Preview

An Improved Agrobacterium-Mediated Transformation Protocol for Recalcitrant Elite Indica Rice Cultivars

Better Essays
Open Document
Open Document
2638 Words
Grammar
Grammar
Plagiarism
Plagiarism
Writing
Writing
Score
Score
An Improved Agrobacterium-Mediated Transformation Protocol for Recalcitrant Elite Indica Rice Cultivars
Plant Molecular Biology Reporter 23: 67–73, March 2005 © 2005 International Society for Plant Molecular Biology. Printed in Canada.

Protocols

An Improved Agrobacterium-Mediated Transformation Protocol for Recalcitrant Elite Indica Rice Cultivars
K.K. KUMAR, S. MARUTHASALAM, M. LOGANATHAN, D. SUDHAKAR and P. BALASUBRAMANIAN*
Rice Transformation Laboratory, Department of Plant Molecular Biology and Biotechnology, Center for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore-641003, Tamil Nadu, India Abstract. We report here a high-efficiency transformation protocol for recalcitrant indica rice cultivars IR64 and IR72 with the selectable marker gene hph and the gusA reporter gene. Factors that favor high-efficiency transformation were found to be use of 2-month-old mature seed-derived embryogenic calli, maltose as a source of carbon, a higher concentration of 2,4-dichlorophenoxyacetic acid, and both phytagel and agar as gelling agents. The putative transgenic (T0) plants were analyzed for integration of the transgene through polymerase chain reaction and Southern blotting analyses. Various factors thought to be responsible for increased transformation efficiency are discussed. Key words: Agrobacterium, indica rice, mature seed-derived calli, rice transformation, transgenic rice Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6-benzylaminopurine; MS, Murashige and Skoog; NAA, α-naphthaleneacetic acid; OD, optical density; PCR, polymerase chain reaction. Agrobacterium-mediated transformation protocol for indica Introduction rice cultivars Kumar et al.

Agrobacterium-mediated transformation is routinely used for the transformation of many monocots, including rice. Several reports are available on the Agrobacterium-mediated transformation of japonica and indica rice cultivars. However, the efficiency with which the indica rice cultivars are transformed is very low. Different explant types are used in the Agrobacterium-mediated


References: Aldemita PR and Hodges TK (1996) Agrobacterium tumefaciens–mediated transformation of japonica and indica rice varieties. Planta 199: 612-617. Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, and Nester EW (1974) Agrobacterium tumefaciens DNA and P58 bacteriophage DNA not detected in crown gall tumours. Proc Natl Acad Sci USA 71: 3672-3676. Datta K, Koukolikova-Nicola Z, Baisakh N, Oliva N, and Datta SK (2000) Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems. Theor Appl Genet 100: 832-839. Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW, Muthukrishnan S, and Datta SK (2001) Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars. Plant Sci 60: 405-414. Datta, K, Velazhahan R, Oliva N, Ona I, Mew T, Kush GS, Muthukrishnan S, and Datta SK (1999) Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor Appl Genet 98: 1138-1145. Dellaporta SL, Wood J, and Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19-21. Ditta G, Stanfield S, Corbin D, and Helinski D (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347-7351. Hiei Y, Ohta S, Komari T, and Kumashiro T (1994) Efficient transformation of rice (Oryza Agrobacterium-mediated transformation protocol for indica rice cultivars 73 sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271-282. Jain RK, Davey MR, Cocking EC, and Wu R (1997) Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties. J Exp Botany 48: 751-758. Khanna HK and Raina SK (1999) Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors. Aust J Plant Physiol 26: 311-324. Khanna HK and Raina SK (2002) Elite indica transgenic rice plants expressing modified Cry1A(c) endotoxin of Bacillus thuringiensis showed enhanced resistance to yellow stem borer (Scirpophaga incertulas). Transgenic Res 11: 411-423. Murashige T and Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497. Park SH, Pinson SRM, and Smith RH (1996) T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices. Plant Mol Biol 32: 1135-1148. Rashid H, Yokoi S, Toriyama K, and Hinata K (1996) Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep 15: 727-730. Roberts-Oehlschlager SL, Dunwell JM, and Faulks R (1990) Changes in sugar content of barley anther during culture on different carbohydrates. Plant Cell Tissue Organ Cult 22: 77-85. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Toriyama K and Hinata K (1985) Cell suspension and protoplast culture in rice. Plant Sci 41: 179-183. Tu J, Ona I, Zhang Q, Mew TW, Khush GS, and Datta SK (1998) Transgenic rice variety IR72 with Xa21 is resistant to bacterial blight. Theor Appl Genet 97: 31-36. Zhang S, Song WY, Chen L, Ruan D, Taylor N, Ronald PC, Beachy RN, and Fauquet CM (1998) Transgenic elite indica rice varieties, resistant to Xanthomonas oryzae pv.oryzae. Mol Breed 4: 551-558.
Continue Reading
View Writing Issues

