2012 Annual Report
1a.Objectives (from AD-416):
The primary objective of this work is to develop techniques and methods that will lead to sterile or highly infertile cultivars of invasive or potentially invasive nursery crops.
1b.Approach (from AD-416):
The general approach is to use use biotechnology or genetic engineering to create seedless plants or to develop methods that will lead to the creation of seedless plants. One area of study will be to test gene suppression using the AGAMOUS gene and dsRNA-RNAi. The AGAMOUS gene is a floral homeotic gene that, when suppressed or mutated, can result in flowers that have petals in place of pistils and stamens, and are therefore sterile. The second area of study will be to create transgenic plants using several different gene constructs that may induce male and/or female sterility.
The main goal of this work is to develop methods that will lead to sterile or highly infertile cultivars of invasive or potentially invasive woody nursery crops. As a model, transgenic sweetgum trees were produced and planted in the field five years ago. They continue to grow very well, showing similar or greater growth and fall color than the wild-type controls that were also propagated in vitro and transplanted at the same time. One non-transgenic control has flowered, and we are expecting that, with their obvious vigorous growth this year, the remaining individuals will flower in 2013.
Tissue samples were collected from all of these transgenic trees in summer of 2010 and 2011 for studies of RNA extraction. Leaf samples were collected from all 250 trees in the field trial and RNA was extracted, quantified, and analyzed for quality. cDNA was synthesized from 86 RNA samples. Three of these cDNA samples were then used for qPCR primer testing. PCR primers for five control genes for qPCR were designed based on published sequences. Only one primer set worked in qPCR, and gave two amplicons instead of the expected one. Additional primer design and qPCR optimization will be necessary.
Near the start of this project, we produced an RNAi construct directed against the AGAMOUS homolog in apple. The construct was sent to a cooperator, who produced transgenic Gala apples with it. The Cooperator has reported the recovery of a number of sterile transgenic flowering trees with multiple whorls of petals, as expected for AG inhibition. The Coopertor is now studying gene expression, morphology, and fertility for those lines. These results suggest that RNAi against AGAMOUS could be an effective means for producing sterile crabapples with showy flowers.
We collaborated with a scientist at Oregon State University on a problem analysis of approaches to reducing invasiveness using conventional biotech and transgenic methods. Our resulting manuscript was recently accepted for publication in the journal HortScience.