Cryopreservation of banana meristem clusters (cauliflower-like structures)

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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.

A second type of regenerative meristematic tissue in banana which has been successfully cryopreserved is the highly proliferating (sometimes called cauliflower-like) meristem clusters. This tissue type was originally produced as a starting material to initiate embryogenic cell suspension cultures in bananas (Dhed’a et al. 1991, Strosse et al. 2006, Schoofs 1997).

Two cryopreservation techniques applied to highly proliferating ‘cauliflower-like’ meristem clusters are described below:

Simple freezing method
Droplet-vitrification of ‘cauliflower-like’ meristem clusters

Preparation of plant materials

Meristem cultures of banana cultivars Nakitengwa (AAA highland banana), Williams (AAA group) and Bluggoe (ABB group) on (left) p5 medium (containing 10μM BA), and (right) p4 medium (containing 100μM BA)(bar = 2 cm) (photo: KULeuven/Bioversity)

Production of ‘cauliflower-like’ meristem clusters

To produce this kind of material in all Musa accessions, meristem cultures are transferred to a medium containing a high concentration of BA (P4 medium, see Appendix 1). Every one to two months, the material is subcultured and small clumps of ‘cauliflower-like’ meristems are selected and transferred to fresh medium (Strosse et al. 2006). The high BA concentration (up to 100 μM) in the P4 medium suppresses outgrowth of meristems, thereby favouring the formation of numerous white apical domes (see photo on the right). Repeated subculturing is necessary and can take
4-12 months.

Preculture of meristem clumps

Discussion and perspectives

The quality of ‘cauliflower-like’ meristem clumps may be too poor for use in cryopreservation experiments (meristematic tissue versus corm tissue is too low and/or the explant shows too much blackening). This can be due to the fact that the cultivar belongs to a ‘difficult’ genomic group (for example East African highland bananas and many plantains). Previously, proliferation was only obtained by using BA at extremely high, nearly toxic, concentrations (100 μM). Prolonged culture on 100 μM BA containing media, therefore, often results in a quality decrease (loss of the typical ‘cauliflower-like’ characteristics) of the cultures. Recently, the use of alternative cytokinins like thidiazuron (TDZ) at lower concentration (1 μM) proved to increase proliferation rates (Strosse et al. 2008).

The simple freezing method (which involves sucrose preculture)

This method (Panis et al. 1996) is illustrated below.

The simple freezing method (figure: KULeuven/Bioversity)


Precultured meristem clumps of “Musa schizocarpa”
(photo: KULeuven/Bioversity)

Petri dish containing control (left) and frozen (right) meristematic clumps of the cv. Bluggoe (ABB group), eight weeks after cryopreservation (bar = 1 cm)
(photo: KULeuven/Bioversity)

Re-warming and recovery

Discussion and perspectives

The simple freezing protocol was applied to 36 banana cultivars belonging to 8 genomic groups (Panis et al. 2002). The results were extremely genotype dependent. Best results (up to 70% regrowth) have been obtained with the ABB cultivars like Bluggoe, Cachaco and Monthan. Intermediate results (around 25% regrowth) were reached with AAA dessert and AAB bananas. AAB plantains and diploids generally respond poorly. For all cultivars under investigation belonging to these genomic groups, plants were regenerated and grown in the greenhouse. However, most of the AAA Highland bananas were not able to withstand simple freezing.

With regard to this simple freezing method, blackening, due to the oxidation of polyphenols is often observed when re-warmed meristems are placed on semi-solid medium. This can cause cytotoxic effects and may also result in the recovering clumps being surrounded by an impermeable layer, thereby preventing nutrient uptake for further outgrowth. One method to overcome this problem is to use liquid regeneration media in order to dilute the released polyphenols. This resulted in regeneration percentages that were up to 20% higher

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The droplet vitrification of ‘cauliflower-like’ meristem clusters method (combining simple freezing with droplet-vitrification)

This method (Panis et al. 2000, Agrawal et al. 2004) is illustrated in the figure below. Loading, dehydration, rapid freezing, storage, re-warming and unloading are almost identical to the droplet vitrification described in the page on Cryopreservation of banana apical meristems. Therefore, only the essential (and differential) steps are indicated in detail below.

