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 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 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.

Development of simple and efficient in Planta transformation method for wheat (Triticum aestivum L.) using Agrobacterium tumefaciens

2006 ◽  
Vol 102 (3) ◽  
pp. 162-170 ◽  
Author(s):  
Putu Supartana ◽  
Tsutomu Shimizu ◽  
Masahiro Nogawa ◽  
Hidenari Shioiri ◽  
Tadashi Nakajima ◽  
...  
Keyword(s):  
Triticum Aestivum ◽  
Agrobacterium Tumefaciens ◽  
Triticum Aestivum L ◽  
Transformation Method ◽  
In Planta Transformation ◽  
In Planta

scholarly journals Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.)

2018 ◽  
Vol 22 ◽  
pp. 293-298
Author(s):  
S. I. Mykhalska ◽  
A. G. Komisarenko ◽  
V. M. Kurchii ◽  
O. M. Tishchenko
Keyword(s):  
Triticum Aestivum ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Winter Wheat ◽  
Natural Frequency ◽  
Triticum Aestivum L ◽  
Transformation Method ◽  
In Planta ◽  
Agrobacterium Mediated Transformation ◽  
Pollination Methods

Aim. To optimize the agrobacterium-mediated method of winter wheat transformation (Triticum aestivum L.); to select the conditions and period of inoculation to effectively transfer the genes during pollination. Methods. Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.) during pollination. Results. The conditions for agrobacterium-mediated transformation method of winter wheat during natural (frequency pollination was 1 %) and non-natural (frequency pollination was 4 %) pollination were defined. Conclusions. The possibility of integrating transgenes into the genome of winter wheat plants by the method of Agrobacterium-mediated transformation in planta in the process of forced and natural pollination is demonstrated. It is found that the transformation efficiency to a large extent depends on the plant genotype and the method of carrying out the transformation procedure. The selection of transgenic plants under water deficit conditions allowed to identify the plants with functional transgene. The signs of functioning transgene have been remaining in the next generation of genetically modified winter wheat. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation in planta, transgenic plants, seeds.

scholarly journals Agrobacterium tumefaciens-MEDIATED IN PLANTA TRANSFORMATION METHOD FOR THE SoSPS1 GENE IN CITRUS PLANTS (Citrus nobilis L.)

2020 ◽  
Vol 7 (1) ◽  
pp. 31
Author(s):  
Ni Putu Ayu Erninda Oktaviani Suputri ◽  
Rindang Dwiyani ◽  
Ida Ayu Putri Darmawanti ◽  
Bambang Sugiharto
Keyword(s):  
Tissue Culture ◽  
Agrobacterium Tumefaciens ◽  
Cauliflower Mosaic Virus ◽  
Total Soluble Protein ◽  
35S Promoter ◽  
Nptii Gene ◽  
Main Function ◽  
In Planta Transformation ◽  
In Planta ◽  
Regeneration System

The SoSPS1 gene of sugar cane plants previously subjected to Agrobacterium tumefacienmediated cloning was to be transferred to citrus plants to increase metabolism of sucrose in plant. The T-DNA harbored the SoSPS1 gene under the control of the CaMV 35S promoter from the cauliflower mosaic virus and contained the NPTII gene (kanamycin resistance gene) as a selectable marker for transformant selection. Generally, gene transformation in plants is carried out by tissue culture. However, tissue culture has several disadvantages such as its being time-consuming, its sometimes resulting in somatic mutations and somaclonal variations, and the requirement of sterile conditions in the procedure of gene transfer. In planta transformation is a useful system for those plants that lack tissue culture and regeneration system. The main function of in planta transformation is to recover the advantages of tissue culture as an efficient, quick method, including its ability to produce a large number of transgenic plants and to accumulate a high concentration of total soluble protein in short time. There are two procedures of in planta transformation for the seeds of citrus plants, namely “prick and coat” and “seed tip-cutting and imbibition”. In the prick and coat method, seeds are pricked on their entire surfaces and smeared with a suspension of Agrobacterium tumefaciens. In the seed tip-cutting and imbibition method, on the other hand, seeds are cut at the tip and soaked in a suspension of Agrobacterium tumefaciens. The leaves derived from seeds treatment were taken as samples for DNA extraction and PCR using primers of the NPTII gene (Forward: 5’-GTCATCTCACCTTCCTCCTGCC-3’; Reverse: 5’-GTCGCTTGGTCGGTCATTTCG-3’). This research found that only the seed tip-cutting and imbibition plants amplified along the 550-bp band, while those of the prick and coat method did not. Additionally, the T-DNA was successfully integrated into the genome of the plants treated with the seed tip-cutting and imbibition method but not with the prick and coat.

A simple and efficient in planta transformation method for pommelo ( Citrus maxima ) using Agrobacterium tumefaciens

2017 ◽  
Vol 214 ◽  
pp. 174-179 ◽  
Author(s):  
Yong-yan Zhang ◽  
Dong-min Zhang ◽  
Yun Zhong ◽  
Xiao-jun Chang ◽  
Min-lun Hu ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transformation Method ◽  
In Planta Transformation ◽  
In Planta ◽  
Citrus Maxima

scholarly journals PRICK AND SOAK Agroacterium tumefaciens-MEDIATED IN PLANTA TRANSFORMATION IN TOMATO (Lycopersicon esculentum Mill.)

2018 ◽  
Vol 5 (2) ◽  
pp. 124
Author(s):  
I Putu Wahyu Sanjaya ◽  
Rindang Dwiyani ◽  
I Gede Putu Wirawan ◽  
Bambang Sugiharto
Keyword(s):  
Tissue Culture ◽  
Agrobacterium Tumefaciens ◽  
Saccharum Officinarum ◽  
Plant Genome ◽  
Nptii Gene ◽  
In Planta Transformation ◽  
In Planta ◽  
Tomato Seeds ◽  
Inoculation Time

One of the modern plant breedings through genetic engineering is Agrobacterium tumefaciens-mediated transformation. Agrobacterium tumefaciens-mediated transformation can be performed in vitro or in planta. In planta transformation arises from the weaknesses of the in vitro method such as need high hygiene standard, professional tissue culture experts, and more time to prepare explants and somaclonal variation. In planta transformation is a method to transfer the gene to the plant genome without any tissue culture stages. The aims of this research were to know the possibility of the prick and soak in planta method with the target of tomato seeds and to know the most suitable inoculation time for tomato seeds transformation by prick and soak method the transformation is done by pricking the seeds and soaking them in the A. tumefaciens suspension. The treatments in this study were 1 and 2 days inoculation time to test the efficacy of prick and soak in planta transformation method. Tomato seeds were pricked with a needle on the center once, and then soaked in A. tumefaciens strain LB4404 suspension carrying pKYS-SoSPS1 plasmid with Neomycin Phosphotransferase (NPTII) and Saccharum officinarum Sucrose Phosphate synthase (SoSPS1) genes. Visualization of tomato’s DNA samples after PCR showed that 1-day inoculation sample was positively integrated with NPTII gene and negative in the 2 days inoculation treatment.

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