4. Usefulness of embryogenic cell suspension cultures for the induction and selection of mutants in Musa spp. - Roux, N.S.^[1], A. Toloza^[2], J. Dolezel^[3], B. Panis^[4]

Abstract

In bananas shoot-tip cultures are traditionally used for mutagenesis. The main problem with this type of culture is the presence of chimerism. In contrast, embryogenic cell suspensions allow the handling of large populations under controlled conditions and simultaneously avoid chimerism if embryos have a single-cell origin. In this study, we verified the unicellular origin of somatic embryos by treating embryogenic cell suspensions (ECS) with colchicine and determining the ploidy of regenerated plants by flow cytometry. We have found that none of the plants regenerated from colchicine-treated ECS was mixoploid (chimeric). To optimise a protocol for the irradiation of ECS, several experiments were performed. Flow cytometric analysis of cell-cycle kinetics proved to be more informative than growth curves for the determination of the optimal timing of irradiation. The optimal irradiation dose was determined using three different parameters. Among them, the fresh weight gain and the regeneration capacity indicated that the optimal irradiation dose lies between 50 and 75 Gy for embryogenic cell suspensions of both Williams (AAA) and Three Hand Planty (AAB plantain).

1. INTRODUCTION

Bananas and plantains (Musa spp.) are two of the world's major crops. They are a staple food of millions of people and rank among the top five food commodities. Only 13% of the total banana production, mainly dessert bananas, is produced for export [1]. Hence, bananas and plantains are important components of food security in the tropics. The three main diseases and pests that significantly affect banana cultivation are Fusarium wilt, black Sigatoka and nematodes. These diseases/pests can reduce fruit yield by 50% [2-4]. No fungicides can be applied against Fusarium wilt. For black Sigatoka and nematodes, chemical control strategies exist but are environmentally unsound, hazardous and too expensive for many farmers [5]. Breeding of resistant cultivars is the only sustainable way to reduce the use of pesticides. Just as evolution is based on genetic variation, so is breeding. Variation is induced by hybridisation and mutation, among other mechanisms. Breeding is difficult in edible Musa spp. because they are polyploid and sterile. Almost all the edible banana and plantain varieties originate through spontaneous mutations [6]. The best example is the spontaneous banana mutant 'Cavendish' originating from Vietnam, which is resistant to Fusarium wilt (race 1) and which replaced 'Gros Michel' in the 1950s and '60s [7]. The discovery of this banana mutant saved the banana industry from collapsing. Consequently, the export trade relies on a very narrow genetic base since only one or two triploid acuminata varieties of the subgroup Cavendish dominate the export market [8]. The banana is probably the best example in the history of agriculture of the pathological danger of monoclone culture. Indeed, without clonal diversification, the trade can hardly be expected to survive indefinitely, and the generation and use of genetic variability may be the only remaining option.

Spontaneous mutations occur at a relatively low frequency. This frequency can be enhanced using physical or chemical mutagenesis. Combination of in vitro mutation techniques (in vitro mutagenesis) raised hopes in the 1980s and '90s. However, not many useful mutants have been obtained so far. This is mainly due to the high degree of chimerism that occurs during mutagenesis of Musa meristems. Depending on the micropropagation method, chimerism can, however be reduced to 8% after four subcultures in vitro. Nevertheless, no propagation system dependent on meristematic tissues eliminated chimerism completely [9]. In several crops, somatic embryogenesis has been shown to be an effective method for eliminating chimeras, since embryos are presumed to regenerate from single cells [10]. This study was initiated with the aim of optimising the in vitro mutagenesis technique to induce genetic variation in Musa, using embryogenic cell suspensions as target material for irradiation. The work was divided into three phases: (a) demonstration of the single cell origin of somatic embryos; (b) determination of the optimal timing for mutagenic treatment; and (c) determination of the optimal dose of gamma irradiation.

2. MAINTENANCE AND REGENERATION OF EMBRYOGENIC CELL SUSPENSIONS (ECS)

The establishment of embryogenic cell suspensions (ECS) using the scalp method [11] consists of three steps: (a) the preparation of highly proliferating material; (b) the induction of embryogenic cell aggregates; and (c) the maintenance of embryogenic cell aggregates in liquid medium. These initial steps were made at the Laboratory of Tropical Crop Improvement, K.U.Leuven, Belgium [12].

