Introduction Rice is a major staple food for higher % of world

population, cultivated in wide range of ecological

environment worldwide, particularly in asia.

It is easy to genetically modify for cereal biology

Rice can be genetically modified either by

Agrobacterium mediated transformation or Biolistic

(Plastid) transformation

Advantage This method can be use to transform all plant species. No binary vector is required. Transformation protocol is relatively simple.

Disadvantage Difficulty in obtaining single copy transgenic events. High cost of the equipment and micro carriers. Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.). Transfer DNA is not protected.

Plastid Transformation

Agrobacterium Transformation Advantage

Disadvantage

One of the major advantage of Agrobacterium mediated transformation is the relative simplicity of the T-DNA loci

Disadvantage of using Agrobacterium for transformation is the host specificity, resulting in low level of transformation in plant species

Seed sterilizationSeed sterilization

Preinduction callus

Preinduction callus

Preculture of callus

Preculture of callus

Callus inductionCallus induction

inoculationinoculation

Culture A. Tumefaciens harboring

pCAMBIA1301 in AB medium

Culture A. Tumefaciens harboring

pCAMBIA1301 in AB medium

Preparation of inoculum

Preparation of inoculum

Co-cultivationCo-cultivation

selectionselection

Shoot regeneration

Shoot regeneration

X-Gluc solutionX-Gluc solution

Examination of expression of

GUS

Examination of expression of

GUS rootingrooting pottingpotting

Material and Methods – Agro bacterium transformation

at 28oC +16 h light +8 h dark cycle

3 weeks

at 28oC In dark 3 days

Co-cultivation

2 mins

3 days

3 days

Continue…

Material and Methods – Biolistic transformation

A schematic protocol for production of fertile transgenic plants using:

1.Biolistic systems,2.Protoplast systems3.Agrobacterium systems

(Datta,et al.)

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

VectorRice 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 p16STB/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 discussionBrowning calli present in a single paper on solid medium

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)

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

According to Terada (2004), the best condition for co-cultivation of rice calli on solid medium are temperature at 25oC, Agrobacterium concentration of OD= 2 and 200µM of acetosyringone

Raineir (1990) reported that rice cells might be capable of producing a certain level of signal molecules that induce the expression of vir genes

There is the unidentified compounds which is produced from rice calli that may enhance transformation efficiency by a different mechanism than acetosyringone (Ozawa, 2010).

In this experiment, 0-15mg/L of acetosyringone at 25oC is suitable

The transgenic plants which carried multiple copies of a transgene were confirmed by using Southern blotting

-Agrobacterium mediated transformation

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

RecommendationUsing other selectable markers such as the

anthranilate synthase alpha-subunit gene, positive selection systems

Identify the compounds produced in rice calli which seem to be critical for high efficiency of Agro bacterium- mediated rice transformation

The amount of embryogenic callus was higher on media containing ABA. This overcomes the permanent injuries caused during Biolistic transformation

ReferencesOzawa 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