2. Cryopreservation of banana tissues: support for germplasm conservation and banana improvement - Panis, B., H. Strosse, S. Remy, L. Sági, R. Swennen

Laboratory of Tropical Crop Improvement
Catholic University of Leuven
Kasteelpark Arenberg 13
B-3001 Leuven
Belgium

Abstract

The world's largest banana collection (1141 accessions) is stored at the International Musa germplasm collection at the INIBAP (International Network for the Improvement of Banana and Plantain) Transit Centre at K.U.Leuven. In vitro proliferating shoot tips are currently maintained under slow-growth conditions at reduced temperatures and light intensity. However, for the long-term conservation of germplasm of banana, cryopreservation of meristem cultures is considered to be the only practicable solution. Three cryopreservation protocols for meristem cultures have been developed. This paper gives an overview of pros and cons for each cryopreservation method.

The initiation of embryogenic cell suspension cultures of banana is still difficult and time-consuming, irrespective of the starting material used. Moreover, once established, these cell suspensions are subject to somaclonal variation and microbial contamination, and a prolonged culture period may result in total loss of morphogenic capacity. Up to now, most successful banana transformation procedures rely on embryogenic cell suspensions. The safe preservation of these valuable suspensions through cryopreservation is thus of utmost importance. A cryopreservation technique has been developed which involves cryoprotection followed by slow freezing and plunging into liquid nitrogen. Currently, 51 independent cell lines and 15 different cultivars are in safe long-term liquid nitrogen storage. Recently, banana cell suspensions were recovered after 5 years storage in liquid nitrogen. Their competence for Agrobacterium-mediated transformation was tested. Both transient â-glucuronidase frequency and stable transformation frequency were unaffected.

1. INTRODUCTION

Seed preservation, the most convenient method to preserve plant germplasm, is not applicable to edible (seedless) banana cultivars. Field collections lose germplasm (genetic erosion) because of pests, diseases and adverse weather conditions, and their maintenance is labour-intensive and expensive. Therefore, in vitro collections have been established. The maintenance of such in vitro banana collections is labour intensive even under reduced growth conditions, and there is always the risk of losing accessions due to contamination or human error. Moreover, in vitro material is subject to somaclonal variation. Cryopreservation or freeze-preservation at ultra-low temperatures (-196°C) is the method of choice, since under these conditions biochemical and most physical processes are arrested. As such, plant material can be stored for unlimited periods. Currently, three cryopreservation protocols are available for shoot-meristematic tissues of banana: (i) simple freezing of proliferating meristem cultures using a sucrose preculture [1]; (ii) vitrification of apical meristems [2]; and (iii) vitrification of sucrose-precultured meristem cultures [3]. Very recently it has been shown that cryopreservation could also be used for eradication of viruses [4]. Cryopreservation is also used to conserve tissues with specific characteristics such as medicinal and alcohol-producing cell lines, genetically transformed tissues and transformation-competent tissues.

The most successful methods for improvement of banana through genetic engineering rely on the availability of embryogenic cell suspensions. Embryogenic cell suspensions are so far the only source of regenerable protoplasts in banana [5]. When subjected to electroporation they give rise to high transient expression frequencies of an introduced marker gene [6]. Walled suspension cells are successfully transformed by means of particle bombardment [7] and Agrobacterium [8]. Since plants regenerated from embryogenic cell suspensions are of unicellular origin they are probably also highly suitable for improvement through induced mutation.

An important drawback for the application in banana of improvement techniques based on cell suspensions remains the availability of suspensions of good quality, i.e. homogeneous embryogenic cell suspensions with high regeneration frequency. The initiation of these suspension cultures is difficult and time-consuming, irrespective of the starting material used (immature male flowers, immature zygotic embryos or proliferating in vitro meristems) [9]. Consequently, it is of utmost importance to conserve embryogenic cell suspensions as stably as possible once they are established. This can be achieved through cryopreservation.

Here we present a practical approach for cryopreservation of meristem cultures for long-term storage of banana germplasm. Secondly, an overview is given of the different embryogenic cell lines of banana which are currently stored in liquid nitrogen. Finally, we show that cryopreserved cell suspensions are suitable for Agrobacterium-mediated transformation.

