three pieces of filter paper moistened with 5.5 mL of N6D medium+ 100mg/L of L-cysteine give calli without browning
0-15mg/L of acetosyringone at 25 o C
Transcript of "Oryza sativa"
Introduction <ul><li>Rice is a major staple food for higher % of world population, cultivated in wide range of ecological environment worldwide, particularly in asia. </li></ul><ul><li>It is easy to genetically modify for cereal biology </li></ul><ul><li>Rice can be genetically modified either by Agrobacterium mediated transformation or Biolistic (Plastid) transformation </li></ul>
Advantage <ul><li>This method can be use to transform all plant species. </li></ul><ul><li>No binary vector is required. </li></ul><ul><li>Transformation protocol is relatively simple. </li></ul>Disadvantage <ul><li>Difficulty in obtaining single copy transgenic events. </li></ul><ul><li>High cost of the equipment and micro carriers. </li></ul><ul><li>Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.). </li></ul><ul><li>Transfer DNA is not protected. </li></ul>Plastid Transformation Agrobacterium Transformation Advantage Disadvantage <ul><li>One of the major advantage of Agrobacterium mediated transformation is the relative simplicity of the T-DNA loci </li></ul><ul><li>Disadvantage of using Agrobacterium for transformation is the host specificity, resulting in low level of transformation in plant species </li></ul>
Seed sterilization Preinduction callus Preculture of callus Callus induction inoculation Culture A. Tumefaciens harboring pCAMBIA1301 in AB medium Preparation of inoculum Co-cultivation selection Shoot regeneration X-Gluc solution Examination of expression of GUS rooting potting Material and Methods – Agro bacterium transformation at 28 o C +16 h light +8 h dark cycle 3 weeks at 28 o C In dark 3 days Co-cultivation 2 mins 3 days 3 days
Continue… Material and Methods – Biolistic transformation
<ul><li>A schematic protocol for production of fertile transgenic plants using: </li></ul><ul><li>Biolistic systems, </li></ul><ul><li>Protoplast systems </li></ul><ul><li>Agrobacterium systems </li></ul><ul><li>(Datta,et al.) </li></ul>
Chloroplast from leaves & DNA Isolated Two homologous fragments are amplified and purified Cloned into pBluescript SK (MB) - pSKE & pSKF sequencing vectors are constructed Double digested with Sac II & Bam H I & fragment inserted in pSKF Vector Rice homologous fragment - pREF Pu16S was digested by Sac I Cut was blunted with klenow fragments and again cut with Hind III Modified 16s promoter (150BP) Digested product was cloned into PT393 between xbal site & hind III site Expression vector p16ST PAZ – Digested by Xba I Cut was blunted and again cut with Hind III Product contain Bar sequence & it was purified Inserted into p16ST B/W Sac I – Hind III site Intermediate vector p16STB was formed Cut with Bam H I – DNA fragments Cloned into pREF – b/w Bam H I site Rice chloroplast pRB
Construction of chloroplast transformation vector Source : LIYi-nü etal
Results and discussion <ul><li>Browning calli present in a single paper on solid medium </li></ul><ul><li>The tissue necrosis (browning) calli will be reduced in the medium containing reductants such as ascorbic acid or L-cysteine in a solid co-cultivation (Olhoft, 2001; Enriquez-Obregon,1999 and Potrykus, 1991) </li></ul>Figure 1: (A) Calli co-cultured on a sterilized filter paper placed on 30 mL solid N6D (B) Co-cultured calli on solid medium were subjected to hygromycin selection for 7 days. (C) Co-cultured calli on solid medium were subjected to hygromycine selection for 7 days and stained with X-Gluc solution. (D)Calli co-cultured on three pieces of filter paper moistened with 5.5 mL of N6D medium (E) Co-cultured calli on liquid medium were subjected to hygromycin selection for 7 days. (F) Co-cultured calli on liquid medium were subjected to hygromycine selection for 7 days and stained with X-Gluc solution Three pieces of filter paper moistened with 5.5 mL of N6D medium+ 100mg/L of L-cysteine give calli without browning
<ul><li>According to Terada (2004), the best condition for co-cultivation of rice calli on solid medium are temperature at 25 o C , Agrobacterium concentration of OD= 2 and 200µM of acetosyringone </li></ul><ul><li>Raineir (1990) reported that rice cells might be capable of producing a certain level of signal molecules that induce the expression of vir genes </li></ul><ul><li>There is the unidentified compounds which is produced from rice calli that may enhance transformation efficiency by a different mechanism than acetosyringone (Ozawa, 2010). </li></ul>In this experiment, 0-15mg/L of acetosyringone at 25 o C is suitable
<ul><li>The transgenic plants which carried multiple copies of a transgene were confirmed by using Southern blotting </li></ul><ul><li>- Agrobacterium mediated transformation </li></ul>
transplastomic rice lines - Biolistic transformation Molecular identification of the bar gene in transplastomic rice plants - Chloroplast transformation bar gene was identified in transplatomic rice plants by using Southern blotting - Chloroplast transformation
Recommendation <ul><li>Using other selectable markers such as the anthranilate synthase alpha-subunit gene, positive selection systems </li></ul><ul><li>Identify the compounds produced in rice calli which seem to be critical for high efficiency of Agro bacterium - mediated rice transformation </li></ul><ul><li>The amount of embryogenic callus was higher on media containing ABA. This overcomes the permanent injuries caused during Biolistic transformation </li></ul>
References Ozawa Kenjirou (2009) Establisment of a high efficiency Agrobacterium- mediated transformation system of rice ( Oryza sativa L.). Plant Science , 176 : 522-527 Ozawa Kenjirou and Takaiwa Fumio (2010) Highly efficient Agrobacterium- mediated transformation of suspension-cultured cell clusters of rice ( Oryza sativa L.). Plant Science , 179 : 333-337. Olhoft PM, Somers DA (2001) L-Cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells, Plant Cell Rep . 20 :706–711. Enrı´quez-Obrego´ n GA, Prieto-Samso´ nov DL, de la Riva GA, M. Pe´ rez, G. Selman-Housein, R.I. Va´zquez-Pado´ n (1999) Agrobacterium-mediated Japonica rice transformation:a procedure assisted by an antinecrotic treatment, Plan. Cell Tissue Org. Cult . 59 : 159–168. Potrykus I (1991) Gene transfer to plants: assessment of published approaches and results , Annu. Rev. Plant Physiol. Plant Mol. Biol. 42 : 205–225. LI Yi-nu (2009) Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants, Agricultural Sciences in China. 8(6) 643-651. Hiratsuka J, Shimada (1989) The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Molecular and General Genetics , 217, 185-194 Boynton J E (1988), Chloroplast transformation in Chlamydomonas with high velocity micro projectiles, Science, 240 , 1534-1538
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