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Cryopreservation of banana apical meristems

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Contributors to this page: Bioversity International, Belgium (Bart Panis).

The content of this page was extracted from Panis B. 2009. Cryopreservation of Musa germplasm: 2nd edition. Technical guidelines No. 9 (F. Engelmann and E. Benson, eds). Bioversity International, Montpellier, France.

Cryopreservation of in vitro grown shoot tips of banana through vitrification was originally reported by Thinh and co-workers (Thinh et al. 1999). Using this method, regeneration percentages were often low and unpredictable. Therefore, the technique was further improved and adapted at KULeuven to make it applicable to a wide variety of cultivars (Panis et al. 2005).

This method is illustrated in the figure below.

Cryopreservation of individual meristems (photo: KULeuven/Bioversity)

Preparation of plant materials

Production of robust, rooted in vitro plants of banana cv. ‘Williams’ (photo: KULeuven/Bioversity)

Preparation of in vitro plantlets

All accessions were obtained from the Bioversity Musa germplasm in vitro collection (KULeuven, Belgium). This collection contains edible banana cultivars, as well as wild relatives.

Dissection and selection of apical meristems

Like many other monocots, banana apical meristems are tightly covered with several layers of whitish, tubular, immature leaves.

A skillful and trained technician can isolate a maximum of ten meristems an hour (roughly, six minutes/isolated meristem).

Illustration of meristem isolation. Leaves are removed one by one until the apical dome is visible but still partially covered by 1-2 young leaf primordia
(photo: KULeuven/Bioversity)

Partly-covered apical meristems of banana cv. ‘Williams’ (photo: KULeuven/Bioversity)

Cryopreservation through droplet vitrification

Loading, dehydration and rapid freezing

Transfer of meristems to a droplet of PVS2 solution (of about 15 μl) on a strip of aluminium foil (5×20 mm) with a 2 ml plastic Pasteur pipette (photo: KULeuven/Bioversity)

Storage, re-warming and unloading

Recovery

Four to six weeks following cryopreservation, four types of reactions can be distinguished:
(i) white shoot tips resulting from an immediate death of the tissue without blackening.
(ii) completely or partially black shoot tips, indicating that there was enzymatic reaction following cryopreservation (production and oxidation of polyphenols).
(iii) unorganized callus growth, representing the outgrowth of small isolated areas of the apical dome and/or primordial tissues.
(iv) shoot tip regeneration resulting from the survival of a substantial part of the apical dome (see figures a-d).

Reaction of apical meristems towards cryopreservation 30 days after re-warming (a) No growth; the meristem remains white; (b) Blackening without further growth; apical dome reacts by formation of polyphenolic compounds that oxidize; (c) Callus formation; watery, non-morphogenic callus; and (d) Shoot regeneration (bar = 600 μm)(photo: KULeuven/Bioversity)

One month after re-warming, a 0.5 cm long shoot can be observed (see photo). Calluses never produced shoots.

Recovered shoots from cryopreserved apical meristems of banana cv. ’Williams’ 1 month after re-warming
(photo: KULeuven/Bioversity)

Discussion and perspectives

Different Musa genotypes have been cryopreserved using this protocol (Thinh et al. 1999, Panis et al. 2005). Post re-warming regeneration percentages range between 20% and 85% (with an average of 53%). These recovery percentages are relatively genotype independent. It was, however, observed that genotypes with more B-genome survive significantly better than those with solely the A-genome (Panis et al. 2005). Microscopic observation of the recovery of cryopreserved meristems (Helliot et al. 2003) has revealed that:

(i) the whole dome of the isolated meristem withstands exposure to liquid nitrogen.

(ii) no regenerable post cryopreservation callus is formed. Therefore, it would appear that somaclonal variation is unlikely to occur.

The main limitations to this procedure are as follows:

This droplet vitrification protocol was recently successfully applied to a wide range of plant species such as potato, ulluco, sweet potato, chicory, strawberry, taro, Pelargonium L., date palm, thyme, olive, Byrsonima, snowdrop, apple and hop and might thus be considered as the first generally applicable protocol (Panta et al. 2006, Gallard et al. 2008, Sant et al. 2008, Feki et al. 2011, Sánchez-Romero and Panis 2009, Marco-Medina et al. 2010, Condello et al. 2011, Maslanka et al. 2013, Coutinho Silva et al. 2013).

References and further reading

Banerjee N, De Langhe E. 1985. A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (banana and plantain). Plant Cell Reports 4:351-154.

Condello E, Caboni E, André E, Piette B, Druart P, Swennen R, Panis B. 2011. Cryopreservation of apple in vitro axillary buds using droplet-vitrification. CryoLetters 32:175-185.

Coutinho Silva L, Paiva R, Swennen R, André E, Panis B. 2013. Shoot-tips cryopreservation by droplet vitrification of Byrsonima intermedia A. juss.: a wooden tropical and medicinal plant species from Brazilian Cerrado. CryoLetters 34: 338-348.

Feki L, Bouaziz N, Sahnoun N, Swennen R, Drira N, Panis B. 2011. Palm cryobanking. CryoLetters 32:451-462.

Gallard A, Panis B, Dorion N, Swennen R, Grapin A. 2008. Cryopreservation of Pelargnonium apices by droplet-vitrifications. CryoLetters 29 (3):243-251.

Helliot B, Swennen R, PouMay Y, Frison E, Lepoivre P, Panis B. 2003. Ultrastructural changes associated with cryopreservation of banana (Musa spp.) highly proliferating meristems. Plant Cell Reports 21:690-698.

Marco-Medina A, Casas JL, Swennen R, Panis B. 2010. Cryopreservation of Thymus moroderi by droplet vitrification. CryoLetters.
31 (1):14-23.

Maslanka M, Panis B, Bach A. 2013. Cryopreservation of Galanthus elwesii Hook. apical meristems by droplet vitrification. Cryo Letters, 34: 1-9.

Matsumoto T, Sakai A, Yamada K. 1994. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabi japonica) by vitrification and subsequent high plant regeneration. Plant Cell Reports 13:442-446.

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco cell cultures. Physiologia Plantarum 15:473–497.

Panis B. 2009. Cryopreservation of Musa germplasm: 2nd edition. Technical Guidelines No. 9 (F. Engelmann and E. Benson, eds). Bioversity International, Montpellier, France. Available here.

Panis B, Piette B, Swennen R. 2005. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science 168:45-55.

Panta A, Panis B, Ynouye C, Criel B, Swennen R, Roca W. 2006. Improvement of potato cryopreservation for the long-term conservation of Andean landraces at CIP. 43rd Meeting of the Society for Cryobiology in association with the Society for Low Temperature Biology. Hamburg, Germany, 24-27 July 2006.

Sakai A, Kobayashi S, Oiyama I. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9:30-33.

Sánchez-Romero C, Swennen R, Panis B. 2009. Cryopreservation of olive embryogenic cultures. CryoLetters 30 (5):359-372.

Sant R, Panis B, Taylor M, Tyagi A. 2008. Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasia exculenta var. esculenta) accessions. Plant Cell Tissue and Organ Culture 92:107-111.

Takagi H, Thinh NT, Islam OM and Senboku T. 1997, Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports 16:594-599.

Thinh NT, Takagi H, Yashima S. 1999. Cryopreservation of in vitro-grown shoot tips of banana (Musa spp.) by vitrification method. CryoLetters 20 (3):163-174.

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