Performing Department
Plant Sciences
Non Technical Summary
Alfalfa is a major forage crop grown in the United States and Wyoming, and accounts for the fourth largest commodity. In 2012 alfalfa grown on 17.3 million acres produced 52 million tons of hay with Western states accounting for approximately 40% of total production. Alfalfa weevil (Hypera postica) is the most destructive insect pest of alfalfa in the United States. Both larvae and adults are damaging stages and feed on leaves, terminal shoots and stems. Reduced vigor, crown density and depleted root reserves resulting from insect damage can result in poor regrowth and significantly lower yields in subsequent cuttings. In case of severe insect pressure, plants can be completely denuded resulting in losses up to 30 - 40%. In addition to yield losses, forage quality including protein content and digestibility can be compromised. Weevil damaged alfalfa stands can also be more susceptible to weed infestation over years due to reduced plant vigor. Alfalfa pest management practices include early harvesting and aerial spraying of large acreages, which can be expensive and not effective in completely controlling the pest.The goal of the proposed project is to develop weevil resistant transgenic alfalfa cultivars and add a valuable tool to existing pest management strategies, which currently include biological, cultural and chemical control. Alfalfa weevil resistance in existing cultivars would be improved using genetic engineering/genetic modification (GM) technology. GM technology has been well established and proven in alfalfa with the development of Roundup Ready alfalfa. Extensive studies have also been conducted to test the benefits and limitations of GM alfalfa and its co-existence with organically grown alfalfa. The project will take a holistic approach by using combined expertise of alfalfa growers and researchers with multidisciplinary expertise in areas of agronomy, genetics, molecular biology and integrated pest management. Development of alfalfa weevil resistant cultivars would be a valuable addition to existing biological, cultural and chemical pest management strategies. The project addresses a serious problem facing Wyoming alfalfa growers and will pave the way for the development of improved cultivars.
Animal Health Component
0%
Research Effort Categories
Basic
10%
Applied
20%
Developmental
70%
Goals / Objectives
The purpose of the proposed project is to incorporate insect (alfalfa weevil) resistance in commercial alfalfa cultivars using genetic engineering technology, thus generating new germplasm not currently available and overcoming a major limiting factor in alfalfa production.Specific objectives include:1) Targeted expression of Galanthus nivalis (snow drop) lectin genes in transgenic alfalfa cultivars,2) Greenhouse screening of transgenic plants for enhanced resistance against alfalfa weevil, and3) Determining yield potential and forage quality of the screened transgenic alfalfa plants.
Project Methods
1) Targeted expression of lectin genes in transgenic alfalfa cultivars (Dhekney)Commercial alfalfa cultivars along with the model variety 'Regen-SY' will be genetically engineered with snowdrop lectin genes and screened against insect resistance. The lectin genes will be isolated from the snowdrop plant and modified for better function in alfalfa. These genes will then be transferred to a vector for insertion in alfalfa. Alfalfa tissue cultures will be initiated from seed materials. Tissue culture materials will be engineered with Agrobacterium containing the lectin genes and transgenic plants will be recovered using standard protocols. We will confirm the number of gene copies inserted in plants and quantify the amount of lectin protein made by regenerated transgenic plant lines. Plant lines that produce high levels of lectin proteins will be used for screening in the greenhouse.Objective 2) Screening transgenic plants for enhanced resistance against alfalfa weevil (Jabbour, Dhekney, Padlock Ranch).Transgenic alfalfa will be screened for alfalfa weevil resistance following a modified approach (Shade, Thompson and Campbell 1975). The overall approach will be to compare juvenile survival, development time and pupal size of weevils feeding on transgenic and control commercial alfalfa cultivars. In early May, adult weevils will be collected via sweep net from alfalfa fields near Wheatland, WY. The weevils will be returned to a laboratory environmental chamber where they will be allowed to lay eggs on terminal shoots of potted alfalfa Regen SY plants. Newly hatched first instar larvae, which can be expected to appear 6-8 days post egg-laying, will be transferred individually to small cages on terminal shoots of potted 7-10 week old experimental alfalfa plants. Each cage will consist of a 267 ml waxed paper cup held in place on the shoot with a foam plug; additionally, the cages will have two 3.5 cm x 3.5 cm screened areas for ventilation, and to allow visual inspection of weevil larvae. Larvae will be inspected daily for survival and cocoon formation. Once a cocoon has formed, the weevil pupae will be removed, dried in a drying oven at 68C and weighed to 0.1 mg. We hypothesize that one or more of the transgenic varieties will show enhanced resistance in the form of reduced weevil survival, delayed development and small size.Note: Although initial screening studies will be carried out under greenhouse conditions, promising lines will be tested in field sites known to be affected by the weevil. However, the time-frame for such tests would depend on the initial identification of resistant lines and time taken to obtain USDA-APHIS permits for testing lines under field conditions. 3) Determining yield potential and forage quality of the screened transgenic alfalfa plants (Islam, Padlock Ranch)Promising lines identified in Objective 2 will be used for quality analyses studies. Plants will be harvested 3-4 times depending on their growth and dry matter yield will be determined by oven-drying samples at 60°C for at least 72 hours. Samples will then be ground to pass a 1 mm screen in a Wiley Mill and used for analysis of crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), and in-vitro dry matter digestibility (IVDMD) using both Near-Infrared Reflectance Spectroscopy and wet chemistry analysis to develop NIRS calibration equations for the measured forage quality parameters. Briefly, calibration samples for tissue nitrogen (N) concentration will be determined by dry combustion using a Leco C/N analyzer and CP will be calculated by multiplying the tissue N concentration by 6.25. The ADF and NDF calibration samples will be done using the ANKOM fiber analyzer. Similarly, calibration samples for IVDMD will be analyzed following a two-stage technique and calibration equations for each forage quality parameter will be developed using partial least squares.