Enhancement Of Resistance To Pests And Disease By Introducing Snowdrop Lectin Gene (GNA) In Maize Elite Inbred Lines Via Agrobacterium Tumefaciens Mediated Transformation

According to the report of the Food and Agriculture Organization of the United Nations (FAO), direct yield losses due to pests in the world every year have been estimated at 14% of total cereal production or hundreds of billion dollars, and 12% of total cereal production lost due to diseases. So crop protection is a long term challenge that farmers and researchers have to face. Maize (Zea mays L.) is a widely cultivated crop. Yet, its yield and quality are severely compromised by pests and diseases. So it is a profound work to enhance the resistance of maize to pest and disease. In this study, scutellum-derived embryogenic (Type II) calli from immature embryos were used as materials for Agrobacterium tumefaciens-mediated transformation. The snowdrop lectin (Galanthus nivalis L. agglutinin, GNA) gene gna has been introduced into four elite maize inbred lines DH4866, DH9942, DH9932, and 8902, and a large amount of transgenic maize plants was obtained. Molecular analyses revealed that the gna gene has been integrated into maize genome and inherited into T₁ to T₃ progenies as a Mendelian pattern. The resistance of transgenic plants to pests and diseases were also studied. The main results presented in this dissertation are as follows:1. Agrobacterium tumefaciens-mediated transformation of elite maize inbred lines and molecular analysesEmbryogenic (Type II) calli derived from the immature embryos of four elite maize inbred lines DH4866, DH9942, DH9932, and 8902, were used as donor cells for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain AGLO containing the standard binary vector pWRG815 was used in all experiments and the gene gna under control of the promoter RSs-1 was introduced into maize genome. Transformants were selected on the selective medium containing hygromycin at 20 mg/L. T₀ (plant regenerated from transgenic Type II callus), T₁ and T₂ plants were artificially selfed to obtain the next generation. Molecular analyses such as polymerase chain reaction (PCR), Southern blot and Western blot were performed, and the resultsconfirmed that the gna gene has been integrated into maize genome and inherited to the following generations. Many homologous transgenic lines carrying gna were identified in T2 and T3 generations. Using DNA from untransformed plants and RSs-1-gwar fragment from plasmid pWRG815 as negative and positive controls respectively, Southern blot analyses were performed with transformants and their progenies. It was found that a single copy insertion of RSs-1 -gna fragment happened in most of the transformants. While, in few transgenic cases, multi-copy (2-3 copies) insertions were found also. Western blot was performed on single-copy insertion events using GNA protein and total soluble protein from untransformed plants as positive and negative controls respectively. Semi-quantitative estimations of expression level of GNA protein in leaf tissue sample were undertaken by comparing the GNA band staining intensity of plant samples with that of standards containing known amounts of GNA. We found that the transgenic plants expressed a 12-KD polypeptide corresponding to the GNA protein standard, and the expression level varied from 0.04% to 0.28% total soluble protein. Differences in level of expressed GNA existed among lines from different independent transgenic evens, which was bigger than that appeared among the plants from a line. Genetic analyses by PCR were carried out with single copy insertion lines from Ti to T3 generation. Most of the transgenic lines showed Mendelian segregation pattern of the gna gene. This inheritance was consistent with the integration of the foreign gene (gna) in a single locus. However, few lines tested didn't obey the Mendelian segregation of the gna gene. And, in some cases, the foreign gene was lost in Ti or T2 generation.2. Insect bioassays of transgenic plantsBecause a homopteran is a phloem feeder, protein, especially expressed in the phloem tissue, would be delivered efficiently to the pest, as well as minimizing any potentially undesirable accumulation of the protein in other parts of the transgenic plant. The rice sucrose synthase-1 promoter (RSs-1) can direct phloem-specific expression and it was used in our studies to promote gna gene. PCR positive T2 and T3 plants were used in aphid toxicity analysis. Non-transformed homologous plants were used as controls. The GNA-expression level in the leaf tissue of the tested plants varied from 0.20% to 0.28% total soluble protein. The transgenic plants had shown significant effects on aphids. Although the survival of aphids was not affected, their fecundity was drastically reduced,with a mean reduction of 52%. In the field investigation, data were collected during the filling stage, and the results supported the significant reduction of aphids' fecundity due to GNA-expression. Compared with the non-transgenic controls, the aphid population density on ear leaves was largely reduced. Thus, the plants were protected from aphid compromising.It was reported