Agrobacterium mediated gene transfer, which is one of the most common rice transformation methods, has been extensively used for developing transgenic rice to study. Shanthi4 abstract the possibility of developing transgenic indica rices through agrobacterium mediated transformation in the absence of. Video articles in jove about agrobacterium mediated transformation include inducible, cell typespecific expression in arabidopsis thaliana through lhgr mediated transactivation, generating transgenic plants with singlecopy insertions using bibacgw binary vector, inducing hairy roots by agrobacterium rhizogenes mediated transformation in tartary buckwheat fagopyrum tataricum. Studies on improved agrobacteriummediated transformation in. Ir64 for initial standardization of various factors critical for its enhanced transformation and regeneration. Experiments were conducted to establish an efficient and simple protocol for. Large number of dicots and some monocots and gymnosperms. The protocol outlined below is a modification of that described by hinchee et al. Therefore, optimization of the transformation protocol is important for specific indica rice cultivar to ensure effectiveness of the transformation. Many reports have demonstrated efficient protocols for genetic transformation of rice by agrobacterium mediated methods hiei et al. Agrobacteriummediated transformation of rice using immature embryos or calli induced from mature seed. A protocol for agrobacteriummediated transformation in rice article pdf available in nature protocol 16. Efficient transformation of suspensioncultured rice cells mediated by.
An improved agrobacterium mediated transformation protocol for deep water indica rice. But poor callusing followed by reduced regeneration ability in rice varieties has limited the use of rice transformation 9. We studied several key factors that affected the efficiency of agrobacteriummediated rice transfor mation, in. The procedure permitted the regeneration of transgenic plants from callus in only two months. A simple and highly efficient agrobacteriummediated. The objective of this study is to optimize agrobacterium mediated transformation in soybean by improving the infection efficiency of. Agrobacterium mediated genetic transformation is the most extensively used as it leads to stable and low copy number gene integration in rice. Review of methodologies and a protocol for the agrobacterium. Using this protocol, we have transformed thousands of gene constructs in the past 10 years and edited. In planta agrobacteriummediated transformation of rice. For newcomers it is usually difficult to understand the true importance of various steps or operations comprising a protocol. Pdf a protocol for agrobacteriummediated transformation.
It is a thai commercial rice cultivar represented in the thai rice export market. Transgenic plants dna is modified using genetic engineering techniques. Agrobacteriummediated gene transfer, which is one of the most common rice transformation. Provides advantages in producing plants wich are drought resistant,cold tolerance, pest resistant, etc. Agrobacterium mediated rice transformation from mature seeds kimberly nelsonvasilchik, joel hague, and albert kausch the plant biotechnology laboratory university of rhode island 530 liberty lane west kingston ri 02892 plant material mature rice seeds oryza sativa cv. Agrobacterium mediated transformation agrobacterium strain, viz. Transformation of wheat mediated by agrobacterium tumefaciens 973 leafbleach assay after the to plants were established in soil, leaf samples 57 mm long were taken from the youngest fully ex panded leaves and placed in a 24well culture plate costar, cambridge, ma. Agrobacterium mediated transformation of maize nature. Agrobacteriummediated transformation of rice free download as powerpoint presentation. Development of an efficient agrobacterium mediated. Agrobacterium mediated genetic transformation of indica. In rice, an efficient method for agrobacterium mediated transformation was developed more than 10 years ago, 1 and is now used as a routine protocol in many laboratories.
Jan 10, 2011 experiments were conducted to establish an efficient and simple protocol for regeneration and agrobacterium mediated genetic transformation of an agronomically important indica rice variety bg 250. An efficient gene transformation is a crucial for complementation test to identify the agronomically important genes. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were cocultivated with agrobacterium in the presence of acetosyringonc, which is. Early infection of scutellum tissue with agrobacterium. The rice cultivar used for the transformation was rd6, which is a glutinous rice widely grown in the northeast of thailand. Yadav2, khushi ram3 1the institutes for applied research, bengurion university, p. Plant transformation protocols soybean cotyledonarynode agrobacterium mediated transformation system. Agrobacterium protocols transformation tb ri tobacco rice 4 regrowth tobacco ce 1 inoculation. We report a highly efficient protocol for agrobacterium mediated genetic transformation of s. Chapter 1 agrobacterium rhizogenesmediated transformation. Transformation facility department of agronomy agrobacterium. This protocol could become a powerful tool for functional genomics in sugarcane. On the other hand, foreign dna is inserted in one or more than one nonhomologous sites with the agrobacterium mediated transformation. Agrobacteriummediated genetic transformation of plants.
