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ARS Home » Pacific West Area » Corvallis, Oregon » National Clonal Germplasm Repository » Research » Publications at this Location » Publication #71829
Title: ABSCISIC ACID-RESPONSIVE PROTEIN, BOVINE SERUM ALBUMIN, AND PROLINE PRETREATMENTS IMPROVE RECOVERY OF IN VITRO CURRANT SHOOT-TIP MERISTEMS AND CALLUS CRYOPRESERVED BY VITRIFICATION

Author
item LUO, JIE - OREGON STATE UNIVERSITY
item Reed, Barbara

Submitted to: Journal of Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: The development & modification of cryopreservation protocols is important to enable successful preservation of a broader range of plant species & cultivars. Toxic effects of cryoprotectants [glycerol, dimethyl sulfoxide (Me2SO)] used in slow freezing protocols are minimized by pretreatment with sugars, sugar alcohols, and amino acids introduced in solid or liquid medium. Pretreatment reduces the cell size & the cytoplasm to vasuole ratio, enhances the ability of cells or tissues to take up cryoprotectants during prolonged exposure, or modifies cell walls & membranes to resist dehydration injury & deformation during freezing. Sucrose & proline stabilize membrane bilayers and enzymes during desiccation and freezing. They act in a colligative manner by preventing a toxic level of accumulation of compounds in membranes during dehydration & freezing. Vitrificaiton, a newly developed cryopreservation protocol which uses highly viscous solutions to form a glass at low temperature, is now used to preserve plant cells & tissues in liquid nitrogen. Present vitrification protocols use only limited pretreatment regimes, and modifications to improve regrowth are needed. Natural cryoprotective mechanisms are present in many seeds & plants. The ABA-responsive proteins may possess unique biochemical properties that could protect cells or tissues during environmental stress. The objective of this research was to investigate the effectiveness of an RABP extract from wheat seeds as a pretreatment agent to improve the recovery of cryopreserved meristems and calli. Also, compared the effectiveness of RABP with that of sucrose, proline & BSA.

Technical Abstract: Improved recovery of vitrified currant (Ribes aureum Pursh and R. ciliatum Humb.& Bonpl.) meristems and calli was obtained following two-hour pretreatment in sucrose, proline, abscisic acid-responsive proteins (RABP), orbovine serum albumin (BSA). Two-hour immersion in 0.4 M sucrose liquid NCGR-RIB medium prior to vitrification greatly improved the regrowth of meristems compared to 0,1,3, and 4 hr. immersion. Two-hr immersion of meristems in 5% and 10% proline dissolved in 0.4 M sucrose liquid medium significantly improved regrowth following vitrification. Initial tests with extracts of crude RABP from wheat seeds showed that regrowth of vitrified Ribes apical meristems improved after two-hr immersion pretreatment with the highest survival at 1%. RABP preparations containing equivalent proteins (1% crude or 0.2% dialyzed RABP) had equivalent effects on regrowth, indicating that the effect was from the proteins rather than sugars & other materials in the crude RABP extracts. Pretreatments of meristems and calli with 5% or 10% proline, 1% crude RABP, or 1% BSA in 0.4 M sucrose solutions produced similar results, and there were no significant differences among them. Meristems in pretreatment groups resumed growth 3 days after thawing, and reached the maximum regrowth at 1 week, compared to 2 weeks for non-pretreatment controls.