- Email: [email protected]
A new method for high transgenic shoot regeneration frequency via Agrobacterium tumefaciens
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Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
ment length polymorphism analysis (RFLP) of rDNA amplicons with endonucleases AluI, BaeI, BfaI, HhaI, HindIII, and MboI. RFLP profiles indicated that phytoplasma belonged to 16SrII peanut withces’-broom group. A phylogenetic tree was also constructed using the Neighbor Joining plot option of the Clustal X program. The sequence clustered together with 16SrII group phytoplasmas, Crotalaria witches’-broom phytoplasma (EF656454.1) and Sweet potato witches’-broom phytoplasma (DQ452417.1) available in GenBank. To our knowledge, this is the first report of detection and identification of peanut witches’-broom group phytoplasma infecting sesame plants in Turkey in addition to previously published 16 SrIX group phytoplasma. http://dx.doi.org/10.1016/j.copbio.2013.05.080 Sensitive detection of sesame phytoplasmas of 16SrII and 16SrIX groups in its plant and insect host using real-time PCR Cengiz Ikten 1 , Rustem Ustun 2 , Mursel Catal 1 , Engin Yol 2 , Bulent Uzun 2 1 Department of Plant Protection, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey 2 Department of Field Crops, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey E-mail address: [email protected] (B. Uzun).
Phytoplasmas have been detected by PCR amplification using the conserved 16S rRNA gene. A nested PCR has also been used for diagnosis on plant or insect vector samples for increasing sensitivity. Real-time PCR is an alternative method for pyhtoplasma detection because it is not only its elevated detection sensitivity but also its short analysis time and high automation capability. Recently, 16SrII and 16SrIX groups of phytoplasmas were shown to infect sesame plants. Real-time PCR assays conjugated with Taqman probes have been developed for rapid, sensitive and quantitative detection of sesame phytoplasmas belonging to groups 16SrII and 16SrIX in sesame plants and insect vectors. The phytoplasmas species were amplified using universal primers of P1/P7 and the amplicons were sequenced. Based on the obtained sequences information, primers and probes specific to 16SrII and 16SrIX groups were designed. Those primers and probes specifically amplified 107 bp and 127 bp fragments from the 16S ribosomal DNA, respectively and reacted positively with 16SrII and 16SrIX phytoplasmas. The healthy and no-template controls had a lower or no melting peak. The rapid, sensitive, specific and reliable rtPCR showed its potential to become a powerful tool for early detection and quantification of the phytoplasmas associated with sesame phyllody and will be very useful for sesame breeding programs to identify the resistance sources against the disease. It is also demonstrated that this real time PCR method was highly powerful for detection and identification of phytoplasmas in the vector insect. http://dx.doi.org/10.1016/j.copbio.2013.05.081
New step of plant biotechnology in post-genomics era Olga I Kershanskaya Institute Plant Biology and Biotechnology MES RK, Almaty, Kazakhstan E-mail address: gen [email protected]. The next generation of biotech crops promises to include a broad range of products that will provide benefits to both farmers and consumers, and continue to meet the global agricultural challenges. These products will most likely involve regulation of key endogenous plant pathways resulting in improved quantitative traits such as yield, photosynthesis, and stress tolerance. Genetic engineering of key metabolic pathways is a powerful tool for crop improvement in new step biotech in post-genomics era. To date, successes in genetic improvement of environmental stress resistance have involved manipulation of a single or a few genes involved in signaling/regulatory pathways. The emergence of novel ‘omics’ technologies: genomics, proteomics and metabolomics, is now allowing researchers to identify the genetic behind plant stress responses.The aim of our project is to improve soybean innate resistance to biotic and abiotic stresses via genetic engineering of the phenylpropanoid pathway, namely — introduction into soybean key genes involved in lignin biosynthesis — the compound that is assigned to a broad range of physiological processes participating in plant growth, providing the rigidity to the cell walls, the natural mechanical barrier and defense against