You May Also Find These Documents Helpful

  • Good Essays

    Torn at the Genes Case Study
    • 676 Words
    • 3 Pages

    Torn at the Genes Case Study

    - The most common method is with an agrobacterium. Since bacteria reproduce quickly, it’s easy to create the same favorable gene many times over. The bacterium causes the plant to send it nutrients. They take one piece of the plant’s DNA and put the favorable DNA into the plant.…

    • 676 Words
    • 3 Pages
    Good Essays
    Read More
  • Good Essays

    Wk5 Transformation BackgroundSP15
    • 2055 Words
    • 7 Pages

    Wk5 Transformation BackgroundSP15

    Learning Goals: Insert your uncut unknown plasmid into chemically competent DH-5 E.coli cells and use antibiotic resistance to confirm the success of the transformation. You should familiarize yourself with the various methods of transformation and the advantages/disadvantages of each type. You should also understand how heat shock transformation works and how chemically competent cells make this type of transformation possible. For this transformation antibiotic markers associated with foreign pieces of DNA will be used to help verify that the DNA of interest was successfully inserted into the vector.…

    • 2055 Words
    • 7 Pages
    Good Essays
    Read More
  • Good Essays

    AP Bio pGLO Transformation Formal Lab Report
    • 1330 Words
    • 5 Pages

    AP Bio pGLO Transformation Formal Lab Report

    Genetic transformation is one of the most important processes in biotechnology. Essentially, genetic transformation involves the process where a cell (in this lab, a bacterial cell) takes up foreign DNA from its surroundings and incorporates it into its own DNA. This gene transfer is accomplished with the aid of a plasmid, a…

    • 1330 Words
    • 5 Pages
    Good Essays
    Read More
  • Good Essays

    Aglo Transformation Lab Report
    • 463 Words
    • 2 Pages

    Aglo Transformation Lab Report

    Plasmids are small circular autonomously replicating pieces of DNA that can be found inside of a prokaryotic bacterial cell. By barrowing a cell’s polymerase they replicate their own DNA. They are easy to extract from the bacterial cells due to their size. Plasmids are helpful for cloning foreign genes because of their ability to express antibiotic resistance as well their ability to be modified to express proteins of interest. A pGLO plasmid contains genes for the green florescent protein (GFP) as well as the gene for ampicillin resistance known as beta-lactamase. It also contains a gene regulation system (operon) that has the ability to control expression of the GFP gene in transformed cells known as araC. The source of GFP is naturally founds within a…

    • 463 Words
    • 2 Pages
    Good Essays
    Read More
  • Good Essays

    Pglo Transformation Lab Report
    • 263 Words
    • 2 Pages

    Pglo Transformation Lab Report

    The purpose of this experiment was understand the process of transformation and the effects on gene expression. The pGLO(-) culture had growth on the LB medium, while the LB amp and LB amp + ara mediums had no growth. It was expected that the LB medium had growth on the plate because it served as a control. The LB amp and LB amp + ara had no growth or glow under UV light because they were not successfully transformed and still contained the antibiotic ampicillin that prevented the growth of E. coli (3).…

