scholarly journals An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation

2021 ◽  
Vol 11 ◽  
Author(s):  
Karthik Kesiraju ◽  
Shaily Tyagi ◽  
Soumyadeep Mukherjee ◽  
Rhitu Rai ◽  
Nagendra K. Singh ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Apical Meristem ◽  
Transformation Method ◽  
In Planta ◽  
Specific Pcr ◽  
Putative Transformants ◽  
Somaclonal Variations ◽  
Promising Tool ◽  
Culture Independent ◽  
Screening Efficiency

Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.

scholarly journals Tissue culture-based Agrobacterium-mediated and in planta transformation methods

2017 ◽  
Vol 53 (No. 4) ◽  
pp. 133-143 ◽  
Author(s):  
M. Niazian ◽  
S.A. Sadat Noori ◽  
P. Galuszka ◽  
S.M.M. Mortazavian
Keyword(s):  
Tissue Culture ◽  
Transformation Method ◽  
Gene Transformation ◽  
In Planta Transformation ◽  
In Planta ◽  
Agrobacterium Mediated Transformation ◽  
Advantages And Disadvantages ◽  
Culture Independent ◽  
Transformation Methods ◽  
Mutant Lines

Gene transformation can be done in direct and indirect (Agrobacterium-mediated) ways. The most efficient method of gene transformation to date is Agrobacterium-mediated method. The main problem of Agrobacterium-method is that some plant species and mutant lines are recalcitrant to regeneration. Requirements for sterile conditions for plant regeneration are another problem of Agrobacterium-mediated transformation. Development of genotype-independent gene transformation method is of great interest in many plants. Some tissue culture-independent Agrobacterium-mediated gene transformation methods are reported in individual plants and crops. Generally, these methods are called in planta gene transformation. In planta transformation methods are free from somaclonal variation and easier, quicker, and simpler than tissue culture-based transformation methods. Vacuum infiltration, injection of Agrobacterium culture to plant tissues, pollen-tube pathway, floral dip and floral spray are the main methods of in planta transformation. Each of these methods has its own advantages and disadvantages. Simplicity and reliability are the primary reasons for the popularity of the in planta methods. These methods are much quicker than regular tissue culture-based Agrobacterium-mediated gene transformation and success can be achieved by non-experts. In the present review, we highlight all methods of in planta transformation comparing them with regular tissue culture-based Agrobacterium-mediated transformation methods and then recently successful transformations using these methods are presented.

scholarly journals In-planta Agrobacterium-mediated Stable hva1 and EPSPS Integration into Potato var. Agria Genome

2021 ◽  
Author(s):  
Darush Choobineh ◽  
nafiseh mahdi nezhad ◽  
Ali Niazi Niazi ◽  
Baratali Fakheri ◽  
Abbasali Emamjomeh
Keyword(s):  
Tissue Culture ◽  
Transgene Expression ◽  
Direct Injection ◽  
Crop Improvement ◽  
Constitutive Expression ◽  
Transformation Method ◽  
Bacterial Suspension ◽  
Potato Seed ◽  
In Planta ◽  
Culture Independent

Abstract BackgroundAfter wheat, maize, and rice, potato is not only an important food crop but also a substantial source of income throughout the world. Developing a practical and effective transformation method for cultivars that are recalcitrant in tissue culture is vital. Hva1 encodes the protein of the LEA III superfamily that involves in reactions to abiotic stresses, which holds considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.ResultsHere, a protocol has been designed for an Agrobacterium-mediated transient transformation in tissue culture-independent conditions in-planta. The protocol establishes for hva1 and EPSPS transformations by direct injection of the bacterial suspension into the potato tuber sprout to encode resistance to cold and against glyphosate herbicide. A two-stage selection was involved using 1% and 2% Glyphosate to eliminate the chimeric and non-transformed plants. Ultimately, the protocol enabled confirmation of gene integration into the plant, transgene expression of the gene and transgene expression, which was made possible by competitive PCR reaction, RT-PCR, and ELISA, respectively. In this research, the transformation efficiencies acquired in potatoes (up to 46%) were higher than those reported using conventional Agrobacterium-mediated approaches in previous studies. ConclusionsThe constitutive expression of the integrated T-DNA neither slowed down the growth rate nor affected potato tuberization significantly. The hva1 gene was expressed successfully leading to the accumulation of the hva1 protein in transgene-generated tubers. This study is the first report on a successful transformation of potato in-planta whereby Agrobacterium can be directed at potato seed sprouts through injection.

scholarly journals Constitutive Overexpression of the Plasma Membrane Na+/H+ Antiporter for Conferring Salinity Tolerance in Rice

2016 ◽  
Vol 25 (2) ◽  
pp. 257-272 ◽  
Author(s):  
Farida Yasmin ◽  
Sudip Biswas ◽  
GM Nurnabi Azad Jewel ◽  
Sabrina M Elias ◽  
Zeba I Seraj
Keyword(s):  
Tissue Culture ◽  
Plasma Membrane ◽  
Indica Rice ◽  
Rice Variety ◽  
Segregation Ratio ◽  
Transformation Method ◽  
Cellular Level ◽  
Salt Tolerant ◽  
In Planta ◽  
Rice Transformation

