Regeneration of lentil (lens culinaris medik) and genetic transformation by using agrobacterium tumefaciens-mediated gene transfer

Akçay, Ufuk Çelikkol
In this study, the effects of different plant growth regulators on regeneration responses of various lentil explants through direct and indirect organogenesis and through somatic embryogenesis from calli and cell suspension cultures were investigated. Shoot regeneration was obtained in low frequencies from longitudinal embryonic axis explants and nodal buds of epicotyls, however whole plant regeneration was unsuccessful. Conditions provided for indirect organogenesis resulted only in swelling of hypocotyls and root directed ends of internodes and weak callus formation on leaves which were followed by tissue browning and necrosis. In somatic embryogenesis studies, the explants longitudinal embryonic axis and cotyledonary petioles produced soft and friable calli on MS media with Gamborg’s vitamins supplemented with 0.75mg/L 2,4-D+0.5mg/L BA. The highest average number of embryos per explant, 12.36 was observed on media containing 0.75mg/L BA +0.5mg/L 2,4-D for cotyledonary petiole explants, whereas 3mg/L BA+1mg/L NAA was the only hormone combination that allowed embryo development to some extent, in both explants. Somatic callus failed to regenerate despite globular embryo formation and embryo development to some extent. Combination of sonication treatment with Agrobacterium transformation of three lentil explants; cotyledonary nodes, half cotyledons and cotyledonary nodes with intact shoots, had no effect on the improvement of transient gus gene expression on explants. Sonication treatment was also unable to form localized wounds on the petiole axils. The best gus gene expression on the axil region was obtained when cotyledonary nodes and KYRT1 strain were used in combination with vacuum infiltration and scalpel wounding of the axils. Gradual selection and repeated removal of regenerated shoots between selection cycles increased the number of gus expressing shoots significantly. The regenerated shoots were grafted on root stocks and whole plant regeneration was achieved in greenhouse conditions. By the use of the optimized Agrobacterium-mediated transformation protocol, 4 independent lines were obtained with 2.3% transformation efficiency. Southern blot analysis confirmed the integration of the gus gene into the genome of lentil plants. T0 plants were fertile and all plants showed Mendelian segregation of the gus gene in 3:1 ratio to their progenies except one line which carries three copies of the gene. Reverse transcription PCR has confirmed the expression of the genes in T0 and T1 generations. T0 plants and the following three generations strongly expressed gus gene uniformly in their tissues and the PCR amplifications of both gus and npt-II genes was successful through generations.


In vitro induction of growth and development of common juniper(Juniperus communis L.) from shoot and bud explants
Koçer, Zeynep Ahsen; Kaya, Zeki; Department of Biotechnology (2005)
The objective of the study was to investigate the optimum conditions for in vitro regeneration of common juniper (Juniperus communis L.) by using indirect organogenesis approach. Throughout the study; callus induction, organogenesis, improved organogenesis and root induction experiments were performed sequentially. It was found that explant position, genotype, gender, treatments and sampling time had significant effects on callus induction rate in common juniper. The results of treatments indicated that IBA...
Genetic transformation of lentil ( Lens culinaris m. cv.Sultan.1) with a transcription factor regulator (MBF1c) and analysis of transgenic plants
Kamçı, Hamdi; Çelikkol Akçay, Ufuk; Kamçı, Hamdi; Department of Biotechnology (2011)
Agrobacterium mediated genetic transformation of lentil Sultan 1 cultivar with MBF1c and evaluation of transgenic plants was aimed. The study was initially based on optimized protocol with Agrobacterium tumefaciens KYRT1 strain and pTJK136 binary plasmid. Based on this protocol and transient marker gene expression in embryo apex, 15% stable transformation efficiency was aimed. However limited knowledge about pTJK136 and problem with curing KYRT1 leaded us to use Agrobacterium tumefaciens C58C1 strain and al...
Optimisation of agrobacterium mediated gene transfer and micrografting systems in lentil (lens culinaris medik)
Kamçı, Hamdi; Öktem, Hüseyin Avni; Department of Biotechnology (2004)
In this work Agrobacterium (KYRT1::pTJK136) mediated gene transfer to lentil (Lens culinaris Medik.) embriyo apex and regeneration through micro-grafting in lentil was studied. In micro-grafting two different types root stock stem height and root stock preparations were optimized. According to the results half stem length was found to be more successful then the full. Also lentil root stock was more successful then the chickpea root stock. The types of root stock preparations studied were designated as Z an...
Transformation of tobacco (Nicotiana tabaccum) with antimicrobial pflp gene and analysis of transgenic plants
Tuncer, Taner; Öktem, Hüseyin Avni; Department of Biotechnology (2006)
The objective of this study was to transform sweet pepper ferredoxin-like protein (PFLP) gene, which has antimicrobial properties, to tobacco and investigate the disease resistance abilities of transgenic tobacco. This protein interacts with another protein, harpin that is produced by the bacteria which is invading the plant tissues, and stimulates hypersensitivity response in plants, thus the spreading of disease is limited. Gene transfer was achieved to tobacco by Agrobacterium- mediated method and with i...
Optimization of regeneration and agrobacterium mediated transformation of sugar beet (Beta vulgaris L.)
Baloğlu, Mehmet Cengiz; Yücel, Ayşe Meral; Department of Biology (2005)
In this study, optimization of a transformation and regeneration system via indirect and direct organogenesis in cotyledon, hypocotyl, petiole, leaf and shoot base tissues of sugar beet (Beta vulgaris L. cv. ELK 345 and 1195) was investigated. Two different germination, three different callus induction and shoot induction medium was used for indirect organogenesis of sugar beet cultivar ELK 345. Except cotyledon, other explants (hypocotyl, petiole and leaf) produced callus. However no shoot development was ...
Citation Formats
U. Ç. Akçay, “Regeneration of lentil (lens culinaris medik) and genetic transformation by using agrobacterium tumefaciens-mediated gene transfer,” Ph.D. - Doctoral Program, Middle East Technical University, 2008.