Update:
To enhance soybean cyst nematode (SCN) resistance in transgenic soybean by modifying gene silencing strategies we have been working on advancing the transgenic lines we have produced. For the first round of crossing, all seeds (about 150 seeds) were planted and are being tested for GOI by PCR, currently gDNA from half of plants were extracted and tested by PCR. There was one plant (Prp17X K11) positive for GOI, which contained intact expression cassette. Additional K11-2363B seedlings were planted for back crossing with this F1 positive plant.

We have performed a second round of crossing with our transgenic soybean with hpRNAi_Y25 and Prp17 to K11-2363B and K12-2333 separately is underway. Approximately 20 plants were used in the crossings and currently pods are forming in these plants. A third round of crossing underway as well as field test plots of a number of events..

As an alternative approach to SCN resistance we are continuing to transform soybean cultures with two genes that together should disrupt SCN mating. We were having initial difficulty in recovering events with one of the genes and suspected that the gene may have a lethal phenotype. To investigate this potential problem we introduced these constructs into Arabidopsis. Two Arabidopsis thaliana events were confirmed to contain the genes and demonstrated that the gene was not lethality to plants. Since then we have confirmed six of our soybean events to contain both constructs and we are currently regenerating these cultures. We are also continuing exploring the possibility to introduce these genes into a bacterium as an alternative delivery method. We are working out the transformation and expression methodologies for this bacterium with our two genes.

Work with the defensin genes was focused on generating plasmids for Agrobacterium transformations. These will be used for hairy root assays and to transform Arabidopsis as a more rapid evaluation of the defensing genes. Constructs were verified by sequencing and we are using those in transformation experiments.

For the eIF knocking down project, more T3 seeds of “400Awash” are available for bioassay test with soybean mosaic virus infection. A bioassay will be set up to test if the transgenic lines have any improved resistance to SMV.

For host-derived RNAi targeting Fusarium virguliforme, the fungus that causes soybean sudden death disease, we have isolated gDNA samples from transgenic callus to confirm presents of the full-length transgenic sequence. To date only one positive by PCR was identified but we are still screening cultures.