Cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips

Mathew L1,2, McLachlan A1, Jibran R1, Burritt DJ2 and Pathirana R1

  1. The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Palmerston North 4442, New Zealand.
  2. 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.