Bioscience Journal | 2022 | vol. 38, e38007 | ISSN 1981-3163 1 Khadija AHANSAL1,2 , Rabha ABDELWAHD2 , Sripada Mahabala UDUPA3 , Hanane AADEL1 , Fatima GABOUN2 , Mohammed IBRIZ1 , Driss IRAQI2 ¹ Laboratory of plant, Animal and Agro-industry Production, Department of Biology, Faculty of Science, Ibn Tofail University Kenitra, Morocco. ² Biotechnology Unit, National Institute of Agronomic Research, Morocco. ³ ICARDA-INRA Cooperation Research Project, International Center for Agriculture Research in the Dry Areas, Morocco. Corresponding author: Khadija Ahansal Email: kh.ahansal@gmail.com How to cite: AHANSAL, K., et al. Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of Moroccan durum wheat. Bioscience Journal. 2022, 38, e38007. https://doi.org/10.14393/BJ-v38n0a2022-54513 Abstract Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens. Keywords: Agrobacterium tumefaciens. Drought Tolerance. Genetic Transformation. HVA1 Gene. Triticum turgidum L. 1. Introduction The Mediterranean countries' main food crop is durum wheat (Triticum turgidum L. var. durum). Various biotic and abiotic stresses limit durum wheat production in these countries. As a result, the yield levels are low in many of the countries including Morocco. Durum wheat production in Morocco is reducing year after year, due to low rainfall (drought) and biotic stresses. For instance, Morocco produced 1.3 million tons of durum wheat during the 2018-19 season (Reuters 2018; Usda-Fas 2019) which is 43% less than the previous year. This year again durum wheat production is expected to reduce further by 60% compared to the previous year due to these stresses (World-Grain 2020). As a consequence, Morocco is not self-sufficient EFFECT OF TYPE OF MATURE EMBRYO EXPLANTS AND ACETOSYRINGONE ON AGROBACTERIUM-MEDIATED TRANSFORMATION OF MOROCCAN DURUM WHEAT https://orcid.org/0000-0001-9045-7415 https://orcid.org/0000-0001-7365-8960 https://orcid.org/0000-0003-4225-7843 https://orcid.org/0000-0001-7939-4003 https://orcid.org/0000-0003-2018-8031 https://orcid.org/0000-0002-9024-912X https://orcid.org/0000-0002-6453-4066 Bioscience Journal | 2022 | vol. 38, e38007 | https://doi.org/10.14393/BJ-v38n0a2022-54513 2 Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of Moroccan durum wheat in durum wheat production. Among abiotic stresses, drought and salinity stresses are important in many countries including Morocco. In addition to classical plant breeding, genetic transformation had been shown to be useful in enhancing tolerance to drought in wheat. Since the first transgenic wheat using the microprojectile bombardment on bread wheat (Vasil et al. 1991; Vasil et al. 1992) and durum wheat (Bommineni et al. 1997) using isolated scutella as explant, GUS as a reporter gene, and bar as a selectable marker, several improvements have been made in the durum wheat transformation protocol (Iraqi et al. 2005; He et al. 2010; Hakam et al. 2016). However, the first successful Agrobacterium tumefaciens mediated transformation for durum wheat using mature and immature embryos was reported by Patnaik et al. (2006) and Wu et al. (2007). Several studies demonstrated that Agrobacterium-mediated transformation has been a better alternative to biolistic for low copy insertions of T-DNA with minimal rearrangements and degree of gene-silencing phenomenon (Cheng et al. 2004; Jones et al. 2005; Shrawat and Lörz 2006) and improved stability of expression over generations than the free DNA delivery methods (DAI et al. 2001). The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like durum wheat requires the identification and optimization of factors (genotype, type of explant, optimized conditions of inoculation and co-cultivation, selection media, vector, and Agrobacterium strain) affecting T-DNA delivery and plant regeneration have been explored in a quest to achieve higher transformation efficiency (Wu et al. 2003). In our study, barley HVA1 gene encoding a late embryogenesis abundant (LEA) protein, which confers osmotolerance for seed embryos as an adaptive response during cellular dehydration in seed was used to transform durum wheat. This gene has appeared as an attractive candidate for the engineering of drought and improving growth characteristics including an enhanced biomass yield under water deficit conditions. The aim of this study is to examine the combined effects of several factors influencing Agrobacterium- mediated transformation such as genotype, type of explant, and acetosyringone concentration to define the optimum conditions using mature embryos as starter explants for introducing the barley HVA1 gene into Moroccan durum wheat varieties. 