data were analyzed using SAS statistical analysis package (SAS Inc. Cary, USA), and were compared via Duncan’s multiple range test at P ≤ 0.05.
In this study the effect of different hormones and genotypes on Espand direct shoot regeneration was investigated. There are only few reports on tissue culture studies in Espand. The results of this study proved that hormone treatments were necessary for shoot regeneration. Shoots induced on hypocotyl explants through direct regeneration on MS containing various concentration of BA alone and in combination with NAA. No organogenesis was occurred on control medium (MS medium without BA and NAA) and medium supplemented with NAA alone. Of course all three ecotypes regenerated shoots directly in almost all treatments but Amol showed the highest. Combination of 0.01 mg/l NAA and 2 mg/l BA was the best treatment. For Amol ecotype the highest shoot regeneration (93.33%) was obtained when MS medium containing 2 mg/l BA+ 0.01 mg/l NAA was applied.
However, the lowest rate of regeneration frequency (6.66%) for Yazd cultivar was obtained in MS medium supplemented with 1 mg/l BA. Ehsanpour and Saadat (2002) also used hypocotyl segments as explant and they achieved a regeneration frequency of 87% on MS medium consisting of BA, Kinetin and NAA. Saini and Jaiwal (2000) used cotyledonary node, cotyledons and hypocotyls as explant and maximum frequency of regeneration (85%) was observed on cotyledonary node explants. It was found that many factors such as genotype, exogenous growth regulators, explant type and, culture conditions have capability to influence on biochemical pathways controlling the endogenous cytokinin content (Krikorian 1995). The significant effect of ecotype, BA, NAA and BA×NAA showed by analysis of variance. Saito et al. (1992) showed Genotype is an effective factor on plant regeneration frequency. Chen et al. (2004) revealed that genotype can indicate different regeneration capacities of same explants. BA alone induced Espand shoot regeneration. It has been used in shoot regeneration of an Indian ecotype (Goel, Singh and Saini 2009). The addition of NAA in combination with BA has been shown to be effective for shoot regeneration of Espand. NAA showed different effect on regeneration in different levels of BA and only in combination with 2 mg/l BA could give the highest frequency of regeneration. Khawar et al. (2005) showed MS containing BA and NAA was optimal for shoot regeneration when shoot apices and first axillary buds were used as explants.
Kanamycin sensitivity of Espand was tested during 4 weeks. The number of regenerated shoots decreased significantly with increasing kanamycin concentration. The concentration of 25 mg/l was the proper one as only less than 10% of explants could survive (data not shown). Out of 60 explants inoculated with Agrobacterium 23 shoots were regenerated on selection medium with a transformation frequency of 38%. PCR analysis of all putative shoots amplified the GUS gene fragment. From 128 T1 plants, 100 remained green on selection medium while 28 died. From 100 living seedlings, 37 were completely green while 63 were light green which showed they are possibly heterozygote for the transgene.
Here we report a successful regeneration and transformation protocol in harmel. PCR confirmed the introgression of β-glucuronidase gene and histochemical GUS assay of transgenics showed GUS activity. T1 segregation analysis confirmed the Mendelian ratio of 1:2:1 genotypic classes of the transgene. Also GUS assay of some T1 seedlings showed the stability of the transgene.
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