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.
At such ultra-low temperature, plant cell biological activities and metabolism are held on, eliminating the need to regularly rejuvenate or regenerate the plant material. Cryopreservation is considered as the most reliable technique for long-term storage (LTS) of clonal plant genetic resources (Popov et al., 2005). It avoids the disadvantages of continuous plant tissue ageing (plasticity loss), time and labour consuming of the in vitro conservation system (Benson, 2008). 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 potentially helps in the elimination of pathogens (e.g. virus contaminants).
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 ice crystals) such as droplet-vitrification, encapsulation-dehydration, preculture-dehydration, and encapsulation-vitrification. Therefore, cryopreservation method should be simple, economical, reproducible 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.
IITA cryobanking strategy and procedure
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-methods and most importantly confirmed unique and true-to-type. This latter validation is done using SNP markers, generated from DArT sequencing, for fingerprinting (see DNA fingerprinting).
Once an accession is “qualified” to be cryobanked, meeting all the 4 criteria, it will be cryobanked through 240 meristems. One hundred and twenty (120) meristems are conserved in the LTS tank (cryobank), 84 stored as safety duplication in another tank and 36 are culture in vitro (rewarmed for regrowth) to estimate the viability of the conserved material. The accession is considered cryobanked only if its viability is validated by a regrowth rate superior or equal of 11/36.
Genebanks are important to preserve the genetic diversity available among old landraces and wild progenitors of modern crops and also a key source of important biotic/abiotic stresses genes that were lost during domestication and in modern breeding. The knowledge of genetic diversity is a key factor to address conservation program of crop resources and to guide the use of germplasm in breeding. The number of deposited accessions in genebanks, however, are continuously growing. Currently, genebanks are facing various resource constraints, in which, slow germplasm characterization has been pointed out as a major cause because of its highly expansive, time consuming and labor intensive. Many genebanks have deficit in phenotypic characterization and evaluation data of all genebank accessions especially for complex traits like of biotic, abiotic and nutritional traits which required multi-environment and multi-year data. This lack of information drastically limits to possibility of the effective use of crop genetic resources stored in genebanks by researchers, breeders and farmers also. Molecular marker technology can be useful to speed up the characterization, evaluation and utilization of genetic resources, because molecular markers are also considered as descriptors that offer reproducible complementary information to the classical morphological descriptors and phenotypic data used in characterization and agronomic evaluation of genebank accessions (de-Vicente et al., 2004).
Molecular markers reflect the actual level of genetic variation existing among genotypes at the DNA level; hence, they provide a more accurate estimate of such variation than do either phenotypic or pedigree information. Currently, molecular research is a routine practice with DNA manipulation including non-model crops and has made huge impact in crop improvement, biotechnology and diversity evaluation. There is still huge potential with gene bank accessions which genetic value is unknown. This value is possible to be explore with the available advance next generation sequencing technology for high throughput analysis of plant genomes with potential for use in genetic diversity, gene discovery, genetic fidelity confirmation (between field and in-vitro collection) and duplicate identification in stored genetic resource. Because of development in high-throughput next-generation sequencing, single nucleotide polymorphism (SNP) markers are commonly used to describe genetic diversity, as they are present in a large number and merge excellent attributes such as wide genomic distribution, high re- producibility and co-dominant inheritance. To explore detailed molecular information of the IITA genebank accessions, we have been initiated SNP genotypic characterization using DArTseq-GBS (Diversity array technology genotype-by-sequencing) for clonal crop of cassava, yam with its core-collection and also other orphan legumes grain crops Africa yam bean and Bambara groundnut. The generated molecular information will use with current genomic and analytic technology to mine natural genepool heritage and also to confirm genetic fidelity (between field and in-vitro collection) and remove duplicates accessions from genebank collection for effective use of crop genetic resources stored in gene banks by breeding programs.
Facilities and equipment
Practically, 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 (-130 to -150°C) 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 (Picture).
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 (Temporary storage tank)
- A CHART LN tank (MVE 1879P-190 of 80k samples capacity) for the long-term conservation (LTS tank) (Picture)
The LN storage tanks are associated with a XiltriX® (IKS International B.V.) monitoring system to control the LN, temperature and incidence reporting futures (Picture).
More so, cryopreservation implementation includes other equipment and consumables such as:
- Cryo-ready PPE: Glows, Ephron, and cryo goggle protection (Picture)
- 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 2017. For yam germplasm, research is ongoing to set an optimal protocol for cryobanking.
IITA cryobank is aiming to store 1000 cassava + 500 yam accessions in 2022.