2. Material and Methods Plant material Amria, Chaoui, and Marouane, three Moroccan durum wheat varieties, were used for genetic transformation. The seeds of these varieties were procured from the Experimental Research Station of INRA, Marchouch, Morocco. Seeds were disinfected for one minute with 70 % ethanol (v/v), then immersed in 20 % commercial bleach (with 0.1 % Tween 20) for 20 minutes, followed by three rinses with sterile distilled water. Inoculation of mature embryos with Agrobacterium cells was performed using three different methods under aseptic conditions: - Embryogenic calli (EC) were prepared by culturing the intact mature embryos on the induction medium for 15 days in the dark at 25 °C before inoculation with Agrobacterium. - The mature embryo halves (MEH) were prepared by cutting mature embryos into two halves along the embryonic axis and used directly for inoculation with Agrobacterium. - The mature embryo pieces (MEP) were prepared by gently scrapping mature embryos into small pieces with a sharp blade and cultured on the induction medium for 7 days in the dark at 25 °C before inoculation with Agrobacterium. The prepared explants were incubated in MS medium (Murashige and Skoog 1962) supplemented by 100 mg/L Myo-inositol, 150 mg/L asparagine, 20 g/L saccharose, and 2 mg/L picloram. Agrobacterium mediated gene transfer in mature embryos of wheat Agrobacterium tumefaciens strain EHA101 harboring pTF101.11 plasmid, containing the HVA1 gene and bar gene which confers resistance to the phosphinothricin (PPT) described by Abdelwahd et al. (2013) were used in this study. For the preparation of the bacterial suspension culture, 1 mL of A. tumefaciens (EHA101) culture was allowed out from a -80 °C glycerol stock culture and precultivated on Yep solid medium containing 50 μg/mL Bioscience Journal | 2022 | vol. 38, e38007 | https://doi.org/10.14393/BJ-v38n0a2022-54513 3 AHANSAL, K., et al. spectinomycin at 28 °C for 1 to 2 days. Single colonies of the bacteria (EHA101) were grown overnight (48 h) on a shaker at 28 °C and 250 rpm in YEP liquid medium containing appropriate antibiotics. The bacterial cell suspensions were centrifuged at 4,500 x g for 10 minutes. The supernatant was eliminated, and the pellet was resuspended in inoculation medium WWC described by (Wang et al. 2009) with different concentrations of acetosyringone (AS) (0, 100, 200, and 400 µM). The cell concentration was adjusted to an OD650 at 0.7. The explant EC, MEH, and MEP were immersed in suspension cells bacteria with AS at different concentrations (0, 100, 200, and 400 µM) for 30 minutes in dark at room temperature. After removing the bacterial suspension, the explants were blotted to dry with sterile filter paper and transferred to fresh co- cultivation medium (i.e., previous induction medium) supplemented by 100 mg/L casein hydrolysate (CH), 2.0 mM MES and 0.75 g/L MgCl2, and maintained in the dark for three days. Subsequently, the embryos were transferred to the same medium plus 300 mg/L of cefotaxime to remove Agrobacterium. After 3-4 weeks in the dark, the calli were transferred onto a regeneration medium (Iraqi et al. 2005) and grown for two weeks. Then, the plantlets were transferred to a selective regeneration medium supplemented with 100 mg/L casein hydrolysate, 3 mg/L of PPT, and 300 mg/L of the antibiotic cefotaxime. DNA analysis Genomic DNA was extracted from leaves of the putative transformed and untransformed control plants using a CTAB DNA isolation method (Udupa et al. 1998). PCR amplification in a 20µL solution containing 50 ng DNA template, 1x Taq DNA polymerase buffer, 200 µM of each dNTP, 0.5 pmol of the respective primers, and 0.6 unit of Taq DNA polymerase revealed the presence of the bar gene, 2x35S promoter, and HVA1 gene sequences in all putative transgenic plants (Promega). 5' GTCTGCACCATCGTCAACC 3' (forward) and 5' GAAGTCCAGCTGCCAGAAAC 3' (reverse) primer pairs were used to detect the bar gene (402 bp). 