NSF Project: Experimental

NSF Project: Experimental

Medicago truncatula Meta Data

Medicago truncatula Cell Cultures

• Callus culture was initiated from roots approximately 6 years ago on SH agar media (Schenk and Hildebrandt 1971).
   
• Root callus culture was maintained on SH agar media plates in the dark at 25C, subculturing approximately every 4 weeks.
   
• Liquid culture was initiated using 5.0g callus culture in 40ml SH media in a 125ml Ehrlenmeyer flask on 11-20-01.
   
• Liquid cultures were maintained in a controlled environment room with temperature maintained at 22C and shaking at ~130rpm in the dark.
   
• Initial cultures (2X40ml) were added to 160ml SH media in a 500ml Ehrlenmeyer flask on 12-07-01 to yield an approximate volume of 250ml culture; this is designated as 1P, or first passage.
   
• Subculturing occurred approximately every two weeks, subculturing one flask into ~3 new flasks using the "swirl and dump" technique such that the final culture volume was ~250ml.
   
• Sample cultures were harvested during 7P and 8P to establish a growth curve, harvesting and weighing tissue on Days 0, 4, 5, and 6. Gram fresh weights of the cultures were: Day 0 = 10-12gfwt; Day 4 = 18-20gfwt; Day 5 = 25-28gfwt; and Day 6 = 30-34gfwt.

General Time Course Technique/ Logistics**

• Cell suspension cultures were subcultured on a Friday.
   
• The time course was initiated on a Wednesday (5 days post-subculturing).
   
• The elicitor and placebo were added to the flasks in sterile tissue culture hoods with the exception of time zero which was added at the harvest location as to minimize the time between treatment and actual harvest.
   
• All treatments/placebos were done in triplicate for all time points.
   
• Two sterile tissue culture hoods were used; one for the treatment and one for the placebo/control.
   
• Two people were in each hood, one adding the treatment/placebo and the other adding the labels and replacing caps.
   
• Several people were "runners;" those who transported flasks from the culture room to the culture hood, and those who transported flasks from the culture hood to the harvesting area.
   
• Each harvester had their own station consisting of the filtration apparatus, spatulas, wash solutions, etc.
   
• Through the 6 hour time point, 6 people harvested flasks, one for each flask at each time point to minimize the harvest time difference between replicates. The later time points had a minimum of two and a maximum of 6 people harvesting.
   
• At least one person transported samples from the harvest area to the upright ?80Cs by transporting the frozen samples on dry ice.
   
• Media was harvested from the methyljasmonate and yeast elicitor time courses at 0, 6, 12, 24, and 48 hours. A portion of the media was placed in a ~50ml glass tube with the remainder placed in a Nalgene 250ml/8 oz. HDPE plastic bottle. The tubes and bottles were placed on dry ice and transported and stored in a ?80C upright freezer. This media harvest included the 50ml of 25% MS salt wash.

**The UV treated time course has several significant differences in it as compared to the other two treatments; these differences will be discussed in the UV time course section.

Sample Identification Code
M = methyljasmonate
Y = yeast elicitor
U = UV irradiation
C = control
E = induced
m = minute
H = hour
I, II, III = replicate one, two, or three
(M,Y, or U) = the internal control (ie, (M) in the yeast elicited time course would be a methyl jasmonate internal control elicited or induced).

Examples: Y3HEII = Yeast elicitor time course, 3 hour elicited, replicate two

Y3HCII = Yeast elicitor time course, 3 hour control, replicate two

Y(M)3HCIII = Yeast elicitor time course, 3 hour methyl jasmonate ethanol

control, replicate three

Y(M)3HEI = Yeast elicitor time course, 3 hour methyl jasmonate treatment, replicate one

Harvesting Technique

• Cultures were vacuum filtered through a 300u Nitex nylon membrane in a buchner funnel using an Ehrlenmeyer side-arm flask and house vacuum.
   
• The cells were rinsed in the funnel under vacuum with 50ml of 25% strength MS salts (GibcoBRL Murashige & Skoog salt mixture, catalog no. 11117-074) as per recipe on container.
   
• Culture cells were then distributed equally between four 50ml red cap Sarstedt tubes and frozen in liquid nitrogen.
   
• Samples were then stored at ?80C, with the 4 tubes being divided equally between two -80C upright freezers.

* IMPORTANT NOTE: One or two autoclave loads of media flasks (40 - 80 flasks) did not autoclave properly. Subsequently, the majority of these flasks had a bacterial contamination which was not caught until the day before the time course (Tuesday, May 21, 2002). Fortunately, not many of these flasks were used to subculture, and, therefore, we had enough flasks to complete the UV time course as originally planned. A couple of contaminated flasks were found at the beginning of the time course and are noted on the original control time sheet. The downside is that, since we did lose some flasks, we had to reduce our internal control treatments to two replicates instead of the three replicates we normally used in the previous two time courses; also, three of the 24 flasks used for the internal control treatments were questionable. These three samples are marked with a "W" on each of their tubes in case there are any problems relating to these samples. The samples are: 1) U(Y)24HCI; 2) U(Y)24HEI; and 3) U(M)48HCII.

References

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