Rapid and efficient agrobacteriummediated transformation in rice springerlink. Oct 31, 20 hiei y, ohta s, komari t et al 1994 efficient transformation of rice oryza sativa l. Efficient and fast production of transgenic rice plants by. Agrobacterium mediated transformation protocol agrobacterium mediated callus transformation and regeneration agrobacterium strain gv3101 carrying pcambia3 was grown to an optical density of 1. The protocol simplifies medium compositions and transformation steps and can be easily followed by a lab technician with little tissue culture experience.
The optimization of several steps in tissue culture allowed the rapid regeneration of plants and increased the rate of transformation up to 29%. However, because current approaches rely on culture systems, routine protocols have been established only in japonica rice, especially those varieties with higher regeneration potential. However, because current approaches rely on culture systems, routine. An improved protocol for efficient transformation and regeneration of. Jun 21, 2007 maize may be transformed very efficiently using agrobacterium tumefaciens mediated methods.
A major advantage of agrobacterium mediated transformation is that a small number of copies often one or two of relatively large segments can be. The number of insertions varies from one to numerous, but on average in the following protocol, three to several insertions were observed. For agrobacterium mediated transformation of plant tissues with no wounding, as could enhance the transformation efficiency of both monocot and dicot crops, for example, wheat raja et al. The agrobacterium mediated transformation is the method of choice in most cases due to certain inherent advantages such as its hightrans.
Agrobacterium mediated transformation of indica rice under acetosyringone free conditions n. In different plant species different gene transfer protocol are applicable. Scribd is the worlds largest social reading and publishing site. The in vitro response is highly genotypedependant, so it was difficult to find a uniform agrobacteriummediated transformation system for indica rice. Agrobacterium mediated gene transfer to cereal crop. Nov 02, 2015 agrobacterium mediated transformation in rice 1. Transformation of rice mediated by agrobacterium tumefaciens. Soto1,2, ewen mullins2 and beata dedicova1,3 abstract while agrobacterium.
Amsod was the gene of interest used in this study for increased tolerance towards salinity tolerance. Pdf an efficient and universal agrobacteriummediated. Transformation efficiency, selection and plasmid backbone. The tdna transfer is represented according to updated knowledge on this process, although many of involved mechanisms have not been elucidated yet and. We have transformed embryoderived cultures of two rice cultivars using several different agrobacterium mediated gene transfer systems. Among the rice group, such as indica, japonica and javanica the indica rice.
Basic steps in transformation of plant cells by agrobacterium tumefaciens. Agrobacteriummediated transformation of banana musa. An efficient and reproducible agrobacterium mediated. Doetsch na, favreau mr, kuscuoglu n, thompson md, hallick rb 2001. An in planta, agrobacterium mediated transient gene. Application of plant biotechnologyplant transformation 2010. Transfer on culture to 200ml yep in a sterile 500ml flask. Here we describe an efficient and fast protocol for producing transgenic japonica rice plants using the agrobacterium mediated transformation method. Furthermore, the protocols utilize supervirulent strains of agrobacterium developed by. Transformation protocols using agrobacterium factors influencing transformation efficiency 5. Agrobacteriummediated genetic transformation tzfira and citovsky 149 figure 2 the role of host factors and cellular processes in the agrobacteriummediated genetic transformation of plant cells.
An improved protocol for agrobacterium mediated transformation of rice is described. Agrobacterium is a common soil bacterium with natural capacity for transkingdom transfer of genetic information by transferring its tdna into the eukaryotic genome. Agrobacterium mediated transformation of arabidopsis thaliana using the floral dip method. Kumar kk, maruthasalam s, loganathan m, sudhakar d, balasubryamaniam p. Plant transformation facility department of agronomy agrobacterium mediated transformation of japonica cv. Agrobacterium mediated genetic transformation following protocol developed by hiei et al 2. This method was developed for the winter wheat cultivar florida but with minor modifications has also been used to successfully transform the spring wheat varieties fielder and cadenza. Protocols plant transformation nebraska center for. Agrobacteriummediated rice transformation from mature. Development of an efficient agrobacterium mediated transformation protocol for sri lankan rice variety bg 250 r. Steps for agrobacterium mediated transformation and reg openi. Gene targeting using the agrobacterium tumefaciens.
