Hereditary And Phenotypic Analysis Of Transgenic Tomato With Interleukin-4 Gene And Its F₁ Hybrids

The present paper focuses on the analysis of il-4 gene transgenic tomatoes in T₅, T₆ generation and F₁-hybrids. The seed emergence ratio was recorded, PPT resistance selection was performed, PCR and PCR-southern testing was carried out, the phenotypic character was investigated. Artificial crossing was operated between different varieties of transgenic plants. To the F₁-hybrids and their parents'inbreed offspring, ELISA test and Western blot analysis was performed. The heredity rules of il-4 gene in high generation transgenic tomatoes was revealed, the difference of il-4 protein content was analyzed. The goal is to find a varieties in which il-4 gene has a high content, and could inherit stably.In our series of experiments, 1639 seeds of T4 generation transgenic tomatoes was planted, and 1060 plants of T₅ generation was obtained, among which 532 plants show PPT resistance character. There are three varieties of T₅ generation plants, whose PPT resistance separation ratio matches the Mendelian rule: resistance plants: non-resistance plants = 3:1. The positive rate of il-4 gene PCR amplification is 4.15%. In the next generation, 2090 seeds of T₅ generation transgenic tomatoes was planted, while 1015 plants of T₆ generation was obtained, among which 485 plants show PPT resistance character. There is only one variety of T₆ generation plants, whose PPT resistance separation ratio fulfills the Mendelian rule. The positive rate of il-4 gene PCR amplification after PPT selection in T₆ generation is 5.32%, while the rate after non-PPT selection in T₆ generation is 9.27%. In F0-hybrids, 187 seed was planted, and 117 plants of F₁-hybrids was obtained. The positive rate of PCR amplification is 7.96%. The PCR-southern testing results demonstrate the existence of il-4 gene in the genome of the high generation plants. Finally, the phenotypic character of T₆ generation transgenic plants and normal plants was recorded and analyzed. There is remarkable difference between T₆ generation and normal plants in height, No. of fruits. Most plants of F₁-hybrids have less fruits and seeds/fruit, compared to T₆ generation plants. However, a few plants demonstrate heterosis. There are 44 plants of T₆ generation, inbreed offspring of parents of F₁-hybrids is PCR positive, among which there are 16 plants (36.36%) positive in ELISA test. The quantity of IL-4 protein is 32.49-352.51ng/FW, occupies 0.03-0.25%. In different varieties of transgenic plants, there is difference in the quantity of IL-4 protein. In the same varieties, the quantity of IL-4 protein is also different in different plants. There are 9 plants of F₁-hybrids positive in PCR test, among which 8 plants (89%) is positive in ELISA test. The quantity of IL-4 protein is 26.46-643.85ng/FW, occupies 0.02-0.51%. Among three combinations of hybrids, two combinations of F₁-hybrids have a higher content of IL-4 protein than their parents'inbreed offspring. Western blot analysis further confirm that IL-4 protein could inherit between generations.