2. MATERIALS AND METHODS

2.1. Cryopreservation of meristem cultures

2.1.1. Simple freezing of proliferating meristem cultures

Highly proliferating meristem clumps are cultured for 2 weeks on a proliferation medium enriched with 0.4 M sucrose. Surviving/growing meristem clumps are then excised, transferred to 2 ml cryotubes and plunged into liquid nitrogen for storage. Rapid thawing takes place in a warm water bath at 40°C. Meristem clumps are recovered in a liquid regeneration medium.

2.1.2. Vitrification of sucrose precultured meristem cultures

Sucrose precultured meristem cultures are treated for 20 min with a loading solution (LS) which contains 2 M glycerol and 0.4 M sucrose at room temperature. Then they are subjected for 2 h to ice-cooled PVS2 solution [30% (3.26 m) glycerol, 15% (2.42 m) ethylene glycol (EG), 15% (1.9 m) DMSO and 0.4 M sucrose [10]], transferred to 2 ml cryotubes and plunged into liquid nitrogen. Rapid thawing takes place in a warm water bath at 40°C. Directly after thawing, the toxic PVS2 solution is removed and replaced by the deloading solution (DS) for 15 min at room temperature. The deloading solution consists of 1.2 M sucrose dissolved in MS medium (pH 5.8). Meristem clumps are recovered on semi-solid regeneration medium.

2.1.3. Vitrification of apical meristems

One millimetre-sized meristems are excised using a binocular microscope from in vitro plants that had been cultured for 1 month on 6% sucrose. The most essential but also most difficult step is the excision of tiny meristem tips of the correct size (a base diameter of 0.5-1 mm). The apical domes may only be covered for two-thirds by the youngest leaf primordium. Tips that are slightly damaged or are not at the correct stage (the meristem is covered too much or too little by leaf primordia) are excluded. The tips are treated for 20 min with the loading solutions, followed by a 15 min treatment with PVS2 at 0°C. Freezing takes place in 2 ml cryovials. After storage meristem tips are rapidly rewarmed in a warm water bath (40°C) and treated for 20 min with the deloading solution. Recovery takes place on semi-solid regeneration medium.

A more detailed description of the three cryopreservation methods is presented in the INIBAP Technical Guidelines 5, Cryopreservation of Musa germplasm [11].

2.2. Cryopreservation of embryogenic cell suspensions and transformation

2.2.1. Freezing

Suspensions are initiated from different explants: from immature male flowers [12], and from proliferating meristem cultures [13]. Suspensions are cryopreserved using slow freezing (1°C/min) in the presence of 7.5% dimethylsulphoxide (DMSO) and 140 g/l sucrose. See [11] for a more detailed description of cryoprotection, freezing, thawing and recovery of Musa germplasm.

2.2.2. Post-thaw observations and re-initiation of embryogenic cell suspensions

Two months after thawing, Petri dishes are screened for the presence of embryogenic callus. If embryogenic callus is absent, the corresponding tubes in the liquid nitrogen tank are discarded. If present, the regrowing callus is transferred to liquid maintenance medium and regularly subcultured.

2.2.3. Transformation

Embryogenic cell suspensions of banana were transformed according the method described by Pérez Hernandez [14]. In this experiment, a suspension line of the plantain cultivar Three Hand Planty [THP (7+8)] was used. This suspension was established from proliferating meristem cultures in July 1994 and frozen and stored in liquid nitrogen at different time intervals (10 April, 1996; 27 September, 1996; and 22 May, 1997). On 21 December, 1999 the suspensions were thawed and plated on semisolid media. Later on, liquid cell suspension cultures were re-established from regrowing embryogenic calli. These three cell lines, together with a control (unfrozen) suspension of the same cell line, were subjected to transformation on the 9 November, 2000.

The Agrobacterium tumefaciens strain EHA101 contains the pFAJ3000 plasmid. This plasmid has an intron-interrupted gusA gene (reporter gene) under the control of a CaMV 35S promoter, and the neo gene coding for neomycin (selectable marker) phosphotransferase driven by the nopaline synthetase promoter (nos). Transient gene expression in banana cells is visualized after 6 days of co-cultivation with Agrobacterium as the number of blue spots after a 4-hour treatment with X-Gluc. After 3 months of culture on selective medium (semisolid suspension maintenance medium supplemented with 50 mg/l geneticin), independent putative transformed cell clumps are counted and transferred to regeneration medium. After 3 months, the number of regenerated plants was counted.

