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An Efficient Technique for Obtaining Solanum nigrum L. Plants Superior In Their Alkaloid Contents Via Transformation by Agrobacterium rhizogenes R1601

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... Hairy root cultures (HRCs) inoculated with A. rhizogenes have been used to treat a wide range of plants [5]. For example, Transgenic Solanumnigrum L. plants could emerge spontaneously from these induced roots [6]. Moreover, the roots induced by A. rhizogenes 1601 harbouring Riplasmid also stimulated the formation of transgenic callus from sugar beet plants [7]. ...
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Protoplasts represent a unique experimental system for the circulation and formation of genetically modified plants. Here, protoplasts were isolated from genetically modified hairy root tissues of Brassica oleracea L. induced by the Agrobacterium rhizogenes strain (ATCC13332). The concentration of enzyme solutions utilized for protoplast isolation was 1.5 % Cellulase YC and 0.1 % Pectolyase Y23 in 13% mannitol solution, which resulted in high efficiency of isolation within 8 hours, in which the protoplast yield was 2 × 104 cells ml-1 and the percentage of viability was 72%. Each protoplast has one nucleus with a nucleation of 48%. A polymerase chain reaction (PCR) assay verified the presence of rol B and rol C genes in hairy root tissues by detaching a single bundle of DNA replication from these roots using a specific pair of primers. The current study demonstrated that A. rhizogenes strain (ATCC13332) is a vector for the incorporation of T-DNA genes into cauliflower plants, as well as the success of the hairy roots retention of rol B and rol C genes transferred to it.
... The rapid growth of transformed hairy roots and its dense content of root hairs, may due to the length of apical meristem for these roots compared with untransformed roots (20), as well as, to the increased rates of cell division (21). These transformed hairy root represent an efficient alternative approach to regenerate into plants as occur in Solanum nigrum (22) or through callus-derived from hairy roots (23). ...
