In vitro genebank is an ex-situ method of conservation which is basically conservation of valuable genetic diversity of a particular crop outside their original or natural habitat (in situ conservation or field banks). In vitro conservation involves maintenance of explants in a sterile, pathogen-free environment and preferentially applied to clonal crop that produce recalcitrant seeds, or do not produce seeds. This modern technique has already been applied for multiplication, storage and collection of germplasm of more than 1000 species, in general more economical and less risky in a long-term perspective; as compared to field collections. The conservation is mainly done through Medium Term Storage (MTS), with particular culture conditions to slow down the plant growth (slow growth conditions): growth regulator retardation, minimal containment, temperature between 16 and 20 °C ±2, photoperiod of 12h light / 12h dark. In vitro conservation can be associated with Long Term Storage (LTS), or cryopreservation, at ultra-low temperature using cryogenic techniques (in liquid Nitrogen at -196oC) (Cryopreservation).
The IITA Genetic Resources Center (GRC) is based at IITA headquarter in Ibadan, Oyo state, Nigeria. IITA GRC consists of three sections. One of which is the in vitro genebank section (others are seed bank section and field bank section). At IITA, in vitro conservation is being used for vegetatively propagated crops such as cassava (Manihot exculenta, crantz), Yam (Dioscorea spp.) and Plantain / Banana (Musa spp.). The principle of in vitro genebank is to makes use of its advantages to safely duplicate the genetic diversities of these staple vegetative propagated crops already conserved on the field bank.
In vitro conservation bears many advantages for clonal crops maintenance. It firstly leads to the reduction of genetic loss risks (frequent in the field banks) and constitutes a viable alternative to complement and reduce the large size required for field banks. In vitro tissue culture is a complementary conservation system that gives the possibility to clean the germplasm from diseases and pest via meristem culture and/or thermo-treatment, in collaboration with IITA Germplasm Health Unit (GHU). The advantages of in vitro conservation over field conservation is also the security of the conserved germplasm, and is an easier system for germplasm exchange, in collaboration with IITA Germplasm Health Unit (GHU) and Nigerian Agricultural Quarantine Services (NAQS), to ascertain that the germplasm is transferred in line with standard phytosanitary regulations. Plant tissue culture is a powerful tool for safer and faster way to multiply large quantity of material for distribution, duplication in other genebanks and international exchange (easier plant material transport); and also for breeding purposes.
In the in vitro genebank of IITA GRC, both the improved lines and landraces of the above mentioned crops are conserved for breeding and research use. As at December 2013, in vitro genebank of IITA has more than 2000 accessions of cassava, as many yam accessions and more than 500 accessions of Plantain/banana. The germplasm conserved is available and almost the entire collections are free. Request can be made through the contact persons.
The facilities available in in-vitro GRC includes competent personnel who are well trained in plant tissue culture technique and standard necessary equipment for plant tissue culture propagation and conservation.
MEDIA PREPARATION FOR IN VITRO PLANT TISSUE CULTURE
In vitro culture implies maintaining plant tissues in artificial conditions for conservation or multiplication purposes. The success of in vitro culture depends on several parameters. Amongst them is the culture medium, i.e., a substrate from where the tissue will extract the critical elements for its growth, such as minerals, water, growth regulators, antioxidants, vitamins, and carbon source. The optimal composition and form of the medium depend on the crop, explant type, and the purpose of the culture (regeneration, proliferation, shoot initiation, rooting, callus production …). Extreme accuracy in the composition of the media is needed for successful in vitro culture.
MERISTEM EXCISION AND CULTURE FROM APICAL OR AXILLARY BUDS
Using sterile forceps, place a shoot on a sterile surface (paper towel or petri dish) under the light of a stereomicroscope (photo 20).Gently cut and remove the white leaf sheets, one by one from the outside without damaging the next internal one. To do this, use either a sterile scalpel (blade no. 11) or a needle (Fig21).When the meristematic dome becomes visible, covered by the 1 or 2 internal leaves primordial (photo 23AD), the excision is completed and the meristems can be cut at its base Meristems are then transferred to meristem regeneration culture medium (see culture medium preparation procedure) either in test tubes or petri dishes which are sealed with parafilm.
Each culture vessel is labelled with the accession number, date of introduction, and line number (number of cuttings from the accession from which the meristem was excised) using long-lasting marker.
GERMPLASM IN VITRO CONSERVATION (MEDIUM-TERM STORAGE
As described in the in vitro introduction procedure, IITA’s in vitro system generally starts with a meristem introduction step. Once in vitro, meristems are expected to evolve into fully developed plantlets. This generally requires several subcultures, i.e., the selection of the active part of the growing tissue and its transfer onto a fresh medium at a relatively high temperature (25oC). Once the plantlets are fully developed, i.e., show roots and stems with at least 2 nodes they are transferred to in vitro slow growth conditions (i.e., a relatively lower storage temperature at IITA: 18 ± 1oC for yam and banana/plantain, 19 ±1oC for cassava). Five plantlets of yam and 10 plantlets of banana/plantain are maintained in slow growth condition for GRC and breeding material (Fig27)
During storage, the in vitro plantlets progressively exhaust their culture medium and overgrow their test tubes, i.e., they reach the top of the cap. As a result, they start showing necrotic signs (drying medium, dry leaves, etc.…). When such observations are made it is time to subculture the plantlets. Depending on the crop, and maintenance conditions in IITA, subculture is needed every 6–18 months (cassava), 18– 30 months (yam) and 3–6 months (banana/plantain). While maintained in vitro, contaminated plantlets are occasionally observed. This can be due either to the presence of undetected endogenous bacteria in the meristem at the time of introduction or to poor laboratory practices (contamination with fungi, bacteria, mites at some stage of the in vitro process). Any contaminated plantlets need to be removed and destroyed as soon as they are observed to avoid possible contamination of the rest of the collection. (Fig28)
SORTING FOR SUBCULTURING
Whatever the accession, once all plantlets shows obvious sign of necrosis or number of seedlings maintained in the bank is lower than three, it is sent for subculture (renewal of the stock).