5' GCACAATCCCACTATCGTTCGC 3' (forward) and 5' TCCGTCCACTCCTGCGGTTC 3' (reverse) primer pairs were used to detect the 2x35S (390 bp) gene. Both primers (bar and 2x35S) were amplified in a thermocycler (Master Cycler, Eppendorf, Germany) with an initial denaturation at 94 °C for 4 minutes, followed by 30 cycles of 1 minute at 94 °C, 1 minute at 58 °C, 2 minutes at 72 °C, and a final 10 minute extension at 72 °C. The primer pairs used to detect the HVA1 gene (290 bp) were 5' AGCTAGATCGTGTGAGACGAAG 3' (forward) and 5' CCTGCGCCGTCTCGTACGTCTT 3' (reverse), HVA1 primers were amplified using an initial 3 minute denaturation at 94 °C, followed by 35 cycles of 30 seconds at 94 °C, 30 seconds at 55.5 °C, 75 seconds at 72 °C, and a final 10 minute extension at 72 °C. PCR products were separated on 1% (w/v) agarose gel and 8% (w/v) polyacrylamide gel and stained with ethidium bromide. The size of the amplified transgene product was compared to that of the positive control. Statistical analysis A completely randomized design (RCBD) was used with 3 genotypes (Amria, Chaoui, and Marouane), 4 concentrations of AS (0, 100, 200, and 400 µM), and three types of mature embryo explants (ME, MEH, and MEP). Each test was performed three times. For each treatment, we used 100 explants per replication. The percentage of callus induction, plantlets regeneration, and resistant plantlets to selective agent phosphinothricin (PPT) were counted. Analysis of Variance (ANOVA) was performed using the General Linear Model (GLM) procedure in SAS (SAS Institute 1985). Fisher's Least Significant Difference (LSD) test was used to explore further and compare the mean of treatments (SAS Institute 1985). 3. Results The experiments were conducted to evaluate the effect of the factors influencing the efficiency of T- DNA delivery in durum wheat known by recalcitrance to Agrobacterium-mediated transformation. These factors include genotype, four different concentrations of AS (0, 100, 200, and 400 µM), and different explants (embryogenic calli, mature embryo halves, and mature embryo pieces). Bioscience Journal | 2022 | vol. 38, e38007 | https://doi.org/10.14393/BJ-v38n0a2022-54513 4 Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of Moroccan durum wheat Effect of genotype on the regeneration and transformation efficiency The mature embryos have been used for evaluating the callus induction and regeneration after infection by Agrobacterium and transformation response in three durum wheat Amria, Chaoui, and Marouane. 3600 explants were infected by Agrobacterium cells for each variety and the analysis of variance clearly showed a significant difference between varieties (Table 1) on callus induction and regeneration after infection with bacteria cells. The callus induction (79.13%) and plantlets regeneration (9.37%) (Table 1) for the Amria variety were higher compared to Chaoui and Marouane. Although the results of plantlets resistant to PPT showed no significant difference between varieties, the transformation efficiency for variety Chaoui (0.06%) was lower than Amria (0.25%) and Marouane (0.11%). In fact, Amria and Marouane were the most sensitive varieties to Agrobacterium infection than Chaoui (Table 1). Table 1. Effect of genotype on callus induction, regeneration and transformation efficiency in durum wheat. Varieties Callus induction (%) Plantlets regeneration (%) Plantlets resistant to PPT (%) Transformation efficiency (%) Amria 79.13a* 9.37a 1.12a 0.25a Chaoui 75.42b 7.24b 1.05a 0.06b Marouane 73.59b 9.35ab 1.38a 0.11a LSD 2.193 2.1287 1.0631 0.165 *The values followed by the same alphabet are not significantly different at alpha =0.05 according to the Least Significant Difference (LSD) test. Effect of acetosyringone concentration on callus induction, regeneration plant, and transformation efficiency The three explants (embryogenic callus, mature embryo halves, and mature embryos pieces) were inoculated for 30 minutes in the presence of Agrobacterium cells with different concentrations of AS 0, 100, 200, and 400 µM. The presence of virulence inducers such as AS in the inoculation media is necessary to improve the efficiency of T-DNA transfer in durum wheat because of its resistance to A. tumefaciens infection. The results revealed that different concentrations of AS had no significant effect on callus induction and plantlets regeneration. However, the inoculation media with 100 and 400 µM gave the highest percentage of plantlets resistant to PPT (1.68%) and (1.56%) respectively compared to 0 and 200 µM (Table 2). The results of transformation efficiency showed a significant difference between the four AS concentrations, inoculation medium containing 100 and 400 µM gives higher transformation efficiency (0.26%) compared to 200 µM (0.04%) (Table 2). Table 2. Effect of acetosyringone