| Project Title: | Selection for Charcoal Rot
Resistance in Soybean |
| Project Leader: | Nancy A.
Reichert, MAFES Mississippi State University Mississippi State, MS |
| Other Participants: | N/A |
No funds were requested in 1998 to conduct this research, since I had MSPB funds from another project which paid for a Research Assistant to refine our genetic transformation protocols. (Unfortunately, she took another position in May, 1998.) The research proposed above should be considered a new project, since Dr. Sciumbato will also play an integral role in it. However, we will build on the research results obtained the last two years on this project (reported below).
1. Determined that cell-free fungal culture filtrates contained elicitors of infection.Both autoclaved (CF-A) and filter-sterilized (CF-F) severely inhibited seedling germination, even at a concentration of 10% (v/v). Because of this, fungal culture filtrates can be utilized in screenings to select soybean seedlings/tissues that may contain varying degrees of tolerance or resistance to charcoal rot.
M. phaseolina cultures were obtained from Dr. Sciumbato and purified. Fungal cultures were grown in liquid PDA media for up to 14 days then harvested. Liquid filtrates were filtered through eight layers of cheesecloth and Whatman #1 paper 4-5 times, then filter-sterilized (CF-F; 0.2 µm filter) or autoclaved (CF-A) to obtain cell-free fungal culture filtrates.
Crawford soybean seeds were surfaced disinfested with a mild bleach solution then rinsed with sterile distilled water (sddw). Seeds were placed in test tubes containing different percentage (0% - 100%; v/v) of fungal CF sterilized by the two methods mentioned above (sddw made up the remainder). Nine days post-inbibition/germination, it was noted that seed germination and hypocotyl elongation were affected by the presence of fungal filtrate, regardless of the concentration used. CF-treated seedlings displayed stunted roots and shorter hypocotyls. This strongly implied the presence of a disease elicitor (most likely phaseolinone) in both autoclaved and filter-sterilized fungal culture filtrates. Therefore, both CF types could be used to conduct massive seedling screenings (in vivo) to determine if any individuals contained inherent tolerance/resistance to charcoal rot. Also, both types of CF should also be able to be used in vitro for the selective regeneration of soybeans displaying tolerance/resistance to charcoal rot.
2. Determined that CF could be used to select soybean regenerants when incorporated into shoot induction media.
Crawford hypocotyl section explants were placed on shoot initiation media containing 25 -75 % (v/v) CF-A or CF-F. Even at 25 %, the toxin(s) severely decreased the shooting responses. This was expected and indicated the selection system should work. Nearly 100 soybean hypocotyl explants were placed on media containing CF. Less than 50% responded by producing shoots, but the response was delayed. (Normal shooting response is up to 100% of the explants within 8 weeks.) However, many developing shoots died. Unfortunately, no shoots were maintained for rooting or further studies. However, we believe that incorporation of CF into regeneration media should enable the selection of mutants that may have tolerance/resistance to charcoal rot.
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