pathogen penetration. Obtained results: first, gene constructs of key genes involved in lignin biosynthesis — PAL, CCR — prepared for introduction into soybean; secondly, optimized germ-line genetic transformation technique for soybean transformation; thirdly, molecular confirmed soybean transgenes of T1–T2 generations with valuable genes; fourthly, biochemical confirmation of increased lignin biosynthesis, metabolic profiling. So, transition is achieved from Genome to Phenome in post-genomics era. http://dx.doi.org/10.1016/j.copbio.2013.05.083 A new method for high transgenic shoot regeneration frequency via Agrobacterium tumefaciens Mustafa Yıldız, Behrouz Alizadeh, Ramazan Beyaz, Cansu Telci Kahramanogulları, Murat Aycan University of Ankara, Faculty of Agriculture, Department of Field Crops, Diskapi, Ankara, Turkey E-mail address: [email protected] (M. Yıldız). In the current study, routinely used method which includes transferring explants onto co-cultivation medium after bacterial inoculation for a while, was compared to three different inoculation methods. In every three methods, 7-day-old sterile seedlings having cotyledon leaves and no root system, were dried under air flow in sterile cabin for 35 min and the effects of different inoculation periods and different volumes of bacterial solution added to liquid inoculation medium on gene transfer frequency were compared. The reason why 7-day-old seedlings having cotyledon leaves were dried under air flow in sterile cabin was to enable bacterial solution to penetrate rapidly towards inner cells of the seedling by increased osmotic pressure of seedlings and by this way to increase the number of cells inoculated and consequently to increase the transgenic shoot frequency. After inoculation, hypocotyl segments — 0.5 cm in length — were excised and left to co-cultivation for 2 days. In the study, sterile seedlings grown from seeds of flax (Linum usitatissimum L.) cvs. ‘Madaras’, ‘Clarck’ and ‘1886 Sel.’ and ‘GV2260 GUS INT’ line of Agrobacterium tumefaciens were used. At the end of the study, the highest results were obtained from the application
Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
in which 35-min-dried flax seedlings with cotyledons were inoculated with 500 l of bacterial solution added to liquid regeneration medium for 20 min.
S41
of BAP and NAA on regeneration and rooting from leaf explant of E. purpurea under in vitro conditions. http://dx.doi.org/10.1016/j.copbio.2013.05.086
http://dx.doi.org/10.1016/j.copbio.2013.05.084 Progressive development of plant biotechnology in Belarus Alexander Kilchevsky Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Belarus E-mail address: [email protected]. The main trend in development of plant biotechnology in Belarus is study on structural and functional organization of plant genomes, development of genetic and cell engineering issues, working out of genomic and cellular biotechnologies for improving the efficiency of plant breeding. The technologies for producing haploids of potato, wheat, triticale and sugar beet were developed in the field of cell engineering. In the field of transgenesis were produced plants as follows: potato resistant to Y-virus, fungal diseases and insects; rape and fiber flax resistant to glyphosate; clover with increased productivity and cranberries with improved taste qualities. The methods of marker-assisted selection (MAS) were developed for 70 genes of resistance and quality of 8 crops (potato, tomato, wheat, apple, rape, soya, barley, flax). The systems of genetic certification were tested on the basis of molecular markers for 10 agricultural crops. More of 170 plant cultivars mentioned above were certified. The results obtained are introduced into practice of breeding institutions of the Republic of Belarus by the innovative center at the Institute of Genetics and Cytology — Republican Centre of Genomic Biotechnologies. http://dx.doi.org/10.1016/j.copbio.2013.05.085 Effect of BAP and NAA plant growth regulators in in vitro regeneration and rooting of Echinacea purpurea (L.) Moench Sam Mokhtarzadeh 1 , Farzad Niyazpour 2 , Farzad Nofouzi 3 , Mohsen Mirzapour 3 , Khalid Mahmood Khawar 3 , Nese Kirimer 1 1 Department of Pharmacognosy, Pharmacy Faculty, University of Anadolu, Eskisehir 26470, Turkey 2 Veterinary Faculty, University of Tabriz, Tabriz, Iran 3 Department of Field Crops, Faculty of Agriculture, Ankara University, Ankara 06110, Turkey E-mail address: [email protected] (S. Mokhtarzadeh).