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Hairy roots were induced on leaf explants excised from sugarbeet (Beta vulgaris) plants when inoculated by Agrobacterium rhizogenes R1601. Paper electrophoresis proved the presence of agropine, the unusual amino acid in these roots which demonstrated the transformation of these tissues. Transformed hairy roots were easily grown on agar-solidified Murashige and Skoog, 1962 (MS) medium containing 0.5mgl-1 2,4dichlorophenoxy acetic acid (2,4-D) or B5 medium supplemented with a mixture of 0.7 mgl-1 Benzyl Adenine (BA) and 0.7 mgl-1 (2,4-D). Callus produced from these hairy roots grown on MS medium was of friable and white in color, while brown in color on B5 medium. Calli were grown efficiently in liquid B5 medium provided with a mixture of 1.0 mgl-1 kinetine (Kin.) and 1.0 mgl-1 2,4-D. These results may contribute in transformation of sugarbeet plants. 456 ‫اﻟﻛﺎﻟس‬ ‫ﺗﻛوﯾن‬ ‫اﻟﻣﻌدل‬ ‫اﻟﻣﺷﺗق‬ ً ‫اﺛﯾﺎ‬ ‫ور‬ ً ‫اﺛﯾﺎ‬ ‫ور‬ ‫اﻟﻣﺣوﻟﺔ‬ ‫اﻟﺷﻌرﯾﺔ‬ ‫اﻟﺟذور‬ ‫ﻣن‬ ‫اﻟﻣﺗﻛوﻧﺔ‬ ‫ﻋﻠﻰ‬ ‫أ‬ ‫اﻟﺳﻛري‬ ‫اﻟﺑﻧﺟر‬ ‫اق‬ ‫ور‬ Beta vulgaris ‫ﺑ‬ ‫ﺑﻛﺗرﯾﺎ‬ Agrobacterium rhizogenes 1601 ‫ﺑﻼزﻣﯾدات‬ ‫ﻋﻠﻰ‬ ‫اﻟﺣﺎوﯾﺔ‬ Ri ‫اﻟﻧﻌﻣﺔ‬ ‫ﺷﻌﯾب‬ ‫ﻗﺗﯾﺑﺔ‬ ‫اﻟﻣﻼح‬ ‫ﻗﺎﺳم‬ ‫اﺣم‬ ‫ﻣز‬ ‫اﻟ‬ ‫اﻟﺗﻘﻧﯾﺎت‬ ‫وﺣدة‬ ‫ﺣﯾﺎﺗﯾﺔ‬ ، ‫اﻟﺣﯾﺎة‬ ‫ﻋﻠوم‬ ‫ﻗﺳم‬ ، ‫اﻟﺗرﺑﯾﺔ‬ ‫ﻛﻠﯾﺔ‬ ، ‫اﻟﻣوﺻل‬ ‫ﺟﺎﻣﻌﺔ‬ ‫اﻟﺧﻼﺻﺔ‬ ‫ﻋﻠﻰ‬ ‫اﻟﺷﻌرﯾﺔ‬ ‫اﻟﺟذور‬ ‫ﺗﻛوﻧت‬ ‫اق‬ ‫أور‬ ‫اﻟﺳﻛري‬ ‫اﻟﺑﻧﺟر‬ ‫ﻧﺑﺎﺗﺎت‬) Beta vulgaris (، ‫ﻣن‬ ‫اﻟﻣﻔﺻوﻟﺔ‬ ‫ﻧﺎﻣﯾﺔ‬ ‫ﻧﺑﺎﺗﺎت‬ ‫اﻟﺣﻘل‬ ‫ﻓﻲ‬ ، ‫اﻟﻣ‬ ‫ﻓﻲ‬ ‫اﻗﻊ‬ ‫و‬ ‫ﺑﺑﻛﺗرﯾﺎ‬ ‫اﻟﻣﻠﻘﺣﺔ‬ Agrobacterium rhizogenes R1601. ‫ﻧﺗﺎﺋﺞ‬ ‫أﻛدت‬ ‫و‬ ‫اﻟ‬ ‫ﺗرﺣﯾل‬ ‫اﻻﻛروﺑﯾن،‬ ‫ﻋن‬ ‫ﻟﻠﻛﺷف‬ ‫اﻟﻛﻬرﺑﺎﺋﻲ‬ ‫اﻟ‬ ‫ﺣﺎﻣض‬ ‫اﻷ‬ ‫ﻣﯾﻧﻲ‬ ‫ﻏﯾر‬ ‫اﻻ‬ ‫ﻋﺗﯾﺎدي،‬ ‫ﻧﺟﺎح‬ ‫ﻫذﻩ‬ ‫ﻓﻲ‬ ‫اﺛﻲ‬ ‫اﻟور‬ ‫اﻟﺗﺣول‬ ‫اﻷﻧﺳﺟﺔ‬. ‫و‬ ٕ ‫ا‬ ‫ﻧﻣوﻩ‬ ‫ﺑﺳﻬوﻟﺔ‬ ‫اﻟﺟذور‬ ‫ﻫذﻩ‬ ‫ﻣن‬ ‫اﻟﻣﺗﻛون‬ ‫اﻟﻛﺎﻟس‬ ‫اﻣﺗﺎز‬ ‫ﻋﻠﻰ‬ ‫وﺳط‬ MS ‫ﻋﻠﻰ‬ ‫اﻟﺣﺎوي‬ ‫اﻟﺻﻠب‬ 0.5 ‫ﻟﺗر‬ ‫ﻣﻠﻐم‬-1 2,4-D ‫و‬ ‫وﺳط‬ ‫ﻋﻠﻰ‬ B5 ‫اﻟﺻﻠب‬ ‫اﻟ‬ ‫ﺑـ‬ ‫ﻣﺟﻬز‬ 0.7 ‫ﻣﻠﻐم‬ ‫ﻟﺗر‬-1 BA ‫و‬ 0.7 ‫ﻣﻠ‬ ‫ﻐم‬ ‫ﻟﺗر‬-1 2,4-D ‫و‬ ‫أ‬ ‫ﺗﺻف‬ ‫اﻣﻪ‬ ‫ﺑﻘو‬ ‫وﻟ‬ ‫اﻟﻬش‬ ‫وﻧﻪ‬ ‫وﺳط‬ ‫ﻋﻠﻰ‬ ‫اﻻﺑﯾض‬ MS ‫وﻟوﻧﻪ‬ ‫اﻟﺑﻧﻲ‬ ‫وﺳط‬ ‫ﻋﻠﻰ‬ B5. ً ‫ﺳﻠوﻛﺎ‬ ‫ﻧﻣوﻩ‬ ‫ﻓﻲ‬ ‫اﻟﻛﺎﻟس‬ ‫وﺳﻠك‬ ً ‫ﻣﻣﺎﺛﻼ‬ ‫ﻋﻠﻰ‬ ‫وﺳط‬ B5 ‫اﻟﺳﺎﺋل‬ ‫ﻋﻠﻰ‬ ‫اﻟﺣﺎوي‬ 1.0 ‫ﻣﻠﻐم‬ ‫ﻟﺗر‬-1 Kin. ‫و‬ 1.0 ‫ﻣﻠﻐم‬ ‫ﻟﺗر‬-1 2,4-D. ً ‫ﺟدا‬ ‫ﻣﻬﻣﺔ‬ ‫اﻟﻧﺗﺎﺋﺞ‬ ‫ﻫذﻩ‬ ‫ﻓﻲ‬ ‫اﻟﺣﯾوﯾﺔ‬ ‫اﻟﺗﻘﺎﻧﺔ‬ ‫ﻣﺟﺎﻻت‬ ‫ﻟ‬ ‫ﻠﻧﺑﺎﺗﺎت‬ ‫ﻻن‬ ً ‫ﺧﻠوﯾﺎ‬ ً ‫ﺧطﺎ‬ ‫ﺗﻌد‬ ‫اﻟﻣﺗﻛوﻧﺔ‬ ‫اﻟﻛﺎﻟس‬ ‫ارع‬ ‫ﻣز‬ ‫ﻓﻲ‬ ‫ﻣﻬﻣﺎ‬ ‫إ‬ ‫ﻧﺗﺎج‬ ‫ﺳﻛري‬ ‫ﺑﻧﺟر‬ ‫ﻧﺑﺎﺗﺎت‬ ً ‫اﺛﯾﺎ‬ ‫ور‬ ‫ﻣﺣوﻟﺔ‬ ‫ﻣن‬ ‫ﻣﺑﺎﺷرة‬ ‫ﻫذا‬ ‫اﻟﻛﺎﻟس‬ .
... The rapid growth of transformed hairy roots and its dense content of root hairs, may due to the length of apical meristem for these roots compared with untransformed roots (Meyer, et al., 2000). The development of transformed hairy roots on various plant species are involving different time, like 15 days of inoculation in Solanum nigrum (Al-Mallah and Salih, 2006), and 4 weeks in Beta vulgaris (Al-Mallah and Al-Nema, 2012). ...