One or two tubes are taken for each accession (note that in the case of breeding material 1–8 tubes will be taken). Also note that, where possible, at least one plantlet of each accession should remain in the bank. The tubes are recorded as having been “sent for subculture” in the inventory system.
One barcode label of each accession selected for subculture is transferred to the first test tube (head tube) containing fresh medium and where the first new micro-cutting will be placed for that particular accession. All the test tubes behind the head tube will be used for the same accession.
- Pull out of the test tube the plantlet ready for subculture using long, sterile forceps and drop it on the sterile paper towel (photo 32).
- Holding the plantlet with forceps, cut off the roots, remove the older leaves (photo 33) and cut the stem into micro-cuttings using a scalpel (blade no. 10). Each micro-cutting must carry at least one bud. To avoid mis-orientation, the upper part of the stem should be shorter than the lower one, especially for cassava.
- Place the micro-cutting on a sterile paper towel set on sterile aluminium paper foil away from the rest of the plantlet to be discarded (photo 34).
- Open a new test tube containing fresh multiplication medium (where the old bar code was stuck if this is the first nodal cutting of the series), expose its mouth to the flame for 2–3 seconds and plant the lower part of the stem in it, using long forceps in a one-movement action. The operator must avoid as much as possible touching the inner parts of the tube.
- Before closing the tube, expose the tube’s mouth briefly to the flame, close the tube with a plastic cap, and seal the tube with parafilm.
- Important: Subculturing should be done quickly so as to avoid dehydration of the material during processing.
- Once all subculture operations for the selected accessions are completed by the operator, subculture information is recorded in the inventory system. For subculture operations, the date of subculture and number of micro-cuttings obtained are captured and recorded in the inventory system. (See inventory system procedure.)If the accession is transferred to the bank for the first time, the accession origin (meristem or nodal cutting), health status, line number, location in the store, and
- introduction date are also recorded. This latter information will follow the accession during its lifetime.
- Transfer the tubes to the growth chamber (temperature 25–27oC, photoperiod 12/12, 38 μmol.m-2.s-1) for growth and rooting
- One week after subculture, the tubes are screened for contamination and necrosis. Any in the two categories are discarded as described above and the inventory is updated.
NEW TRANSFER/REPLACEMENT OF OLD MATERIAL IN THE GENE BANK
Once micro-cuttings show a well-developed root system and stem (around 6 weeks after sub culturing in the conditions described above) they are transferred to the bank area (photo 27). For each accession, the old plantlets are replaced by the new one at their initial location with a maximum of 5 tubes per accession for gene bank material and 10 tubes per accession for breeders’ material. The newly transferred plantlets are recorded in the inventory system as “bank update.” The old or extra ones are recorded as extra and will be discarded. The storage conditions of the in-vitro gene bank are as follow: 12 h light per day, 43μmol.m-2.s-1, 18 ºC for yam and banana/plantain, 19–20 ºC for cassava.
IN VITRO INTRODUCTION FOR MEDIUM TERM STORAGE
The establishment of the plant material in the in vitro culture system is called in vitro culture. It involves two main steps: In vitro introduction (explants are transferred aseptically from field or screen house into a culture container) and multiplication (where an adequate environment is provided for the explants to evolve into small plantlets). One application of in vitro culture is in vitro conservation where tissues are maintained in slow growth conditions (medium-term storage). At IITA, three collections are maintained in vitro (yam, cassava, and banana/plantain). The initial explants used are preferably apical or axillary meristems, and meristem-derived plantlets are maintained in slow growth conditions. Occasionally, nodal cuttings are used as initial explants but they are not preferred as their level of endogenous pathogens is much higher than for meristems.
The transfer from controlled in vitro growth conditions to the natural environment (field or screen house) is traumatic for in vitro plantlets as they have to adapt to severe environmental changes (non-aseptic conditions, new temperature, light, and hygrometric conditions, no carbon supply, i.e., need to become photosynthetic, etc.). The progressive change of environment before the transfer to field conditions is called acclimatization or hardening.
IN VITRO GERMPLASM DISTRIBUTION/RECEPTION
In vitro collections of the clonally propagated crops conserved at IITA by the GRC are distributed worldwide under the Standard Material Transfer Agreement (SMTA) for research for food and agriculture.
Germplasm distribution is initiated by an official request, most of the time through e-mail containing information such as the requestor’s name, affiliation, the purpose of use, and a confirmed list of the material requested. Once a request is received, the secretary in the GRC sends a Standard Material Transfer Agreement (SMTA) and a standard letter (informing the requestor about the procedure), prior further processing. These data are centralized by the secretary and are uploaded to the on-line system. The request is forwarded to the proper GRC officer for processing by the GRC head
For international requests, the request is put on hold until the recipient has provided an import permit or confirmed that he will be able to provide one. Once the import permit has been received, a phytosanitary certificate request is sent to GHU. Both the import permit and phytosanitary certificate must accompany the germplasm during shipment.