3. RESULTS

3.1. Cryopreservation of meristem cultures

Forty-four banana accessions obtained from the International Musa Germplasm collection at the INIBAP Transit Centre at K.U.Leuven were screened for their response towards one or more cryopreservation methods. The results are grouped according to the genomic constitution and cryopreservation method used (Table 1). We observed that the frequency of post-thaw regeneration depends on (a) genomic group, (b) cryopreservation protocol and (c) cultivar, despite high variations between repetitions. ABB cultivars respond very well to the simple cryopreservation protocol, whereas for AAB, AAA, some AAB plantains and AB bananas, vitrification of proliferating cultures is highly efficient. The response to vitrification of apical meristems is cultivar-independent.

Table 1 Response (% post-thaw regeneration) of different banana cultivars according to the genomic constitution towards different cryopreservation protocols

^a AABpl: Plantain; AAAh: East African Highland banana
^b Not determined

A calculation was made of the number of accessions that can be cryopreserved per person per year according to the cryopreservation method (Table 2). The time required is very dependent on the cryopreservation method. The most labour-intensive method (vitrification of apical meristems excised from rooted in vitro plants) would thus only be applied to cultivars which are difficult to cryopreserve with the other two methods (for example AAA highland bananas).

Table 2 Comparison of three cryopreservation protocols for Musa

^a Calculated time needed to prepare the culture media and meristem cultures followed by cryopreservation. For each cultivar three repetitions would be made with three cryotubes per repetition containing 10 (vitrification of individual meristems) to 20 (simple freezing and vitrification of proliferating clumps) explants. For each repetition a representative sample would be thawed and regenerated.

^b 1 year = 220 working days (8 h/day).

^c Starting from two tubes until this accession is safely stored in liquid nitrogen (three repetitions).

Since the start of our cryopreservation work, a total of 51 accessions have now been placed 'safely' in long-term storage in liquid nitrogen using both vitrification and simple freezing.

3.2. Cryopreservation of embryogenic cell suspensions and transformation

The most important parameter for post-thaw regrowth of embryogenic cell suspensions is their quality. Most suspensions which contain a high proportion of embryogenic cells (cells which are isodiametric and characterised by a relatively large nucleus, small multiple vacuoles and tiny starch and protein grains) survive cryopreservation. 'Less ideal' suspensions which contain a low proportion of embryogenic cells or in which 'embryogenic' cells are highly vacuolated or contain big starch grains seldom survive the freezing process. Since these 'less ideal' suspensions are also less favourable for transformation, not much effort is made to store them in liquid nitrogen. Currently, 51 cell lines belonging to 15 banana cultivars, representing 745 cryotubes, are safely stored in liquid nitrogen (Table 3).

Since transformation requires embryogenic cell suspensions in liquid media, regrowing calluses were reinitiated in liquid media. Most of the cell suspensions thus obtained displayed embryogenic characteristics comparable with the non-frozen controls. Different embryogenic cell lines of the cultivars Williams and Three Hand Planty, which had been stored in liquid nitrogen up to 4.5 years, were thawed and reinitiated. As expected, regeneration capacity was not reduced by the long storage period.

Table 3 Number of cryotubes containing embryogenic banana cell suspensions that are currently stored in liquid nitrogen

AABp: Plantain; AAAh: East African Highland banana

We checked whether the storage of embryogenic cell suspensions in liquid nitrogen has an effect on their transformation capacity. The cell line THP (7+8) [Three Hand Planty (AAB plantain)] was subjected to Agrobacterium-mediated transformation. Prolonged storage in liquid nitrogen (2.5-3.5 years) does not affect the transient gus expression of re-initiated embryogenic cell suspensions (Table 4).

Stable transformation data were also gathered. For this, the number of independent putative transformed cultures after selection on 50 mg/l geneticin, as well as the frequency of these cultures giving rise to shoots, were counted (Table 5). For 2 out of 3 cryopreserved suspension cultures, the number of independent putative transformed cultures is not significantly different from the non-frozen suspensions. THP (7+8), cryo 22/05/1997, results in a significantly lower number of surviving clumps despite its comparable transient GUS expression. This might be due to the inferior physiological state of this suspension at the time of transformation. However, the calculated average of the independent putative transformed cultures of the three cryopreserved cell lines (40.7) is almost identical to that of the non-cryopreserved suspension (42.0). To test whether cryopreservation had indeed affected the stable transformation frequency, this re-initiated suspension should be tested again. More important is that all suspensions showed a comparable number of transformed shoots.

