Introduction Of The Puroindoline A And B Genes Into Durum Wheat (Triticum Turgidum L. Var. Durum) Using Particle Bombardment

Wheat grain hardness is one of the most important factors determining milling and processing quality of wheat and is mainly controlled by Puroindoline a (PINA) and Puroindoline b (PINB) proteins which are encoded by two highly linked genes Pina and Pinb located on the short arm of chromosome 5D. Either the lack of Pina or the mutation of Pinb results in increasing the grain hardness. In this study, the foreign wild-type Pina and Pinb genes were introduced by particle bombardment into four cultivars (Luna, Ofanto, Venusia and Latino×Lira) of durum wheats (Triticum turgidum L. var. durum) which lack Pina and Pinb. The results obtained from the experiments are as follows. 1) The effects of five different plant growth hormones on rescuing two aged bread wheat (Triticum aestivum L.) cultivars (Xiang 83-2661 and Xiangmai 5) and four aged durum wheat (Triticum turgidum L. var. durum) cultivars (Luna, Ofanto, Venusia and Latino×Lira) were studied by in vitro mature embryo cultures on MS medium, using single factor and orthogonal experimental designs. Single factor and orthogonal experimental results showed that GA3 could signaficantly enhance the germination efficiency of the aged wheat seeds tested, and MS medium containing 2.5mg/L GA3 and the combinations of 2.0 mg/L 2,4-D, 1.1 mg/L NAA, 0.5 mg/L 6-BA and 4.0 mg/L GA3 could increase the germination efficiency from 29.2% to 32.5%. 2) The protocols for both bacterial transformation and large-scale plasmids preparation have been optimised. The pCaI-Neo, pUbiPina and pUbiPinb were transformed into XL1-Blue strain of E coli. respectively, using the optimal protocols. The plasmids were extracted by alkaline lysis method and purified by phenol-chloroform. The plasmids were measured by UV spectrophotometer and then were analysed by enzyme restriction digestion followed by gel electrophoresis. The results proved that the prepared plasmids were the original plasmids with high quality and yields. 3) The bombardment conditions were optimised by GUS transient expression test. The result showed that the combination of 900 Psi bombardment pressure and 6 cm bombardment distance was suitable for the durum wheat transformation. The plasmids pUbiPina, pUbiPinb and pCaI-Neo containing Pina, Pinb and Neo genes were introduced into four donor durum wheats of Luna, Ofanto, Venusia and Latino×Lira through cotransformation with the optimal condition by particle bombardment. The results of tissue culture showed that the scutella bombarded from four durum wheat cultivars (line) had the high embryogenesis frequencies of 99.4%, 100.0%, 95.2% and 97.0% respectively.