Eastern purple coneflower or purple coneflower (Echinacea purpurea L.) family Asteraceae, genus Echinacea is an important herbaceous perennial species of flowering plants that grow to 120 cm at maturity. It blooms during spring and summer and is evaluated as an ornamental and medicinal plant. A review of literature suggests development of numerous cultivars for flower quality and medicinal characteristics. It has anti-depressant and immune system stimulating characteristics. Optimum in vitro and ex vitro culture techniques are unknown in Turkey and there is need to optimize these techniques for these plants. It is hoped that development of in vitro protocols for Echinacea sp. can play an important role in establishing of this novel and useful germplasm in Turkey. It will also help in rapid multiplication, biotransformation and genetic transformation for an enhanced phytochemical production. This study reports effects of different concentrations
Study of callus induction and regeneration of Papaver somniferum L. Fereydoon Bondarian 1 , Sepideh Maziar Bahreini 3
Torabi 1 , Mansour
Omidi 2 ,
1 Department of Plant Breeding, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Agronomy & Plant Breeding, University of Tehran, Karaj, Iran 3 Department of Agricultural Biotechnology, Science and Research Branch, Islamic Azad University. Tehran, Iran E-mail address: [email protected] (F. Bondarian).
Papaver somniferum L. (opium poppy) is an herbaceous, annual and diploid plant that is very important because of pharmacological and strategic view in the world. The limitation of this plant farming, hard and law amount extraction of this plant alkaloids cause studying about tissue culture of Papaver somniferum L. for getting high level of these components. This plant has a special group of alkaloids named benzylisoquinolin is due to be secondary metabolites such as morphine, codeine and papaverine is a great value in pharmacy. In this research tissue culture of this plant to achieve the best hormonal levels for callus induction and regeneration examined from seed way culture. Small samples of leaves, roots and hypocotyls of 30–45 days from seedlings, and were transferred to different media with hormonal treatments. Best of hormone treatment for callus induction is 2 mg/l 2,4-D and 0.25 mg/l BAP, 4 mg/l 2,4-D and 0.5 mg/l BAP and best hormonal levels to regeneration includes 1 mg/l BAP and 0.1 mg/l 2,4-D. http://dx.doi.org/10.1016/j.copbio.2013.05.087 Gynogenesis induction in Allium tuberosum L. Fevziye Celebi Toprak, Arzu Kaska, Ali Ramazan Alan Pamukkale University, Plant Genetics and Agricultural Biotechnology Application and Research Center (PAU BIYOM), Kinikli, Denizli 20070, Turkey E-mail address: [email protected] (F.C. Toprak). The present study describes production of plantlets using immature flower buds of Allium tuberosum cultured on various gynogenesis induction media. The gynogenesis induction experiments were carried out by culturing about 21 thousand immature flower buds collected from 30 donor plants during the summer of 2012. In the induction experiments, buds were cultured on BDS, B5 and MS based media with varying amounts of sugar and growth regulators. Gynogenic plantlets started to emerge from cultured buds about 3 months after culture initiation. Gynogenic plantlets were obtained from all media types tested although frequencies of gynogenic plantlet production in various media were significantly different. Induction experiments provided a total of 6100 gynogenic plantlets. Analysis of nuclei samples isolated from gynogenic plants with flow cytometry showed that majority of the regenerants had nuclear DNA contents similar to donor plants. The gynogenic lines were transferred to in vivo successfully. Gynogenic lines are being characterized and compared with donor lines for various features in the greenhouse. http://dx.doi.org/10.1016/j.copbio.2013.05.088
Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