concentration on callus induction, regeneration and transformation efficiency in durum wheat. AS (µM) Callus Induction (%) Plantlets Regeneration (%) Plantlets Resistant to PPT (%) Transformation efficiency (%) 000 77.52b* 7.74b 0.94ab 0b 100 76.17b 8.56b 1.68a 0.26a 200 75.12b 9.67b 0.54ab 0.04b 400 75.38b 8.65b 1.56a 0.26a Control 81.30a 20.83a 0b 0b LSD 2.9401 2.8343 1.405 0.1912 *The values followed by the same alphabet are not significantly different at alpha =0.05 according to the Least Significant Difference (LSD) test; AS: Acetosyringone. Effect of explant on callus induction, regeneration, and transformation efficiency Among the three explants used, embryogenic calli showed the highest values on callus induction (100%) in comparison to MEH and MEP explant. However, the highest regeneration rate was obtained using mature embryo halves as explant (10.6%) (Table 3). The selection results indicated that the plantlets resistant to selective agent PPT differed significantly among explants (p<0.001). In fact, embryogenic calli Bioscience Journal | 2022 | vol. 38, e38007 | https://doi.org/10.14393/BJ-v38n0a2022-54513 5 AHANSAL, K., et al. recorded the highest value (2.41%) of resistant plantlets followed by mature embryos pieces with 0.79% and mature embryo halves with 0.36%. While transgenic plants were obtained only when using embryogenic calli as started explant with a transformation efficiency of 0.42%. The results suggested that embryogenic callus was the best explant to be used as a receptive tissue from the mature embryos for A. tumefaciens infection. Table 3. Effect of explant on callus induction, regeneration and transformation efficiency in durum wheat. Explant Callus induction (%) Plantlets regeneration (%) Plantlets resistant to PPT (%) Transformation efficiency EC 100a* 7.688b 2.406a 0.415a HME 63.257b 10.6a 0.354b 0b MEP 64.882b 7.676b 0.785b 0b LSD 2.1931 2.1287 1.0631 0.3918 *The values followed by the same alphabet are not significantly different at alpha =0.05 according to the Least Significant Difference (LSD) test. Durum wheat is known for its low regeneration capacity. The transformation by Agrobacterium reduced further callus induction and plantlets regeneration for all varieties compared to the control. The results revealed that the Amria genotype was a more sensitive genotype to Agrobacterium infection than the others when the embryogenic callus was used as starting materials for the transformation. The highest transformation efficiency was observed in the variety Amria (2.33%) at 400 µM of AS followed by 100 µM (0.8%) and 200 µM (0.33%) concentration of AS. Moreover, the transformation efficiency was also highest in variety Chaoui (0.33%) and Marouane (1.33%) using 100 µM as a concentration of AS (Table 4). The callus induction and regeneration were significantly influenced by the type of explant (Table 5). Table 4. Effect of explant embryogenic callus (EC) and their interaction with genotype and acetosyringone (AS) concentration on plantlets regeneration, plantlets resistant to PPT, and transformation efficiency in durum wheat. AS (µM) Number of EC infected Plantlets regeneration (%) Plantlets resistant to PPT (%) Transformation efficiency (%) Amria 0 300 3.56de* 0.79c 0c 100 300 8.54cde 1.97bc 0.3bc 200 300 5.27cde 3bc 0.33bc 400 300 13.92bc 7.52ab 2.33a Control -- 23.75a 0c 0c Chaoui 0 300 5.01de 1.97bc 0c 100 300 3.42de 1.18c 0.66bc 200 300 5.52cde 2.77bc 0c 400 300 1.52e 0c 0c Control -- 22.25ab 0c 0c Marouane 0 300 11.67cd 1.18c 0c 100 300 23.57a 11.5a 1.33ab 200 300 4.73de 0c 0c 400 300 5.52cde 0c 0c Control -- 26.25a 0c 0c LSD 8.8924 6.2446 0.4147 *The values followed by the same alphabet are not significantly different at alpha =0.05 according to the Least Significant Difference (LSD) test; AS: Acetosyringone. Bioscience Journal | 2022 | vol. 38, e38007 | https://doi.org/10.14393/BJ-v38n0a2022-54513 6 Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of Moroccan durum wheat Table 5. Analysis of variance for effects of variety, explant, acetosyringone concentration (ConAS) and their interaction on callus induction, plantlets regeneration (%), plantlets resistant to PPT, and transformation efficiency (%) in durum wheat. Callus induction (%) Plantlets regeneration (%) Plantlets resistant to PPT (%) Transformation efficiency (%) Explant 540.73*** 5.45** 8.36*** 11.93*** Variety 12.29*** 3.43* 0.05 2.01 Explant*variety 8.11*** 3.94** 0.71 2.01 Concentration of AS (ConAS) 1.47 0.97 1.07* 0.04 Explant*ConAS 1.73** 4.42*** 2.14* 2.45* Variety*ConAS 2.76* 1.01 2.44* 1.89 Explant*variety*ConAS 2.32** 2.15* 3.22*** 1.89* *Significant at p<0.05; **Significant at p<0.01; ***Significant at p<0.001; ConAS: Concentration of Acetosyringone. Molecular analysis of transgenic plants and inheritance of transgenes PCR analysis was performed to confirm the presence of the bar gene, 2x35S promoter, and HVA1 gene in genomic DNA isolated from the putative transgenic plants T0. 