Droplet vitrification protocol of meristem clumps (figure: KULeuven/Bioversity)

Loading, dehydration and rapid freezing

Meristem clumps in a droplet of PVS2 solution (of about 15 μl) on a strip of aluminium foil (5×20 mm)
(photo: KULeuven/Bioversity)

Storage, re-warming and unloading


Regenerating shoots from one control and three frozen proliferating meristems of the cultivars ‘Kisubi’ (AB group) ‘Dominico Harton’ (AAB plantain), ‘Bluggoe’ (AAB group) and ‘Williams’ (AAA group) three months after cryopreservation (photos: KULeuven/Bioversity)

Discussion and perspectives

It has been found that post re-warming regrowth percentages of sugar pregrown meristems are higher compared to those grown on normal P4 medium. Sucrose preculture seems to increase tolerance of meristems not only towards the PVS2 solution but also towards the damaging events taking place during the cooling process. When comparing the results from the droplet vitrification of ‘cauliflower-like’ meristem clusters with those obtained using the simple freezing method for the same cultivar, an increase in viability percentages for almost all cultivars is observed. The increase in post-thaw regeneration for the ABB bananas is limited. Recovery remains between 50 and 70%. For AAA dessert and AAB bananas, the increase of regeneration percentages amount to 30-50%, while for plantains 20-30% is reached. AAA Highland bananas which proved to be recalcitrant towards cryopreservation using simple freezing give 0-20% survival using the droplet vitrification method.

For most plant species, optimal dehydration of meristematic tissues with PVS2 is obtained after 10-30 min. at room temperature (Takagi 2000). Among the exceptions are shoot apices of sweet potato and pineapple which need to be treated with PVS2 for 100 min. and 7 hrs., respectively (Plessis and Steponkus 1996, Gonzalez-Arnao et al. 1998). The duration of this treatment has to be optimized case by case since enough dehydration must take place to avoid the formation of lethal ice crystals during freezing. At the same time care has to be taken to prevent the treatment with the potentially toxic solution from irreversibly damaging the tissue. In the case of sucrose precultured proliferating banana cultivars, it has been observed that optimal post re-warming regeneration percentages are generally obtained after a 2 or 2.5 hr. PVS2 treatment. Survival after 3 hrs for most cultivars is considerably lower, probably due to the toxicity of this highly concentrated solution.

References and further reading

Agrawal A, Swennen R, Panis B. 2004. A comparison of four methods for cryopreservation of meristems in banana (Musa spp.). CryoLetters 25:101-110.

Dhed’a D, Dumortier F, Panis B, Vuylsteke D, De Langhe E. 1991. Plant regeneration in cell suspension cultures of the cooking banana cv. ‘Bluggoe’ (Musa spp., ABB group). Fruits 46 (2):125-135.

Gonzalez-Arnao MT, Ravelo MM, Villavicencio CU, Montero MM, Engelmann F. 1998. Cryopreservation of pineapple (Ananas comosus) apices, CryoLetters 19: 375-382.

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, Schoofs H, Thinh NT, Swennen R. 2000. Cryopreservation of proliferating meristem cultures of banana. In: Engelmann F, Takagi H, editors. Cryopreservation of tropical plant germplasm. Current research progress and application. Japan International Research Center for Agricultural Sciences, Tsukuba, Japan / International Plant Genetic Resources Institute, Rome, Italy. pp. 238-243.

Panis B, Strosse H, Van den Hende S, Swennen R. 2002. Sucrose preculture to simplify cryopreservation of banana meristem cultures. CryoLetters 23:375-384.

Panis B, Totté N, Van Nimmen K, Withers LA, Swennen R. 1996. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose. Plant Science 121:95-106.

Plessis P, Steponkus PL. 1996. Effect of preculture in a sucrose-containing medium on the sugar content of potato shoot-tips, Cryobiology 33: 654-655.

Schoofs H. 1997. The origin of embryogenic cells in Musa. Dissertationes de Agricultura 330. Katholieke Universiteit Leuven, Belgium. 257 pp.

Strosse H, Schoofs H, Panis B, André E, Reyniers K, Swennen R. 2006. Development of embryogenic cell suspensions from shoot meristematic tissue in bananas and plantains (Musa spp.). Plant Science 170:104-112.

Strosse H, André E, Sági L, Swennen R, Panis B. 2008. Adventitious shoot formation is not inherent to micropropagation of banana as it is in maize. Plant Cell Tissue and Organ Culture 95:321-332.

Takagi H. 2000, Recent developments in cryopreservation of shoot apices of tropical species. In: Engelmann F, Takagi H, editors. Cryopreservation of tropical plant germplasm. Current research progress and application. IPGRI, Rome, Italy. pp. 178-193.

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