ment length polymorphism analysis (RFLP) of rDNA amplicons with endonucleases AluI, BaeI, BfaI, HhaI, HindIII, and MboI. RFLP profiles indicated that phytoplasma belonged to 16SrII peanut withces’-broom group. A phylogenetic tree was also constructed using the Neighbor Joining plot option of the Clustal X program. The sequence clustered together with 16SrII group phytoplasmas, Crotalaria witches’-broom phytoplasma (EF656454.1) and Sweet potato witches’-broom phytoplasma (DQ452417.1) available in GenBank. To our knowledge, this is the first report of detection and identification of peanut witches’-broom group phytoplasma infecting sesame plants in Turkey in addition to previously published 16 SrIX group phytoplasma. http://dx.doi.org/10.1016/j.copbio.2013.05.080 Sensitive detection of sesame phytoplasmas of 16SrII and 16SrIX groups in its plant and insect host using real-time PCR Cengiz Ikten 1 , Rustem Ustun 2 , Mursel Catal 1 , Engin Yol 2 , Bulent Uzun 2 1 Department of Plant Protection, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey 2 Department of Field Crops, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey E-mail address: [email protected] (B. Uzun).
Phytoplasmas have been detected by PCR amplification using the conserved 16S rRNA gene. A nested PCR has also been used for diagnosis on plant or insect vector samples for increasing sensitivity. Real-time PCR is an alternative method for pyhtoplasma detection because it is not only its elevated detection sensitivity but also its short analysis time and high automation capability. Recently, 16SrII and 16SrIX groups of phytoplasmas were shown to infect sesame plants. Real-time PCR assays conjugated with Taqman probes have been developed for rapid, sensitive and quantitative detection of sesame phytoplasmas belonging to groups 16SrII and 16SrIX in sesame plants and insect vectors. The phytoplasmas species were amplified using universal primers of P1/P7 and the amplicons were sequenced. Based on the obtained sequences information, primers and probes specific to 16SrII and 16SrIX groups were designed. Those primers and probes specifically amplified 107 bp and 127 bp fragments from the 16S ribosomal DNA, respectively and reacted positively with 16SrII and 16SrIX phytoplasmas. The healthy and no-template controls had a lower or no melting peak. The rapid, sensitive, specific and reliable rtPCR showed its potential to become a powerful tool for early detection and quantification of the phytoplasmas associated with sesame phyllody and will be very useful for sesame breeding programs to identify the resistance sources against the disease. It is also demonstrated that this real time PCR method was highly powerful for detection and identification of phytoplasmas in the vector insect. http://dx.doi.org/10.1016/j.copbio.2013.05.081
New step of plant biotechnology in post-genomics era Olga I Kershanskaya Institute Plant Biology and Biotechnology MES RK, Almaty, Kazakhstan E-mail address: gen [email protected]. The next generation of biotech crops promises to include a broad range of products that will provide benefits to both farmers and consumers, and continue to meet the global agricultural challenges. These products will most likely involve regulation of key endogenous plant pathways resulting in improved quantitative traits such as yield, photosynthesis, and stress tolerance. Genetic engineering of key metabolic pathways is a powerful tool for crop improvement in new step biotech in post-genomics era. To date, successes in genetic improvement of environmental stress resistance have involved manipulation of a single or a few genes involved in signaling/regulatory pathways. The emergence of novel ‘omics’ technologies: genomics, proteomics and metabolomics, is now allowing researchers to identify the genetic behind plant stress responses.The aim of our project is to improve soybean innate resistance to biotic and abiotic stresses via genetic engineering of the phenylpropanoid pathway, namely — introduction into soybean key genes involved in lignin biosynthesis — the compound that is assigned to a broad range of physiological processes participating in plant growth, providing the rigidity to the cell walls, the natural mechanical barrier and defense against pathogen penetration. Obtained results: first, gene constructs of key genes involved in lignin biosynthesis — PAL, CCR — prepared for introduction into soybean; secondly, optimized germ-line genetic transformation technique for soybean transformation; thirdly, molecular