Cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips
- The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Palmerston North 4442, New Zealand.
- Department of Botany, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
Cryopreservation is a reliable and cost-effective method for the long-term preservation of clonally propagated plants. The number of clonally propagated species conserved by cryopreservation is increasing as vitrification-based methods are developed; droplet vitrification is becoming the preferred method for many species, as it ensures fast freezing and thawing rates. We investigated whether cold, antioxidant and osmotic pre-treatments could maintain the structural integrity of cells, thus aiding in developing a droplet vitrification protocol for kiwifruit, using Actinidia chinensis var. chinensis ’Hort16A’ as a model. Cold acclimation of donor plantlets at 4°C for 2 weeks followed by sucrose pre-culture of shoot tips and supplementing all media used throughout the procedure with ascorbic acid (0.4 mM) resulted in 40% regeneration after cryopreservation. Transmission electron microscopy was used to examine meristematic cell structure at every critical step of droplet vitrification. After treatment in vitrification solution, meristematic cells from cold-acclimated plantlets pre-treated with sucrose and ascorbic acid exhibited severe plasmolysis and some disruption of membrane and vacuoles. In contrast, cells without pre-treatments exhibited minimal changes even after exposure to vitrification solution. However, after cryopreservation and recovery, all shoot-tip cells not pre-treated showed rupturing of the plasma membrane, loss of cytoplasmic contents and organelle distortion. By comparison, most pre-treated shoot-tip cells from cold-acclimated plantlets retained their structural integrity after cryopreservation, suggesting that only dehydrated and plasmolysed cells can withstand cryopreservation by vitrification.