98 plants were obtained from all treatments, 9 plants obtained from mature embryos pieces, and 14 plants from mature embryo halves. No plant among those has integrated the HAV1 gene into their genome. Among 75 plants issue from embryonic callus, 14 showed amplification with bar gene, 2x35S promoter, and HVA1 gene (Figure 1). Using embryogenic call as explant the data indicated that transgenic plants expressing HVA1 gene were successfully produced via Agrobacterium mediated transformation of Moroccan durum wheat with a transformation efficiency of 18.6% calculated by (Number of HVA1 gene-positive plants/Total PPT resistant plants) x 100 and a transformation efficiency of 0.42% calculated as (Number of HVA1 gene-positive plants/Total number of callus infected) x 100. Figure. 1 PCR analysis of genomic DNA samples of T0 transformants of durum wheat: A – using primers specific to 2x35S promoter (390 bp); B – using primers specific to bar gene (402 bp); and C – using primers specific to HVA1 gene (290 bp). The lanes M, molecular weight marker (50 bp); 1, plasmid (pTF101.1); 2, non-transformed control; 3, Water control; and lanes 4–8, PCR products of transgenic plants. Among the 14 transgenic T0 plants, 9 plants were found to be fertile, PCR amplification was further undertaken to screen for the presence of bar gene, 2x35S promoter, and HVA1 gene in the T1 progeny. In 5 of 9 T1 plants, bar gene, 2x35S promoter, and HVA1 gene were integrated (Figure 2). 2 3 4 5 6 7 8 1 M A 300 bp 400 bp 2 3 4 5 6 7 8 1 M 400 bp B C 2 3 4 5 6 7 8 1 M 300 bp Bioscience Journal | 2022 | vol. 38, e38007 | https://doi.org/10.14393/BJ-v38n0a2022-54513 7 AHANSAL, K., et al. Figure. 2 PCR analysis of genomic DNA samples of T1 transformants of durum wheat: A – using primers specific to 2x35S promoter (390 bp); B – using primers specific to bar gene (402 bp); and C – using primers specific to HVA1 gene (290 bp). The lanes M, molecular weight marker (50 bp); 1, plasmid (pTF101.1); 2, non-transformed control; 3, Water control; and lanes 4–8, PCR products of transgenic plants. 4. Discussion Durum wheat is considered as one of the most recalcitrant species for in vitro regeneration and Agrobacterium-mediated transformation, mainly due to the low efficiency of plant regeneration under tissue culture conditions. Since the first transgenic bread wheat plant by Agrobacterium-mediated transformation using immature embryos (Cheng et al. 1997), more research focused on transgenic wheat via Agrobacterium had been given. However, Agrobacterium-mediated transformation of wheat is known to be affected by different factors including genotype, explant, AS concentration. The present investigation was initiated to develop an efficient gene delivery protocol for durum wheat using three explants (embryogenic calli, mature embryo halves, and mature embryo pieces) derived from mature embryos of Moroccan durum wheat varieties of Amria, Chaoui, and Marouane; and four different concentrations of AS (0, 100, 200 and 400 µM). Our results showed that the plantlets' regeneration and transformation efficiency was significantly influenced by explant. The transgenic plants were obtained only when using embryogenic calli as explant and no transgenic plant was recorded using mature embryo halves and mature embryos pieces. This is not in line with the results of Wang et al. (2009) where mature embryo halves explant was generally more suitable for transformation, with a frequency of 0.12% to 1.79%, for bread wheat varieties (Yumai 66, Lunxuan 208 and Bobwhite). Other studies had also shown that the frequencies of resistant plantlets were higher using MEH (0.53 to 11.62%) compared to EC (0 to 6.93%) (Aadel et al. 2018). Patnaik et al. (2006) successfully generated transgenic durum wheat through Agrobacterium mediated transformation with the frequency of 1.28% and 1.54% using the constructs LBA4404 (pBI101/Act1) and LBA4404 (pCAMBIA/pin2) respectively with mature embryo-derived callus. In our study, the