confirmed soybean transgenes of T1–T2 generations with valuable genes; fourthly, biochemical confirmation of increased lignin biosynthesis, metabolic profiling. So, transition is achieved from Genome to Phenome in post-genomics era. http://dx.doi.org/10.1016/j.copbio.2013.05.083 A new method for high transgenic shoot regeneration frequency via Agrobacterium tumefaciens Mustafa Yıldız, Behrouz Alizadeh, Ramazan Beyaz, Cansu Telci Kahramanogulları, Murat Aycan University of Ankara, Faculty of Agriculture, Department of Field Crops, Diskapi, Ankara, Turkey E-mail address: [email protected] (M. Yıldız). In the current study, routinely used method which includes transferring explants onto co-cultivation medium after bacterial inoculation for a while, was compared to three different inoculation methods. In every three methods, 7-day-old sterile seedlings having cotyledon leaves and no root system, were dried under air flow in sterile cabin for 35 min and the effects of different inoculation periods and different volumes of bacterial solution added to liquid inoculation medium on gene transfer frequency were compared. The reason why 7-day-old seedlings having cotyledon leaves were dried under air flow in sterile cabin was to enable bacterial solution to penetrate rapidly towards inner cells of the seedling by increased osmotic pressure of seedlings and by this way to increase the number of cells inoculated and consequently to increase the transgenic shoot frequency. After inoculation, hypocotyl segments — 0.5 cm in length — were excised and left to co-cultivation for 2 days. In the study, sterile seedlings grown from seeds of flax (Linum usitatissimum L.) cvs. ‘Madaras’, ‘Clarck’ and ‘1886 Sel.’ and ‘GV2260 GUS INT’ line of Agrobacterium tumefaciens were used. At the end of the study, the highest results were obtained from the application
Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
in which 35-min-dried flax seedlings with cotyledons were inoculated with 500 l of bacterial solution added to liquid regeneration medium for 20 min.
S41
of BAP and NAA on regeneration and rooting from leaf explant of E. purpurea under in vitro conditions. http://dx.doi.org/10.1016/j.copbio.2013.05.086
http://dx.doi.org/10.1016/j.copbio.2013.05.084 Progressive development of plant biotechnology in Belarus Alexander Kilchevsky Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Belarus E-mail address: [email protected]. The main trend in development of plant biotechnology in Belarus is study on structural and functional organization of plant genomes, development of genetic and cell engineering issues, working out of genomic and cellular biotechnologies for improving the efficiency of plant breeding. The technologies for producing haploids of potato, wheat, triticale and sugar beet were developed in the field of cell engineering. In the field of transgenesis were produced plants as follows: potato resistant to Y-virus, fungal diseases and insects; rape and fiber flax resistant to glyphosate; clover with increased productivity and cranberries with improved taste qualities. The methods of marker-assisted selection (MAS) were developed for 70 genes of resistance and quality of 8 crops (potato, tomato, wheat, apple, rape, soya, barley, flax). The systems of genetic certification were tested on the basis of molecular markers for 10 agricultural crops. More of 170 plant cultivars mentioned above were certified. The results obtained are introduced into practice of breeding institutions of the Republic of Belarus by the innovative center at the Institute of Genetics and Cytology — Republican Centre of Genomic Biotechnologies. http://dx.doi.org/10.1016/j.copbio.2013.05.085 Effect of BAP and NAA plant growth regulators in in vitro regeneration and rooting of Echinacea purpurea (L.) Moench Sam Mokhtarzadeh 1 , Farzad Niyazpour 2 , Farzad Nofouzi 3 , Mohsen Mirzapour 3 , Khalid Mahmood Khawar 3 , Nese Kirimer 1 1 Department of Pharmacognosy, Pharmacy Faculty, University of Anadolu, Eskisehir 26470, Turkey 2 Veterinary Faculty, University of Tabriz, Tabriz, Iran 3 Department of Field Crops, Faculty of Agriculture, Ankara University, Ankara 06110, Turkey E-mail address: [email protected] (S. Mokhtarzadeh).
