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STUDIES ON TRANSGENE EXPRESSION IN INDICA RICE

Riaz, Naveeda (2001) STUDIES ON TRANSGENE EXPRESSION IN INDICA RICE. PhD thesis, University of the Punjab, Lahore.

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Abstract

Rice is the major staple food for about 2.5 billion people; almost all of them live in developing countries. To maintain an adequate supply of rice for tremendous annual increase in population is a formidable challenge to the scientific community. Development of at transgenic plants to protect crop losses due to insect attack is a technically feasible and otherwise viable strategy. Biolistic procedure was used for indica rice transformation using home made gun. Scutellum derived calli of rice variety Basmati 370 were used as target tissue to bombard DNA coated tungsten particles. Different parameters of biolistic gun i.e. distance, age of explant and osmotic treatment were optimized using GUS reporter gene. Maximum blue spots (27 ± 11.3 per plate) were observed when distance between filter unit and target tissues was 21 cm. Transient GUS expression increased 4 times when calli were treated with high osmoticum medium (0.4 M mannitol) 4 hours before bombardment and 16 hours after bombardment. There are concerns that insects could evolve genetic resistance to transgenic plants harbouring single insecticidal gene. One of the strategies to-delay insect resistance build-up, if any, is the concurrent use of multiple genes that target the same pest through different molecular mechanisms. As a part of this strategy, synthetic cry1A(c) and cry2A genes were selected for gene pyramiding in rice. Cry1A(c) and Cry2A proteins recognize and bind to different receptor sites inside the insect midgut. Therefore, a single mutation in insect will not modify two different receptor sites in the insect. A vector pSM6 was constructed, consisting of two complete cassettes, for the expression of cry1A(c) (under Ubi promoter) and cry2A (under 35S promoter) genes, tungsten particles (Sylvania) were coated with pSM6 vector, containing cry1A(c) and cry2A and pROB5 vector containing hygromycin antibiotic marker gene. The gene of interest and marker gene were used in 3:1 ratio and 1.9 % transformation efficiency was observed. All primary transformants were analyzed by dot blot analysis which confirmed the presence of cry1A(c), cry2A and hph genes. Further confirmation was done by Southern blot after hybridization of Hindlll digested genomic DNA blots using cry 1 A (c) and cry2A probes. The expression of insecticidal proteins Cry1A(c) and Cry2A was confirmed by Western blot analysis and by insect bioassay utilizing yellow stem borer and rice leaf folder neonates. The insect mortality was found upto 100% in all transgenic plants. The seeds of line NCB-313 were grown to study stability of Bt genes and inheritance pattern in the next generation. Eighty percent plants were able to grow on hygromycin 50 ug/ml, indicating 3: 1 ratio of hph gene. Progeny plants showed 40 to 100% mortality against rice leaf folder and yellow stem borer. Insecticidal genes were segregated and expressed in progency of transgenic plants in 3: 1 ratio. Expression of Cry1A(c) and Cry2A was determined in different plant parts i.e. leaf, straw, husk and seeds. Expression of Cry1Ac was higher when compared to Cry2A in different parts of the plants, suggesting that ubi promoter was more efficient than 35S promoter. Maximum Cry proteins were present in leaves. Level of Cry1A(c) protein in leaves was 4-16 ug/gm of tissue while Cry2A protein was present at level 0.89-1.45 ug/gm of the tissue. In T2 generation expression of Cry1A(c) protein was 13.0-24.8 ug/gm of tissue while level of Cry2A protein was 0.9-2.7 ug/gm of the leaf tissue. Variation in protein level in T1 and T2 was due 10 differences in growth stages. T2 plants were very young and ELISA of T1 plants was performed at end of growth period. Progeny plants were divided into three categories i.e. with only cry1A(c) or cry2A and those with both genes and the ability to kill agronomically important pests was tested against rice leaf folder and yellow stem borer. Transgenic plants exhibited improved performance against rice yellow stem borer when both genes were expressing in transgenic plants. However mortality of rice leaf folder was almost the same in three different categories of transgenic plants. Progeny plants were infested with yellow stem borer at different growth stages. Damage was recorded in terms of dead hearts after one month. Percentage damage was higher in 4 month old plant as compared to two and three month old plants. Biochemical analysis of transgenic and untransformed plants were done in order to determine their amylose contents and their cooking quality. 20.6 to 25.7 % amylose contents were present in different transgenic lines. These values were not significantly different from that of untransformed plant i.e. 23.7 %. The results of molecular analysis and insect feeding assay have confirmed the stable integration and expression of cry1A and cry2A genes in T1 and T2 progeny plants. According to results of biochemical analyses amylose contents, length to width ratio, stickiness and cooking quality of transformed and untransformed plants are the same.

Item Type:Thesis (PhD)
Uncontrolled Keywords:Transgene Expression, Indica Rice, Basmati 370, Cry1a(C) Genes, Cry2a Genes, NCB-313, Progeny Plants, Bacillus Thuringiensis, Cry Proteins, Transgenic Plants
Subjects:Biological & Medical Sciences (c) > Biological Sciences(c1) > Biology (c13)
ID Code:341
Deposited By:Mr Ghulam Murtaza
Deposited On:26 Jun 2006
Last Modified:04 Oct 2007 21:00

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