Table 4 Transient GUS expression of one control and three cryopreserved embryogenic cell suspension of the cv. Three Hand Planty (AAB plantain) after Agrobacterium-mediated transformation

^a Numbers are not significantly different by the Tukey honest significant difference (HSD) test at an alpha level of 0.05.

Table 5 Stable transformation of one control and three cryopreserved embryogenic cell suspension of the cv. Three Hand Planty (AAB plantain) after Agrobacterium-mediated transformation and selection on 50 mg/l geneticin

^a Numbers followed by the same letter are not significantly different by the Tukey honest significant difference (HSD) test at an alpha level of 0.05

4. DISCUSSION

Cryopreservation of plant material serves two main purposes: conservation of genetic diversity; and conservation of material with specific characteristics. For example, large scale cryopreservation of embryogenic cultures is essential for advanced forestry breeding programs. Approximately 5000 genotypes representing 14 conifer species have been stored [15]. For species like banana, where the establishment of embryogenic cultures is far from routine, meristem cultures are used for safe storage of germplasm. Three protocols for cryopreservation of meristem cultures are presented, which are used routinely at the INIBAP Transit Center. The method of choice mainly depends on the genome configuration of the accession. We have calculated that on average, 50 banana accessions can be cryopreserved per person per year.

Cryopreservation of banana embryogenic cultures is of utmost importance, since it is the only way for safe preservation of transformation-competent tissues. Successful cryopreservation of plant embryogenic cultures has been reported using vitrification [10,16-18], direct immersion in liquid nitrogen [19], encapsulation dehydration [20,21], encapsulation vitrification [22], encapsulation combined with slow freezing [22] and vitrification combined with slow freezing [23]. However, for non-organized tissues like cell suspensions and callus cultures, slow freezing in the presence of cryoprotective agents is still the method of choice. During slow freezing, crystallisation is initiated in the extracellular spaces, since it is believed that cells rarely contain ice nuclei. Since only a fraction of the water in the extracellular solution undergoes transition into ice, the solution becomes more and more concentrated and thus hypertonic to the cell. To restore the osmotic equilibrium, cellular water will leave the protoplast. Slow freezing will thus result in a concentrated cytoplasm which will vitrify upon subsequent plunging into liquid nitrogen. The addition of penetrating substances like DMSO facilitates this process. Vitrification (or glass formation) of the cellular solution is an important requirement for successful cryopreservation, since this is the only way to freeze a solution without the formation of lethal ice crystals.

After cryopreservation, a thawed suspension needs to be viable, able to give rise to an embryogenic cell suspension, be true-to-type, and retain its characteristics. The first two requirements are met for most embryogenic cell lines of banana [24-26]. The true-to-typeness of plants recovered from cryopreserved embryogenic cell suspensions of banana was screened by Côte and co-workers [27]. They proved that there was no difference at the agronomical level between plants produced from cryopreserved embryogenic cell suspensions and control plants. Finally, a cryopreserved suspension needs to retain typical features. It was proven that cryopreservation did not affect the expression of a foreign sam gene in transgenic Papaver somniferum cells [28], had no impact on paclitaxel biosynthesis in suspension cultures of Taxus chinensis [29], and was even beneficial for pyrethrin biosynthesis in Chrysanthemum cinerariaefolium Vis. cells [30]. Also, the production of regenerable protoplasts was not influenced by cryopreservation as shown for rice [31, 32], and Festuca and Lolium species [33]. Moreover, cryopreserved rice callus [34] and maize cell cultures [35] proved to be a constant source of regenerable cell cultures for the production of transgenic plants. Moukadiri and co-workers [19] showed that calluses that were stored in liquid nitrogen demonstrated a higher competence for transformation, as indicated by transient gene expression levels. In banana, comparable levels of transient expression, as well as stable transformation, were obtained in both cryopreserved and non-cryopreserved suspensions. We can therefore that assume that there was no cryoselection.

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