The IITA Genetic Resources Center (GRC) holds international collections of tropical clonal crops such as cassava (Manihot esculenta Crantz), yam (Dioscorea species), Banana and plantains (Musa species). These crops hardly produce seeds (very few or no seed production) which are recalcitrant and/or intermediate heterogeneous seed. That’s why they are propagated clonally, through stem cuttings, tubers and suckers respectively. They cannot be conserved as orthodox seeds; they require other methods of conservation. The conservation and maintenance of the clonally propagated crops is systematically done on the field conditions at IITA. The collections are then duplicated into in vitro slow growth medium-term conservation system to support safer conservation of the genetic diversity and sustainable utilization. Since 2017, cryopreservation conservation method for clonal crop has been started with the cryobanking of cassava germplasm, as supplementary conservation system for longer-term storage.
Cryopreservation is almost systematically associated with in vitro conservation. It’s about maintaining plant material at ultra-low temperature (in liquid Nitrogen at -196 °C) using cryogenic techniques, preserving the biologic structure and/or function of the living systems.
The collections are then duplicated into in vitro slow growth medium-term conservation system to support safer conservation of the genetic diversity and sustainable utilization.
Cryopreservation is an advance conservation method for clonal crop germplasm, which is almost systematically associated with in vitro conservation. It’s about maintaining plant material at ultra-low temperature (in liquid Nitrogen at -196 °C) using cryogenic techniques, preserving the biologic structure and/or function of the living systems by freezing. At such low temperature, plant cell biological activities and metabolism are stopped, eliminating the need to regularly rejuvenate or regenerate the plant. It is presently a supplementary tool to improve conservation of germplasm in a longer-term perspective. Cryopreservation is the most reliable technique for long-term storage of clonal plant genetic resources (Popov et al., 2005). It avois the disadvantages of irreversible loss of totipotent competencies caused by in vitro ageing process (Benson, 2008), and time + labour consuming. Many studies confirmed that it’s economically more competitive compared to other conservation system (Harvengt et al., 2004; Reed et al., 2004a; Keller et al., 2008). The costs of maintaining an accession in cryopreserved storage for the long-term (over 20 years) are considerably lower than those of maintenance in the field or in vitro, particularly when dealing with a large number of accessions (Keller et al. 2013). Cryopreservation helps to overcome many of in vitro maintenance disadvantages such as labour-intensive subculturing, potential elimination of fungal and bacterial contaminants and less somaclonal variation risk. It also ensures the safe long-term conservation of genetic resources. Thus, cryoconservation techniques have been increasingly used for the Long-Term Storage (LTS).
In the last 25 years, several cryogenic techniques have been developed, especially those based on vitrification method (the transition of water directly from the liquid phase into an amorphous or “glassy” phase, whilst avoiding the formation of crystalline ice) such as encapsulation-dehydration, preculture-dehydration, and encapsulation/vitrification. Therefore, the main requirement for using cryopreservation method is that it should be simple, economical, reproductible and should allow relatively high regrowth rate, as advised by Leunufna and Keller (2003). Recently, the latest method called V-cryo plate (VCP) developed by the National Institute of Agrobiological Sciences (NIAS) in Japan was introduced at IITA (Yamamoto et al. 2011). Adaptation and validation of the VCP method was carried out on cassava and yam.
To ensure long-term conservation for IITA international clonal crop collections of around 11,000 accessions, cryopreservation was emphasized on. All the prerequisites preparedness for cryopreservation, which entails setting a suitable conservation strategy, well-trained staff, quality/risk management system and most importantly facilities, consumables and equipment; were taken care.
Concerning the cryobanking procedure itself, any cassava or yam accession to be cryopreserved will first be tested clean (from virus and endophytes), amenable to cryo method and most importantly confirmed unique and true-to-type. This latter validation is done using SNP markers, generated from DArT sequencing, for fingerprinting.
On the technical side, a continuous liquid nitrogen (LN) supply is vital to keep frozen samples (meristems) at -196°C. Any raise up of the temperature beyond the critical limit would surely remove any chance to get recover plant from the conserved meristems. In the Nigerian (African) context where getting LN is costly, discontinued and not secured, the ideal situation is to have an in-house LN generator. Therefore, a 60L/day LN plant (NOBLEGEN: LN60 LIQUID NITROGEN SYSTEM, including a closed-loop water chiller safety kit, oxygen alarm and air vent, a five-year spares kit and a one week hand on training on the maintenance of the LN plant) was acquired by IITA.
The cryopreservation procedures set up at IITA includes Deware flasks: cryopreservation tanks.
- Two ARPEGE 170 Liquid CRYOMEMO of 6k samples each, for safety duplication and temporary conservation and viability validation of any accessions cryopreserved
- A CHART LN tank (MVE 1879P-190 of 80k samples capacity) for the long-term conservation
The LN storage tanks are associated with a XiltriX monitoring system to control the LN, temperature and incidence reporting futures.
More so, cryopreservation implementation includes other equipment and consumables such as:
- Cryo-ready PPE: Glows, Ephron, and cryo goggle protection
- Laminar flow hood [AireGard ES (Energy Saver) NU-201-630E]
- Stereo Microscopes for meristem excision.
- Big fridge for culture medium storage – Esprit 3750 (770) D.
- pH meter (Bench-top conductivity meter, pH meter, pH tester, ELE-522
With the cited equipment and consumables, the implementation of IITA cassava cryobanking has been started in August 2017. For yam germplasm, research is ongoing to set an optimal protocol for cryobanking.
With all this preparation, the IITA cryobank is aiming to have 1000 cassava + 500 yam accessions in 2022.