transformation frequency ranged from 0.33% to 2.33% using embryogenic calli in the three varieties of durum wheat (Amria, Chaoui, and Marouane). This percentage is higher compared to the result obtained by Wang et al. (2009) and Patnaik et al. (2006). Furthermore, the average rate of transformation was higher than those reported for Moroccan durum wheat using mature embryos and biolistic transformation (0.66%; Hakam et al. 2016). Other results revealed a higher transformation efficiency (average 3.1%) of durum wheat cv. Ofanto with a superbinary pGreen/pSoup system (Wu et al. 2007). He et al. (2010) had further improved the transformation efficiency to 6.3% in durum wheat cv. Stewart. The lower value of transformation efficiency obtained in our experiments compared to He et al. (2010) and Wu et al. (2007) could be due to several factors such as the use of a different genotype, explant or transformation conditions. M 1 2 3 4 5 6 7 8 A 400 bp M 1 2 3 4 5 6 7 8 C 400 bp M 1 2 3 4 5 6 7 8 B 400 bp 200 bp Bioscience Journal | 2022 | vol. 38, e38007 | https://doi.org/10.14393/BJ-v38n0a2022-54513 8 Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of Moroccan durum wheat Our results confirm that AS is necessary for the Agrobacterium mediated transformation in durum wheat, which is in line with several other reports in bread wheat (Cheng et al. 1997; Wu et al. 2003; He et al. 2010). In our study, the highest transformation efficiency was observed in the variety Amria (2.33%) at concentration 400 µM of AS. Also, in the case of variety Amira, increase in the concentration of AS, increased transformation efficiency, which is in line with the observation of He et al. (2010) where they suggested that an increase of AS concentration from 200 µM to 400 µM improve final transformation. Another study has also shown that the use of a co-culture medium supplemented with AS at a concentration of 400 µM was the most favorable for T-DNA delivery (Manfroi et al.2015). Aadel et al. (2018) had reported that 200 μM of AS was the optimal concentration for the transformation of bread wheat. In contrary with Rashid et al. (2011), they reported that only 50 µM of AS gives maximum transformation efficiency in wheat. 5. Conclusions In conclusion, we have successfully introduced the barley HVA1 gene into Moroccan durum wheat varieties via Agrobacterium-mediated transformation using embryogenic calli originating from mature embryos as an explant and using Acetosyringone to improve transformation efficiency. The transformation protocol was developed for the first time in Moroccan durum wheat varieties. Authors' Contributions: AHANSAL, K.: conception and design, acquisition of data, analysis and interpretation of data, drafting the article; ABDELWAHD, R.: conception and design, critical review of important intellectual content; UDUPA, S.M.: conception and design, critical review of important intellectual content; AADEL, H.: , acquisition of data; GABOUN, F.: analysis and interpretation of data; IBRIZ, M.: conception and design; IRAQI, D.: conception and design, critical review of important intellectual content. All authors have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: Not applicable. Acknowledgments: The authors would like to thank the funding for the realization of this study provided by ICGEB (International Centre for Genetic Engineering and Biotechnology - Italy), and ICARDA (International Center for Agricultural Research in the Dry Areas - Morocco). References AADEL, H., et al. 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Plant cell reports. 2003, 21(7), 659–668. https://doi.org/10.1007/s00299-002-0564-7 Received: 7 May 2020 | Accepted: 17 September 2021 | Published: 16 February 2022 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 work is properly cited. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x https://doi.org/10.5897/AJB10.770 https://af.reuters.com/article/moroccoNews/idAFL8N1TL51N https://doi.org/10.1111/j.1467-7652.2006.00209.x https://doi.org/10.1007/s001220050899 https://www.fas.usda.gov/data/morocco-grain-and-feed-update-12 https://doi.org/10.1038/nbt0891-743 https://doi.org/10.1038/nbt0692-667 https://www.world-grain.com/articles/13887-moroccos-wheat-production-to-reach-historic-low https://www.world-grain.com/articles/13887-moroccos-wheat-production-to-reach-historic-low https://doi.org/10.1556/CRC.37.2009.1.1 https://doi.org/10.1007/s11248-007-9116-9 https://doi.org/10.1007/s00299-002-0564-7