Ryegrass Fluorescence Testing Why differentiate? Annual ryegrass, - PowerPoint PPT Presentation

Traditional and Genetic Methods Ryegrass Fluorescence Testing

Why differentiate? Annual ryegrass, Lolium multiflorum Perennial ryegrass, Lolium perenne • Forage crop • Rapid growing ability. • Preferred for permanent lawns, • Flowering independent of • Over-winters -does not require photoperiod and vernalization. seeding each year. • About twice the value of annual ryegrass Different Uses and Values

Traditional Method Seedling Root Fluorescence  Distinguish Lolium multiflorum and Lolium perenne  Idea: Annual ryegrass roots fluoresce under UV light. (1930’s)  Needs=filter paper+UV light  Seedlings with fluorescent roots recorded and removed at first count (7d) and final count (14d)

How to:

Tilt boxes in chamber –orientation of roots

Dark box with UV light

Fluorescence of roots under UV light-in dark box

Fluorescence of roots under UV light.

Evalution

Data Sheet Example: Warm Customer ID: 5/28/10 Planted: Final Office: Initial Read: 6/9/10 6/16/10 Read : Date Received: Analyst: Analyst: Sample Lot Seed Componen Rep 3 Rep 4 Total (for Variety Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 No. Size t Purity) HALO Normal fl 1 2 0 1 1 3 4 0 12 Comments Normal : non-fl 86 87 85 89 Abnormal 2 3 5 6 Dead 10 5 6 4 Firm ungerm 0 0 0 0 Rep total - all Normals 88 92 89 90 359 Ave. Normal 89.75 (Test fluorescence) TFL=3.34% total normal fl/total of all normals *100 (Varietal AOSCA value fluorescence) VFL=2.87%

Key:  Report % PRG and % ARG on Purity Report  Pure seed and crop percentages may adjust based on the germination fluorescence test.  ARG = Annual ryegrass ( Lolium multiflorum )  PRG = Perennial ryegrass ( Lolium perenne )  TFl = Test fluorescence (lab determined )  VFl = Variety fluorescence (AOSCA value)  VFlA = Variety fluorescence for annual ryegrass  VFlP = Variety fluorescence for perennial ryegrass  If you are not given the variety name, the variety is not listed by AOSCA, or you are not testing that species, then:  Assume VFlA to be 100%  Assume VFlP to be 0%  SRF =Seedling Root fluorescence, the method

The Equation %VFL (annual) - % TFL % Perennial Ryegrass= %VFL (annual) - % VFL (perennial) x % pure ryegrass (From purity) let’s say it’s So, In our example: 99.5% with 0.50% inert %PRG= 100-3.34 x 99.5 matter 100-2.87 Report of Analysis Pure Seed Complete the equation : Lolium perenne 99.02% Inert matter 0.50% % PRG= 96.66 x 99.5 = 99.02% Other crop 97.13 Lolium multiflorum 0.48% Weed Seed 0.00%

What this means:  If No VFL has been described and accepted by AOSCA, all fluorescent normals are considered annual contamination and go against pure perennial ryegrass %.  If TFL is less than VFL, report no annual in a perennial lot.  If over 5% annual ryegrass, in AOSA=Mixture  L. perenne and L. multiflorum are both reported as pure seed kinds

Seedling Root Fluorescence Test Perennial Ryegrass Referee 2009

Motivation  Determine the uniformity of test results from lab to lab  Review method as described in Cultivar Purity Handbook  Goal:  To help clarify the method and foster uniformity ▪ Lifting vs non-lifting of roots ▪ Intensity of Fluorescence

Referee Setup  Capture Environmental Differences  Production Environment  Lab Environment-Variation of up to 6% in TFL over a period of less than one year ( Sharon Davidson )  Referee Study:  Seven samples-varying in annuality and production area.  Prechill vs. No Prechill  Completed within one month  Cultivar Purity Handbook (version 2008)

Current provisions-Cultivar Purity Handbook (Version 2008)  All fluorescent root traces should be counted regardless of the intensity of fluorescence.  Non-fluorescent seedlings should not be lifted to observe fluorescence.  Fluorescence for abnormal seedlings should not be recorded.

Survey Results  80% of the participants=experienced/very experienced.  42% test 1,000 samples/year or more  58% using most current version 2008-09 of CPH; 42% had older versions

Survey Results Continued  PreChill ▪ 80% labs do prechill (majority being when it’s fresh) ▪ 77% use 10C and 23% use 5C  Media ▪ 47% use filter paper ▪ 29% use blotters ▪ 24%=combination ▪ 80% tilt boxes ▪ 71% use KNO3; 27% water; 2% distilled H2O  Light ▪ 80% = 8 hours light ▪ 13% = 16 hours light ▪ 6% = 12 hours light

Survey Results Continued  Light intensity  67% use 700-1250 lux.  others = 30-40watts  Lux not measured  Length of Test  73% do 1st read at 7 days: 20% at 10 days; 7% don’t do first count  100% do final read at 14 days  Fluorescence  31% remove all seedlings at final count;69% do not  40% look underneath root for path of fluorescence*  94% do not discriminate based on intensity

