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.

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.

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 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.

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 Expression Levels of avrBs3-Like Genes Affect Recognition Specificity in Tomato Bs4- But Not in Pepper Bs3-Mediated Perception

2005 ◽  
Vol 18 (11) ◽  
pp. 1215-1225 ◽  
Author(s):  
Sebastian Schornack ◽  
Kristin Peter ◽  
Ulla Bonas ◽  
Thomas Lahaye
Keyword(s):  
Xanthomonas Campestris ◽  
Transcript Abundance ◽  
Effector Proteins ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Disease Resistance Gene ◽  
In Planta ◽  
Type Iii ◽  
Recognition Specificity ◽  
Bacterial Type

The tomato Bs4 disease resistance gene mediates recognition of avrBs4-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria to give a hypersensitive response (HR). Here, we present the characterization of the Bs4 promoter and its application for lowlevel expression of bacterial type III effector proteins in planta. Real-time polymerase chain reaction showed that Bs4 is constitutively expressed at low levels and that transcript abundance does not change significantly upon infection with avrBs4-containing xanthomonads. A 302-bp promoter fragment was found to be sufficient to promote Bs4 gene function. Previous studies have shown that high, constitutive in planta expression of avrBs3 (AvrBs3 and AvrBs4 proteins are 96.6% identical) via the Cauliflower mosaic virus 35S (35S) promoter triggers a Bs4-dependent HR whereas X. campestris pv. vesicatoria-mediated delivery of AvrBs3 into the plant cytoplasm does not. Here, we demonstrate that, when expressed under control of the weak Bs4 promoter, avrBs3 does not trigger a Bs4-dependent HR whereas avrBs4 does. In contrast, the pepper Bs3 gene, which mediates recognition of AvrBs3- but not AvrBs4- delivering xanthomonads, retains its recognition specificity even if avrBs4 was expressed in planta from the strong 35S promoter. Importantly, Bs4 promoter-driven expression of hax3, hax4 (two recently isolated avrBs3-like genes), avrBs3, and avrBs4 resulted in identical reactions as observed upon infection with X. campestris pv. vesicatoria strains that express the respective avr gene, suggesting that the protein levels expressed under control of the Bs4 promoter are similar to those that are translocated by the bacterial type III secretion system.

scholarly journals A Second T-Region of the Soybean-Supervirulent Chrysopine-Type Ti Plasmid pTiChry5, and Construction of a Fully Disarmed vir Helper Plasmid

2000 ◽  
Vol 13 (10) ◽  
pp. 1081-1091 ◽  
Author(s):  
Karuppaiah Palanichelvam ◽  
Philippe Oger ◽  
Steven J. Clough ◽  
Chung Cha ◽  
Andrew F. Bent ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Ti Plasmid ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
Selection For ◽  
Border Repeats ◽  
The Right ◽  
Binary Plasmid

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadori opines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 coinherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

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