that GNA has detrimental effects on the larval development of the maruca pod borer (Maruca vitrata Fabricius) and the tomato moth {Lacanobia oleracea L.) at all stages. In this study, artificial diet bioassays have shown that GNA has a significant effect on the development and survival of the Asian corn borer (Ostrinia furnacalis Guenee [Lepidoptera : Pyralidae];ACB), when fed at 20 mg GNA per kg diet. The effects of GNA on ACB were shown by reductions in survival (at ca. 24%), the length of the fifth instar larvae (P < 0.01, Student's t-test), pupation success rate (P < 0.05, Student's Mest), and the weight of pupae (P < 0.01, Student's Mest). The larvae period was also significantly prolonged (P < 0.01, Student's Mest). The results indicated that GNA is toxic to ACB. Using non-transformed homologous plants as controls, the resistance of transgenic plants to ACB was studied by transferring neonate larvae to GNA-expressing plants, and the GNA expression levels in the leaf tissue varied from 0.24% to 0.28%. In this study, larval survival dropped by 6.7%¹0.0%, larval duration was prolonged significantly (P < 0.01, Student's Mest), and the length of the fifth instar larvae, pupation success rate as well as pupal weight, were reduced significantly (at P < 0.01, P < 0.05, and P < 0.01 separately, by Student's Mest). Using non-transformed parental plants as controls, field investigations were performed with the same GNA-expressing lines used above. Data were collected during the grain-filling stage. The results showed that the extent and severity of transgenic plants damage were largely reduced and their resistance to ACB was enhanced.These results showed that GNA-expressing maize plants not only enhanced resistance to homopterans, such as aphids, but that these plants also gained some resistance to another major pest, namely ACB. After a series of artificial self-crosses, some homozygous transgenic lines expressing GNA were obtained. Thus, we have obtained new maize materials with resistance to pests for further breeding.3. Disease resistance test of transgenic plantsIn China, maize rough dwarf disease is only transmitted by small brown planthopper (Laodelphax striatellus Fallen;SBPH), a sap-sucking insect of homopteran, in a persistent manner. It was reported that GNA is effective in decreasing the survival, development and fecundity of SBPH and other homopteran pests. In this work, we studied maize rough dwarf disease (MRDD) resistance of GNA-expressing plants. The virus-carrying SBPH artificial inoculation trials were performed, using untransformed homologous plants as controls and the GNA-expression level in leaf tissue of the tested transgenic plants varied from 0.24% to 0.28% total soluble protein. Data were collected during the grain-filling stage. The results showed that the resistance of transgenic plants to MRDD was improved. With the existence of untransformed plants, the GNA-expressing plants reduced efficiently the MRDD infestation percentage (at 34.0%⁵0.0%, significant at P < 0.01, by Student's Mest), as well as increased the relative plant height (significant at P < 0.01, by Student's /-test). The MRDD sensitivity index was also reduced. However, when plants were individually covered in insect-proof meshes and confined the inoculated SBPHs to a single plant, no significant difference was found between GNA-expressing plants and controls in MRDD infestation percentage. The explanation of the improved resistance to MRDD might be that SBPHs prefer to feed on non-transgenic plants, which would reduce its feeding frequency on GNA-expressing plants. Thus the possibility that the GNA-expressing plants were infected by MRDD was significantly decreased. Therefore, effective production practices would favor planting some non-transgenic maize together with the transgenic ones in the field to exert the pest resistance of GNA-expressing plants better, reduce chemical pesticide use, cut down production cost, protect environment and increase yield. Field evaluation of the resistance of transgenic plants to MRDD was carried out at grain-filling stage. In the field, the MRDD infestation percentages of the GNA-expressing lines were 7.7%¹3.0% lower than those of the untransformed controls, and their mean plant heights were 9.9—17.8 cm higher than the control ones'. The results indicated that the resistance of transgenic plants to MRDD was enhanced. Similar results were also obtained from T3 plants. At the meantime, the resistance of transgenic plants to maize dwarf mosaic disease was enhanced significantly also.Elite maize inbred lines DH4866, DH9942, DH9932, and 8902 were used in this transgenic work. Compared with original inbred lines, transgenic plants selected (from T2to T3 generation) had no obvious change in agricultural characteristics such as plant height, ear position and the number of leaves. We did not find any obvious alteration in the combining ability of these progenies from transformed plants after selections also. After a series of artificial selfing, selecting, many homozygous GNA-expressing plants were obtained. These pests and disease resistant maize materials can be prospectively used for further breeding work.