    • 263 Words
    • 2 Pages
    Good Essays
    Read More
  • Good Essays

    Las 432 - Genetically Modified Foods
    • 14577 Words
    • 59 Pages

    Las 432 - Genetically Modified Foods

    Bibliography: 1983-Researchers transfer new DNA into plants, leading to the creation of genetically modified crops.…

    • 14577 Words
    • 59 Pages
    Good Essays
    Read More
  • Good Essays

    Playing God in the Garden - Book Analysis
    • 496 Words
    • 2 Pages

    Playing God in the Garden - Book Analysis

    While the idea of biotechnologically creating plants may pose many questions, conventional ways of farming brings with them many obstacles as well. Pesticides that are sprayed on crops can be extremely toxic. In…

    • 496 Words
    • 2 Pages
    Good Essays
    Read More
  • Powerful Essays

    Miss
    • 8881 Words
    • 29 Pages

    Miss

    When the insertion of a gene to a plant occurs, the plants ability to culminate with the natural minerals could be negated, thus not allowing the regular nutritional value of the plant to develop. An increase in antibiotic resistance in bacteria strains has alarmed health professionals. When incorporating a new gene into bacteria it no longer naturally mutates the antibiotic resistance so scientists have been unable to identify if the targeted plant will incorporate the new gene into its genome. When the GM plant has the respected gene attached to an antibiotic resistant gene a test formulate whether or not the gene was contained by the livelihood or death of the bacteria. The concern of health professionals is that the bacteria once consumed by humans may not be completely digested because of the antibiotic resistant trait.…

    • 8881 Words
    • 29 Pages
    Powerful Essays
    Read More
  • Powerful Essays

    The Ability of Humans to Manipulate Dna Is a Mixed Blessing
    • 1189 Words
    • 5 Pages

    The Ability of Humans to Manipulate Dna Is a Mixed Blessing

    Plants are genetically engineered to be resistant to herbicides, produce their own pesticides, have viral resistance, and to convert nitrogen directly from the soil. But these transgenes can insert into the genetics of relatives through cross-pollination, creating superweeds resistant to herbicides, pests and viruses. This may increase pest’s immunities and antibiotic resistances, transfer of pathogenic genes, the creation of new viruses, and introduce allergens into the food supply. Worse, these problems will be faster growing, disease resistant, and heat, cold, and drought tolerant. These and mutations may cause irreversible disaster. Planting engineered herbicide resistant…

    • 1189 Words
    • 5 Pages
    Powerful Essays
    Read More
  • Good Essays

    Gal4-Gfp Enhancer Trap System
    • 431 Words
    • 2 Pages

    Gal4-Gfp Enhancer Trap System

    Agrobacterium-mediated transformation is used to transform plant cells with a plasmid containing the modified GAL4-VP16 and UAS-GFP genes. The modified GAL4-VP16 gene is positioned adjacent to the Tr border sequence…

    • 431 Words
    • 2 Pages
    Good Essays
    Read More
  • Good Essays

    Week 3 assignment
    • 850 Words
    • 3 Pages

    Week 3 assignment

    From the perspective of six pillars of character, if I were Cindie, I would be honest with Lyndell and tell him what’s been bothering me. Hopefully, by keeping quiet about him leaving early, he will feel that I’m trustworthy enough to tell me his problems, whether it’s drinking or any other problems he is facing in his life. I might be able to help him or at least come up with some ideas to help him. I will also respect him more if he can open up to me and is willing to fix his problems. If he continues to leave early, I would have no choice but to report this situation to the store manager because I would feel responsible if something bad happens to the store while I was on duty alone. Apparently, Lyndell is not a responsible person because he leaves work early and makes Cindie do part of his job. Cindie is responsible because even though it got really busy when 10 Japanese customers came in at the same time one night, she rushed through and was able to satisfy the customers’ needs. It is not fair to Cindie that she has to close up by herself and often work the late shift just because she is the “new kid on the block” (Mintz & Morris, 2011). Everybody should be treated equally (Mintz & Morris, 2011).…