At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway comprising SOS3, SOS2, and SOS1 has been proposed to mediate cellular signaling under salt stress to maintain ion (Na+) homeostasis. In this regulatory pathway, both OsSOS1 encoding plasma membrane and OsNHX1 encoding vacuolar Na+/H+ antiporters are regulated by SOS3?SOS2 protein kinase complex. In the present study, the rice variety BRRI dhan28 - which is popular with farmers and high yielding, but salt sensitive, was transformed with the OsSOS1 gene isolated from salt tolerant Pokkali rice and driven by the constitutive promoter, CaMV35S. The construct was transformed through a tissue culture-independent Agrobacteriummediated in planta transformation method that circumvents the problems associated with tissue culture-based indica rice transformation methods. Integration of the foreign genes (OsSOS1) into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression (SOS1) was also quantified by semi-quantitative RT PCR and real time PCR. Genetic segregation ratio for T1 progenies was calculated and found to follow the Mendelian law of inheritance in case of positive transformants. The transformants were shown to be salt tolerant compared to wild type in molecular analysis as well as physiological screening. Future work will involve transformation of both the OsSOS1 and OsNHX1 genes together; with the expectation for enhancing the tolerance level compared to currently available transgenic rice.Plant Tissue Cult. & Biotech. 25(2): 257-272, 2015 (December)

scholarly journals Optimization of genetic transformation method for an indica rice (Oryza sativa L.) variety Ratnagiri-711

2020 ◽  
Vol 80 (02) ◽  
Author(s):  
G. B. Sawant ◽  
S. G. Bhave ◽  
S. V. Sawardekar ◽  
M. M. Burondkar ◽  
N. B. Gokhale ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Transformation Method ◽  
Transformation Frequency ◽  
Vector System ◽  
Nptii Gene ◽  
Cry Genes ◽  
In Planta ◽  
Germinating Seeds

A study for method optimization of Agrobacterium-mediated genetic transformation for insect resistance was carried out for a rice variety Ratnagiri-711 showing better regenerability. Three different cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa with a vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 were used. The respective genes were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. Scutellum-derived callus bits and embryonic shoot apical meristem of germinating seeds were used as target tissues for callus-mediated and in planta transformation, respectively. Kanamycin screening and PCR analysis was employed for confirmation of presence of transgene. Among five methods of colonization and co-cultivation tried with three cry genes, a callus-mediated transformation method consisting of 20 minutes colonization and 3 days co-cultivation with cry2Aa gene recorded highest transformation frequency (13.79%) but minimum survival (5.27%). On the contrary, considerable transformation frequency (6.35%) with highest survival (79.42%) was observed in an in planta method employing mild injury to embryonic shoot apical meristem of germinating seeds followed by injection of Agrobacterium having cry2Aa gene followed by 15 minutes colonization and then directly sowing in pots. Among three cry genes used, the gene cry2Aa was found most effective showing more transformation frequency.

Tissue culture and genetic analysis of somaclonal variations of Solanum melongena L. cv. Nirrala

2014 ◽  
Vol 9 (12) ◽  
pp. 1182-1195
Author(s):  
Samar Naseer ◽  
Tariq Mahmood
Keyword(s):  
Tissue Culture ◽  
Acetic Acid ◽  
Soluble Protein ◽  
Solanum Melongena ◽  
Total Soluble Protein ◽  
Naphthalene Acetic Acid ◽  
Fresh Tissue ◽  
Somaclonal Variations ◽  
Random Amplification ◽  
Plant Grown

AbstractThe present study was designed to analyze genetically somaclonal variants using biochemical and molecular markers. Efficient tissue culture protocol for Solanum melongena L. cv. Nirrala was developed. Maximum callus induction (100%) was observed for Murashige and Skoog (MS) media supplemented with 2.0 mg L−1 naphthalene acetic acid +0.5 mg L−1 6-benzylaminopurine; and nodal explants gave best callusing response (88.8%) as compared to internodes (88.3%) and leaves (87.7%). The best shooting was induced on nodal and internodal callus in the presence of 2.0 mg L−1 6-benzylaminopurine. Total soluble protein content of callus and regenerated variant plants was estimated for biochemical analysis, and largest amount of soluble protein was found in callus (6.54 mg g−1 fresh tissue) followed by variant plant grown on 2.0 mg L−1 6-benzylaminopurine (5.96 mg g−1 fresh tissue). Random amplification of polymorphic DNA technique was done with five decamer primers (OPC1-OPC5) and maximum polymorphism was detected by OPC 2 (26.99%) among all samples, whereas nodal callus on media containing 1.0 mg L−1 naphthalene acetic acid +1.0 mg L−1 6-benzylaminopurine showed highest polymorphism producing 22 bands, out of which 8 bands were polymorphic. The study shows that this marker system can provide better evaluation of genetic variation induced by tissue culture.

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