Improved agrobacteriummediated transformation of three maize. Till now, a number of commercially valuable crops like tomato, potato, rice, wheat, maize, cotton, soybean, alfalfa, barley, carrot, sugarcane, pepper and broccoli were obtained using agrobacterium mediated transformation ozyigit 2012. Some of the presented protocols are still very popular in certain research lines while the others are only of historical importance. Furthermore, early infection of rice seeds with agrobacterium enhanced the ef. The improved protocol, as reported earlier in the methods section, was applied to.
Protocols for agrobacteriummediated transformation of potato. A highly effective agrobacterium mediated transformation. Hettiarachchi1 department of chemistry faculty of science, university of colombo sri lanka abstract. While the first successful genetic transformation events in cereal species had been based on direct gene transfer, which was associated with a number of disadvantages, the pioneering study of hiei et al. Rapid and efficient agrobacteriummediated transformation.
Rice transformation has been successfully developed via several techniques including electroporation, polyethylene glycol treatment, particle bombardment and agrobacterium mediated method since the late 1980s chen et al. Agrobacterium mediated transformation of rice using. Agrobacterium mediated transformation usually results in the insertion of unrearranged segments of dna into recipient plant. Genetic transformation of rice with agrobacterium requires regeneration of an intact plant from a transformed callus and ironically, shoot regeneration represents a. Urushibara s, tozawa y, kawagishikobayashi m, wakasa k.
Here, we provide comprehensive, highly efficient protocols for agrobacterium tumefaciens mediated transformation of a wide range of rice genotypes. Many approaches are verified for stable introduction of foreign genes into the plant genome. Agrobacterium mediated transformation of rice oryza. Agrobacterium tumefaciens mediated transformation is a preferred method in many plants. Agrobacterium mediated genetic transformation tzfira and citovsky 149 figure 2 the role of host factors and cellular processes in the agrobacterium mediated genetic transformation of plant cells. The type ii clustered, regularly interspaced, short palindromic repeat crispr crisprassociated protein 9 cas9 system is a novel molecular tool for sitespecific genome modification. A protocol for agrobacterium mediated transformation in. Review of methodologies and a protocol for the agrobacteriummediated transformation of wheat article pdf available in plant methods 11. Nipponbare were obtained from usda agricultural research service. The crisprcas9 system was recently introduced into plants by transient or stable transformation. Protocol for artificial microrna mediated overexpression. Studies on improved agrobacteriummediated transformation. The optimized rice agroinfection protocol was also applied with success to sugarcane suggesting that other graminaceous species are amenable to this procedure unpublished. Here, we report gene targeting in rice via the agrobacterium tumefaciens mediated crisprcas9 system.
Genetic transformation of wheat mediated by agrobacterium. Reference for highly efficient protocols for agrobacterium tumefaciens mediated transformation. Agrobacterium mediated transformation is an effective and widely used approach to introduce foreign dna into dicotyledons plants. Steps af have to be performed under dark conditions. Agrobacteriummediated genetic transformation following protocol developed by hiei et al 2. It is primarily used for the production of resistance against bacterial blight in rice rashid et al. Comprehensive and highly practical, agrobacterium protocols, second edition, volumes 1 and 2 offers plant biotechnologists a gold standard collection of agrobacterium mediated transformation techniques for stateoftheart plant genetic engineering, functional genomic analysis, and crop improvement, and for inspiration in developing new methods. Studies on improved agrobacteriummediated transformation in two indica rice oryza sativa l. Agrobacterium, callus induction, indica rice, transformation. Agrobacterium mediated genetic transformation of indica rice. Pdf in planta agrobacterium mediated transformation of rice.
This method was developed for the winter wheat cultivar florida but with minor modifications has also been used to successfully transform the. Nipponbare callus tissue and recovery of transgenic rice plants materials a plasmid agrobacterium tumefaciens strain eha101 hood et al. Agrobacterium protocols volume i kan wang springer. This cultivar is susceptible to insect pests and diseases. A protocol for agrobacteriummediated transformation.