Eastern purple coneflower or purple coneflower (Echinacea purpurea L.) family Asteraceae, genus Echinacea is an important herbaceous perennial species of flowering plants that grow to 120 cm at maturity. It blooms during spring and summer and is evaluated as an ornamental and medicinal plant. A review of literature suggests development of numerous cultivars for flower quality and medicinal characteristics. It has anti-depressant and immune system stimulating characteristics. Optimum in vitro and ex vitro culture techniques are unknown in Turkey and there is need to optimize these techniques for these plants. It is hoped that development of in vitro protocols for Echinacea sp. can play an important role in establishing of this novel and useful germplasm in Turkey. It will also help in rapid multiplication, biotransformation and genetic transformation for an enhanced phytochemical production. This study reports effects of different concentrations
Study of callus induction and regeneration of Papaver somniferum L. Fereydoon Bondarian 1 , Sepideh Maziar Bahreini 3
Torabi 1 , Mansour
Omidi 2 ,
1 Department of Plant Breeding, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Agronomy & Plant Breeding, University of Tehran, Karaj, Iran 3 Department of Agricultural Biotechnology, Science and Research Branch, Islamic Azad University. Tehran, Iran E-mail address: [email protected] (F. Bondarian).
Papaver somniferum L. (opium poppy) is an herbaceous, annual and diploid plant that is very important because of pharmacological and strategic view in the world. The limitation of this plant farming, hard and law amount extraction of this plant alkaloids cause studying about tissue culture of Papaver somniferum L. for getting high level of these components. This plant has a special group of alkaloids named benzylisoquinolin is due to be secondary metabolites such as morphine, codeine and papaverine is a great value in pharmacy. In this research tissue culture of this plant to achieve the best hormonal levels for callus induction and regeneration examined from seed way culture. Small samples of leaves, roots and hypocotyls of 30–45 days from seedlings, and were transferred to different media with hormonal treatments. Best of hormone treatment for callus induction is 2 mg/l 2,4-D and 0.25 mg/l BAP, 4 mg/l 2,4-D and 0.5 mg/l BAP and best hormonal levels to regeneration includes 1 mg/l BAP and 0.1 mg/l 2,4-D. http://dx.doi.org/10.1016/j.copbio.2013.05.087 Gynogenesis induction in Allium tuberosum L. Fevziye Celebi Toprak, Arzu Kaska, Ali Ramazan Alan Pamukkale University, Plant Genetics and Agricultural Biotechnology Application and Research Center (PAU BIYOM), Kinikli, Denizli 20070, Turkey E-mail address: [email protected] (F.C. Toprak). The present study describes production of plantlets using immature flower buds of Allium tuberosum cultured on various gynogenesis induction media. The gynogenesis induction experiments were carried out by culturing about 21 thousand immature flower buds collected from 30 donor plants during the summer of 2012. In the induction experiments, buds were cultured on BDS, B5 and MS based media with varying amounts of sugar and growth regulators. Gynogenic plantlets started to emerge from cultured buds about 3 months after culture initiation. Gynogenic plantlets were obtained from all media types tested although frequencies of gynogenic plantlet production in various media were significantly different. Induction experiments provided a total of 6100 gynogenic plantlets. Analysis of nuclei samples isolated from gynogenic plants with flow cytometry showed that majority of the regenerants had nuclear DNA contents similar to donor plants. The gynogenic lines were transferred to in vivo successfully. Gynogenic lines are being characterized and compared with donor lines for various features in the greenhouse. http://dx.doi.org/10.1016/j.copbio.2013.05.088