1. NASC protocols GROWING ARABIDOPSIS FOR WHOLE PLANT RNA EXTRACTION AT 4 DEFINED GROWTH STAGES OF PLANT DEVELOPMENT Joan Jotham NASC Technician

2. NASC protocols Essential requirements for preparation of plant material for RNA extraction Standardisation of ecotypes chosen Standardisation of growth method Standardisation of stages of morphological development chosen for harvesting material and storage at -70 o C

3. NASC protocols Standard ecotypes / backgrounds N933 (Col-4) NW20 (Ler) N1601 (WS)

4. NASC protocols N933 -(Col 4)-Columbia line Columbia is the ecotype which has been sequenced in the public Arabidopsis Genome Initiative. Derived by Redei from the non-irradiated Laibach Landsberg population. Col-4 was used as one parent for the Lister & Dean recombinant inbred population.

5. NASC protocols NW20- (Ler-0)- Landsberg erecta ecotype Background line for a wide range of mutants (e.g those produced at Wageningen University by Feenstra,Van den Veen and Koorneef) Selected from Landsberg seed following X-ray mutagenesis experiments Ecotype chosen for the private sequencing project by Cereon Genomics, Used as the other parent for the Lister and Dean RI population.

6. NASC protocols N1601 -(Ws-2)-Wassilewskija N1601 (Wassilewskija) was used as the background for the original large T-DNA tagged line population from Ken Feldmann. Consequently, many mutants are available in this background.

7. NASC protocols GROWTH METHODS Growth of seedling material Growth of plants from seeds sown in compost

8. NASC protocols GROWTH OF SEEDLING MATERIAL Sowing Petri dishes -wetted filter paper-60 seeds each Stratification at 5 o C - 72 hours - dark For germination -Plates moved to growth room continuous light (2400 lux.) at 21.6 o C to 22.6 o C at 45%RH to 68%RH Monitoring - Light intensity, temperature, humidity were recorded twice daily. Watering - Filter paper was kept moist. Condensation was removed from lids Seedling growth - Observed and recorded twice daily

9. NASC protocols GROWTH OF PLANTS SOWN IN COMPOST Sowing 13-cm pots filled with Arthur Bowers Multipurpose Compost watered with 0.02% Intercept WG pots enclosed in plastic collars (flower sleeves from Zwapak). Stratification was at 5 o C - 72 hours - dark For germination - Plates moved to growth room under continuous light (2400 lux.) at 21.6 o C to 22.6 o C at 45%RH to 68%RH Collars folded half way down 7 days after sowing to reduce humidity, the compost kept well watered, observations recorded twice daily

10. NASC protocols HARVESTING FOR STORAGE AT - 70 o C Stages of morphological development chosen for harvest of plant material (1) Cotyledons fully opened (1. 0) (2) 4 pairs of rosette leaves formed (1. 08) (3) First flower buds visible (5.10) (4) First flower bud (unopened), petals showing (6. 00) (Reference for growth stages listed in brackets : Boyes et al. Growth Stage- Based Phenotypic Analysis Of Arabidopsis: A Model for High Throughput Functional Genomics in Plants (The Plant Cell. Vol. 13,1499-1510,July 2001)

11. NASC protocols Method used to ensure that harvest was at these precise stages and to reduce circadian influence. Twice daily monitoring of growth stage of plants and of growth conditions Plants were selected for harvest by absolute uniformity of required growth stage and not by chronological time elapsed since sowing A record was kept of time of harvest. It is known that circadian rhythms can control hundreds of genes in Arabidopsis Reference:Orchestrated Transcription of Key Pathways in Arabidopsis by the Circadian Clock: Harmer et al. Arabidopsis Research Articles SCIENCE VOL 290 (2000) and even under constant light conditions we are aware that circadian rhythms may entrain to rhythms of diurnal greenhouse light quality.

12. NASC protocols TIME (IN DAYS) TO PLANT GROWTH STAGES 1 to 4

13. NASC protocols PREPARATION OF PLANTS FOR STORAGE AT -70 o C Whole plants were harvested for each of the four defined growth stages of plants; for all three ecotypes. Plants were wrapped in foil and were then immediately plunged into a container of liquid nitrogen They were then transferred for storage to a freezer at -70 o C until extraction of RNA was performed.