Critical Point Evaluation of Cryopreservation Protocols for Plant Germplasm Conservation
Scientific Cooperation Research Program work
plan:
Critical Point Evaluation of Cryopreservation Protocols for Plant
Germplasm Conservation
Dr. Barbara M. Reed
USDA-ARS
National Clonal Germplasm Repository
33447 Peoria Rd
Corvallis, OR 97333-2521
Telephone 1-541-750-8712 ext. 111
Fax 1-541-750-8717
reedbm@bcc.orst.edu
Materials and Methods
Micropropagated shoots were multiplied and meristems recovered on NCGR-Ribes medium (RIB), which contains the mineral salts and vitamins of Murashige and Skoog (1962) but with only 30% of the normal ammonium and potassium nitrate concentrations, and per liter: 50 mg ascorbic acid, 20 g glucose, 0.1 mg N6-benzyladenine, 0.2 mg gibberellic acid (GA3), 6 g agar (Sigma, Poole, Dorset, UK or Bitek, Difco, Detroit MI, USA), at pH 5.7. Shoots were grown in both locations at 25oC with a 16-h light (25 µmol· m-2· s-1)/ 8-h dark photoperiod. Cold acclimation was 8-h light at 22oC and 16-h dark at -1oC. All cultures were cold acclimated for 1 wk (Reed 1990). After acclimation 0.8 mm apical meristems were excised for cryopreservation.
Vitrification: A technique modified for Ribes was used (Reed and Yu 1995). Meristems from cold-acclimated shoots were pretreated for 2 days under the cold-acclimating conditions described above on RIB medium containing 5% DMSO (v/v). PVS2 cryoprotectant (Yamada et al. 1991) [(v/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in liquid RIB medium with 0.4 M sucrose (RIB medium also contains glucose), at pH 5.7] was dispensed into cryotubes on ice and meristems added and stirred. After 20 min the vials were immersed in LN. Samples were rewarmed for one min in a 45oC water bath and then transferred to a 22 oC water bath for 2 min. The meristems were immediately rinsed in liquid RIB medium with 1.2 M sucrose two times, and transferred to RIB medium for recovery.
Encapsulation-dehydration: A method developed for pear (Dereuddre et al. 1990) was modified for Ribes. Meristems were dissected, transferred to agar plates, encased in alginate beads [3% (w/v) low viscosity alginic acid (Sigma, Poole, UK or St. Louis, USA) with 0.75 M sucrose in liquid RIB medium without calcium, pH 5.7], polymerized 20 min in calcium carbonate solution, and pretreated for 18 h in liquid RIB medium with 0.75 M sucrose. Following pretreatment, the beads were separated on sterile Petri dishes, air dried in the laminar flow hood for 4 h (approx. 20% moisture content), placed in cryotubes, and plunged into LN. Vials were rewarmed at room temperature for 15 min, rehydrated in liquid medium for 5 min, and encapsulated meristems were then plated on RIB recovery medium.
Experimental design and data analysis: Each cryopreservation experiment included 30 meristems distributed into 3 separate cryovials (where n = 30 meristems for each treatment); an additional 5-15 control (unfrozen, cryoprotected) meristems were used for each protocol. Each cryopreservation experiment was repeated three times (n = 90 meristems for each genotype). Assessments of the recovery of the meristems were made weekly for 6 weeks and the phenological stage reached in each case was recorded. Greening, leaf expansion, and shoot production were all required for a meristem to be considered fully recovered from the cryopreservation treatment. Data were analyzed by ANOVA and least squares means.
Drs. Reed and Benson will design critical point evaluations prior to the training sessions. All participants will receive an intensive 2-week training in the techniques to be used. They will implement the techniques in their laboratories in the first set of experiments. Drs. Reed and Benson will visit the participating laboratories to evaluate the transfer of techniques using the critical point evaluations. They will also assist the participants in overcoming any problems they may initially encounter. A mid-program meeting of Drs. Reed and Benson will allow for additional evaluation of the techniques and evaluation of data from the first set of experiments. In the second year each laboratory will perform a second set of experiments using revised protocols. At the end the data will be discussed and evaluated as a group and preliminary manuscripts formulated. At the end of the project Drs. Reed and Benson will perform a final critical point evaluation.
An initial meeting between Dr. Reed and Dr. Benson (March 2001) will be used to establish the first two objectives. Collaborators will train in techniques in May-June 2001. In Spring 2002 Drs. Reed and Benson will visit the cooperating laboratories to evaluate the critical points and assist with any problems. From June 2001 to June 2002 all laboratories will perform initial cryopreservation experiments. Drs. Reed and Benson will meet in June 2002 to discuss data and problems encountered in the first set. From June 2002 to June 2003 a second set of experiments will be run in all laboratories. All participants will meet in June 2003 for final discussions and analysis of data. In September 2003 Drs. Reed and Benson will again visit the cooperating laboratories to evaluate the critical points. A final manuscript will be prepared for publication by February 2004.
Dr. Stan Pluta
Research Institute of Pomology and Floriculture
Skiernievwice, Poland
spluta@insad.isk.skierniewice.pl
Dr. Irina Kovalchuk
Kazakh Research Institute of Horticulture and Viniculture
238-a Gagarin Avenue, Almaty, 480060
Republic of Kazakhstan
karycheva@ratel.kz
Dr. Andreas Meier-Dinkel
Lower Saxony Forest Research Institute
Department of Forest Gene Conservation
Forstamtstrasse 6
D-34355 Staufenberg-Escherode
Germany
nfv-abtc@t-online.de
Dr. Erica Benson
Molecular and Life Sciences
University of Abertay-Dundee
Kydd Building, Bell Street
Dundee DD1 1HG, Scotland, UK
e.e.benson@tay.ac.uk