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Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a shortcut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.
... The rapid growth of transformed hairy roots and its dense content of root hairs, may due to the length of apical meristem for these roots compared with untransformed roots (Meyer, et al., 2000). The development of transformed hairy roots on various plant species are involving different time, like 15 days of inoculation in Solanum nigrum (Al-Mallah and Salih, 2006), and 4 weeks in Beta vulgaris (Al-Mallah and Al-Nema, 2012). ...
Article
Full-text available
Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.
Article
Full-text available
Shoots were regenerated from petioles – derived callus on solidified Murashige and Skoog (MS) medium supplemented with 1.5 mg / l BA only and MS medium containing 4.0 mg / l IBA and 1.0 mg / l BA . The percentage of shoot formation was 30.0 % . The regeneration frequency was reasonable to obtained sufficient shoots by this method . This simple protocol is the first report in tissue culture of this woody plant which encourage other researchers to worke other studies in tissue culture of neem . Moreover , callus culture will be useful to obtain a wide range of industrial plant products .
Research
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Inoculation of leaves and stems explants excised from field-grown Trigonella foenum-graecum L. with engineered Agrobacterium rhizogenes 1601 inoculum of optical density 1.90, 2.06 and 1.96 led to the formation of hairy roots on these explants. The highest percent of infection with inoculum of O.D 1.90 was 20% in leaves, and was 53.3% in stems using inoculum of O.D 2.06. Infection percent was 50% in hypocotyl compared with non-inoculated explants. The results showed that inoculation of seedlings lacking roots were slow in hairy root formation and take long time compared with their formation in intact seedlings inoculated with the same inocula. Results of paper electrophoresis of these root proved the incidence of their genetic transformation. Moreover, these transformed roots grow happily in liquid WP medium with an average fresh weight 5.9g after four weeks of culture, whereas fresh weight of normal roots was 1.02g under the same conditions.
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