Dec 30, 2011 overview of the modified protocol for efficient transformation and regeneration. Agrobacterium mediated transformation of rice is an important method that has been widely adopted by many laboratories. The transformation efficiencies of agrobacterium tumefaciens strains agl1 and eha105 were compared with the. Research article agrobacterium mediated transformation. An efficient and universal agrobacteriummediated transformation protocol in rice article pdf available in journal of plant biochemistry and biotechnology 212 july 2012 with 175 reads. Pdf a protocol for agrobacteriummediated transformation in rice. Different stages of rice transformation with agrobacteriummediated method using mature seeds. Optimization of agrobacterium mediated transformation in. You have free access to this content the plant journal volume 6, issue 2, version of record online. As a result, numerous genetic transformation protocols have been developed to improve and better understand this particular agronomic plant. Pdf a protocol for agrobacteriummediated transformation in. Rice transformation is a challenging task as it is comparatively less amenable to genetic manipulation, es.
They generated many independent transgenic plants, with tdna. Various challenges to infect monocotyledons including rice with agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Rice transformation using agrobacterium tumefaciens is a method of choice due to stable and low copy number integration of transferdna tdna into the plant chromosome and transfer of larger dna segments with defined ends. Factors affecting agrobacteriummediated transformation. However, significant varietal difference was observed in the efficiency of agrobacterium mediated transformation in rice. Agrobacterium protocol for the genetic modification of rice evelyn zuniga. Our intention is to provide the public research community with. Agrobacterium tumefaciensmediated transformation of corn zea. The most critical factor in the transformation protocol is the cocultivation of healthy immature. The culture was centrifuged at 3500 rpm for 10 min and the pellet was re. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an agrobacterium mediated standard binary vector system to target maize immature embryos.
Rice transformation using agrobacterium tumefaciens is a method of choice due to stable and low copy number integration of transferdna. Transgenic plant contains genes inserted artificially called transgenes. Agrobacteriummediated rice transformation from mature seeds kimberly nelsonvasilchik, joel hague, and albert kausch the plant biotechnology laboratory university of rhode island 530 liberty lane west kingston ri 02892 plant material mature rice seeds oryza sativa cv. Agrobacteriummediated genetic transformation of rice oryza sativa l. High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Our results showed that as was not necessary for the floraldip transformation technique developed in rice. Efficient transformation protocols mediated by agrobacterium were reported for rice in 1994 and 1996.
Hiei y, ohta s, komari t et al 1994 efficient transformation of rice oryza sativa l. Callus induction was achieved on modified n6b5 medium supplemented with 2,4 d 2. Tissue culture and agrobacteriummediated genetic transformation. Regeneration and agrobacteriummediated transformation of.
Since the first report of wheat transformation by agrobacterium tumefaciens in 1997, various factors that influence tdna delivery and regeneration in tissue culture have been further investigated and modified. In this study, crucial parameters affecting agrobacterium mediated transformation were optimized to obtain transgenic rice of local rice cultivar indica mr219. Steps for agrobacterium mediated transformation and regeneration of rice calli. Pdf an improved agrobacteriummediated transformation. An agrobacterium tumefaciensmediated corn transformation method based. Although it is only half a dozen years since the current procedure of rice transformation mediated by agrobacterium was published, the economic importance and the accumulation of genomic information has made rice the species of focus in plant biotechnology.
Thus, the transformation method reported here could be used as a standard protocol to transfer another. Pdf agrobacteriummediated transformation of rice is an important method that has been. From tair agrobacterium competent cells you should double up this protocol it is almost the same amount of work and you can thus get some 80 tubes. An improved agrobacteriummediated transformation protocol for recalcitrant elite indica rice cultivars. A protocol for agrobacterium mediated transformation in rice. However, the transformation efficiency in soybean is still low. Agrobacterium mediated transformation usually results in the insertion of unrearranged segments of dna into recipient plant genomes, often at low copy number 6. Agrobacterium mediated transformation in tomato for. Agrobacterium mediated genetic transformation of rice for. An improved protocol for efficient transformation and. Agrobacteriummediated rice transformation from mature seeds.
911 316 978 627 1088 483 65 871 1049 1357 329 1311 1496 1542 953 1065 838 124 1154 523 1395 347 612 119 25 1480 1234 1074 734 218 802