Results: Samples 1 and 2 Growout from DNA on 3,000 Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC SRF (No PC) seeds 1 8 82.25 1.82 86.5 0.29 1 10 92.75 0.81 89.5 0.28 1 6 81.5 2.15 88.5 1.41 1 9 90.5 0.27 1 2 96 1.56 89.75 0 1 5 92.75 1.08 91.25 1.37 1 12 92.75 0.27 93.75 1.33 2 perennial 1 3 87 0.57 94.5 0.53 Estimated ARG = 1 4 84.5 0.89 88.75 2.25 0 annual 0.95% Growout from DNA on 3,000 Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC SRF (No PC) seeds 2 8 94 74.47 96 82.55 2 10 96.75 97.41 98.75 81.01 2 6 94.75 59.1 94.5 56.88 2 9 94.75 59.1 2 2 93.75 38.4 95.25 35.43 2 5 91.25 60 89.5 61.73 2 12 96.25 84.42 95.75 66.58 210 perennial 2 3 90 56.94 95 60 Estimated 2 4 94.5 60.05 94.75 59.1 16 annual ARG=19.41%

Results: Samples 3 and 4 Germ (no Growout from Sample Lab Germ (PC) TFL-PC PC) TFL - No PC SRF (No PC) DNA on 3,000 seeds 3 8 97 98.71 97.5 96.15 3 10 97 99.22 98.25 100 3 6 96 99.74 98 98.47 3 9 98.5 100 3 2 98.25 96.69 94.25 92.57 (Growout of 3 5 98 96.92 97 97.43 NFL) 3 12 97.25 99.23 98.75 99.24 3 perennial 3 3 96 99.74 95.5 98.95 1 Estimated 3 4 98.25 100 97.75 99.49 annual ARG=61.68% Germ (no Growout from Sample Lab Germ (PC) TFL-PC PC) TFL - No PC SRF (No PC) DNA on 3,000 seeds 4 8 84.75 0.59 85.75 0 4 10 87.5 0.29 93.5 0.27 4 6 88 0.57 90.5 0.55 4 9 85.5 0 4 2 96 0.26 93.75 0.27 4 5 91 0.27 87.5 0.29 4 12 93.75 0.8 91.25 0.27 4 3 91.5 0 93 0.54 2 perennial 4 4 89.5 0.56 88.75 0.85 0 annual Estimated ARG=0.14%

Growout Germ Germ (no TFL - No from SRF DNA on 3,000 Sample Lab (PC) TFL-PC PC) PC (No PC) seeds 5 8 94 2.93 96.75 3.1 Results: 5 10 95.75 4.18 96 2.08 5 6 94.75 2.9 93.5 5.08 5 9 94 4.78 5 2 96.75 1.55 96 2.34 Samples 5, 6 5 5 93 4.03 94.25 2.65 13 5 12 96.5 3.89 94.25 2.39 perennial 5 3 94 1.86 95.25 3.41 0 Estimated and 7 5 4 94.75 2.9 96.5 4.15 annual ARG=1.78% Growout Germ Germ (no TFL - No from SRF DNA on 3,000 Sample Lab (PC) TFL-PC PC) PC (No PC) seeds 6 8 90.5 0.28 90 0.56 6 10 91.75 0.27 94.75 0 6 6 92.25 0.54 89.75 0.56 6 9 90.25 0.27 6 2 93.5 0.53 91.25 0 6 5 93 0.27 93 0.27 6 12 94.5 0.26 91.5 0.55 2 perennial 6 3 88.5 0 90.75 0.55 Estimated 6 4 92.75 0.81 91.75 0.27 0 annual ARG=0.18% Growout Germ Germ (no TFL - No from SRF DNA on 3,000 Sample Lab (PC) TFL-PC PC) PC (No PC) seeds 7 8 94.25 0.53 90 0.28 7 10 92.75 0 97 0 7 6 90.25 0 91.5 0.27 7 9 91 0.27 7 2 93 0 91.75 0.27 7 5 89.25 0 92.25 0.81 7 12 93.25 0 90.75 0.28 7 3 89.25 0 89.75 0 No Estimated 7 4 90.25 0.28 92.75 0 growout ARG=0.13%

TFL: Prechill vs No Prechill TFL-PC Perennials Annuals TFL-PC TFL - No PC TFL - No PC 120 6 100 5 80 4 TFL % 60 TFL % 3 40 2 20 1 0 0 Sample Sample Sample Sample Sample Sample Sample

TFL vs VFL (for samples with VFL) 6 TFL-PC TFL-No 5 PC VFL 4 Fluorescence % 3 2 1.72% 1 0.04% 0 Variety=Silver Dollar Variety=Prelude

Germination Across Laboratories No Prechill Prechill 100 100 98 95 96 94 90 92 90 85 88 86 80 84 82 75 0 10 20 30 40 50 60 80 0 10 20 30 40 50 60 70 Sample Sample

Germination Results Between Laboratories- How do we compare? Germination Results-Ryegrass Referee 2009 No Prechill Prechill Sample Average Range Tolerance Within? Average Range Tolerance Within? 1 88.75 14.5 6 No 91 8.5 5 No 2 93.5 7 4 No 94.25 9.5 4 No 3 97.5 3 3 Yes 96.5 4.5 3 No 4 90.5 11 5 No 90 8 6 No 5 95 4 4 Yes 95 4 4 Yes 6 91.5 6 5 No 92.25 5.5 5 No 7 91.75 5.5 5 No 93.25 7.5 5 No

Referee Conclusions  All treatments and interactions among them affected the results of both germination and fluorescence (except prechill vs no prechill)  Goal was to bring about uniformity in the existing test, but  1. Still room for improvement ▪ Education? ▪ Inherent variability each time you test a lot  2. Move on-DNA? One year post institution of SRF  learned that fluorescence not tightly linked to annuality.

Genetic Testing Methods Ryegrass Testing-