    • 850 Words
    • 3 Pages
    Good Essays
    Read More
  • Better Essays

    Recent Trends in the Biotechnology of Chrysanthemum: a Critical Review
    • 1299 Words
    • 6 Pages

    Recent Trends in the Biotechnology of Chrysanthemum: a Critical Review

    Various techniques have been developed which could help breeders to meet the demand of the cut flower industry in the next century. Available methods for the transfer of genes could significantly shorten the breeding procedures and overcome some of the agronomic and environmental problems which would otherwise not be possible through conventional methods. On the other hand, nutritional requirements (mineral nutrients), carbohydrates and other organic compounds (vitamins, amino acids, etc), environmental factors (e.g. light, gaseous environment, temperature and humidity) and treatments with growth regulators have helped in achieving high proliferation rates to allow commercially viable micropropagation. An overview of the regeneration of chrysanthemum by direct and indirect organogenesis, embryogenesis from explains and embryo rescue is presented in this article. In addition, the use of these techniques in association with several biotechnological methods to enrich the genome of chrysanthemum, such as selection of somaclonal variants, screening for various usetul characteristics and genetic transformation, is reviewed. © 1997 Elsevier Science B.V.…

    • 1299 Words
    • 6 Pages
    Better Essays
    Read More
  • Good Essays

    Transgenic Bt Cotton
    • 9372 Words
    • 38 Pages

    Transgenic Bt Cotton

    Dr CD Mayee Dr P Singh Dr AB Dongre Dr MRK Rao Dr Sheo Raj…

    • 9372 Words
    • 38 Pages
    Good Essays
    Read More
  • Powerful Essays

    Influence of Rhizobium on the Growth and Yield of Rice
    • 5081 Words
    • 21 Pages

    Influence of Rhizobium on the Growth and Yield of Rice

    Rice (Oryza sativa L.) is one of the world's most important crops. The present investigation was designed to assess the range of growth-promoting activities of various diazotrophic bacteria on rice seedling vigor, its carryover effect on straw and grain yield, and the persistence of an inoculant strain on rice roots under greenhouse conditions. Growth responses to inoculation exhibited bacterial strain–rice variety specificity that were either stimulatory or inhibitory. Growth responses included changes in rates of seedling emergence, radical elongation, height and dry matter, plumule length, cumulative leaf and root areas, and grain and straw yields. Most notable were the inoculation responses toRhizobium leguminosarum bv. trifolii E11 and Rhizobium sp. IRBG74, which stimulated early rice growth resulting in a carryover effect of significantly ( P = 0.05) increased grain and straw yields at maturity, even though their culturable populations on roots diminished to below detectable values at 60 d after planting. The test strains were positive for indole-3-acetic acid production in vitro, but only some reduced acetylene to ethylene in association with rice under laboratory growth conditions. These studies indicate that certain strains of nonphotosynthetic diazotrophs, including rhizobia, can promote growth and vigor of rice seedlings, and this benefit of early seedling development can carryover to significantly increased grain yield at maturity.…

    • 5081 Words
    • 21 Pages
    Powerful Essays
    Read More
  • Powerful Essays

    Fipronil Rice Literature Study
    • 3269 Words
    • 14 Pages

    Fipronil Rice Literature Study

    Andhra Pradesh Rice Research Institute and Regional Agricultural Research Station, Maruteru-534 122, West Godavari District, Andhra Pradesh, India…

    • 3269 Words
    • 14 Pages
    Powerful Essays
    Read More

Related Topics