2.1. Maintenance of ECS

The age of ECS used ranged from a few months up to 9 years. During this period, they were maintained at the Laboratory of Tropical Crop Improvement, K.U.Leuven. Then they were hand-carried to the Plant Breeding Unit of the FAO/IAEA Agriculture and Biotechnology Laboratory. Different cell lines of the following cultivars were used:

• Williams (AAA)

• Grande Naine (AAA)

• Three Hand Planty (AAB plantain)

• Bluggoe (ABB)

Individual embryogenic cultures originating from different scalps (cell lines) were kept separately. Erlenmeyer flasks were placed on an orbital shaker at 70 r.p.m. (Figure 1). The medium was refreshed every 2 weeks. The ECS were multiplied in ZZ medium (containing zeatin and 2,4 D) [11] until a sufficient number of flasks had been obtained for further experiments.

2.2. Regeneration of ECS

Regeneration of ECS into pro-embryos, embryos and finally plantlets was done in three steps, each involving a different medium: R1 (hormone-free MS), R2 (MS containing 1 mM BAP) and R3 (MS containing 1 mM BAP and 1 mM IAA) respectively [11]. Regeneration took place on semi-solid medium in Petri dishes for R1 and R2 media and in test tubes for R3 medium. Embryos developed in R2 were also regenerated using the Temporary Immersion System (TIS) vessels containing R3 liquid medium (Figure 1).

Figure 1 Maintenance of Williams cell suspension cultures and regeneration of plants from them: (A) Maintenance of cell-suspension culture. (B) Pro-embryos on filter paper in a Petri dish containing the first regeneration medium R1. (C) Embryos in a Petri dish on the second regeneration medium R2. (D) Regenerated plantlets in Temporary Immersion System (TIS) vessels on the third regeneration medium R3 (liquid).

3. THE ORIGIN OF ECS

Biotechnological techniques such as in vitro mutagenesis [13] and genetic transformation [14, 15] depend on the availability of efficient methods for chimera dissociation [9]. Considering this, somatic embryogenesis is the most promising method since somatic embryos have been assumed to be of single-cell origin [16]. In some species, histological studies confirmed the single-cell origin of somatic embryos. They develop either directly from an explant or as secondary embryos at the surface of older somatic embryos [17]. Grapin et al. [18] deduced from cytological studies on somatic embryo ontogenesis in Musa that a unicellular origin was more than likely. However, such studies can only be performed on a few somatic embryos, and thus the extrapolation of these findings to a large number of somatic embryos is risky. Mixoploidy induction with colchicine and ploidy analysis with flow cytometry [19] allowed Roux et al. [9] to monitor the effectiveness of three different micropropagation techniques in dissociating chimeras. This approach was applied in this study to verify the unicellular origin of somatic embryos from ECS.

3.1. Effect of colchicine on ECS

Two cell lines of the variety Williams (AAA Group) (WIL-124C and WIL-124T) were sieved using a 1000-µm pore-size mesh to obtain a fine suspension. The ECS were then subcultured in the maintenance medium ZZ in 100 ml flasks at an initial concentration of around 3% settled cell volume (SCV). The cells were treated with colchicine for 3 days after subculture at a final concentration of 0.05%, 0.1% and 0.2% (w/v) for 24 h. The cells were then washed three times in ZZ medium. After 24 h colchicine treatment, 0.5 ml of ECS at 10% SCV was taken from each flask and transferred to a Petri dish containing the regeneration medium (R1). Nuclear DNA content distribution in the ECS of the two cell lines WIL-124C and WIL-124T was determined using flow cytometry as described previously [17] before transfer to the regeneration medium R1 (Figure 2) and after complete regeneration into plantlets (Table 1).

Figure 2 Frequency distribution of Williams suspension cells with triploid (3x) and hexaploid (6x) nuclear DNA content observed 15 days after colchicine treatment (w/v): (A) WIL-124C and (B) WIL-124T.

Table 1 Ploidy distribution of regenerated plants from colchicine-treated ECS of two triploid cell lines of the cultivar Williams

Even though the two cell lines (Wil-124C and Wil-124T) were initiated from the same accession [Williams (ITC-0365)], the effect of colchicine on polyploidy induction was different. The response to colchicine treatment was clearer for cell line WIL-124C than for WIL-124T, especially when high concentrations were applied (0.1% and 0.2%). The proportion of polyploidised cells increased to about 50% for WIL-124C, but only to 15% for WIL-124T.

3.2. Regenerated plants from colchicine-treated ECS

The embryos were subsequently transferred to R3 medium in test tubes. As soon as green plantlets with shoot and roots were obtained, 0.5 cm² pieces of leaves were excised and their ploidy measured by flow cytometry before acclimatization (Table 1). For both cell lines, WIL-124C and WIL-124T, the majority of the plants regenerated were triploid. ECS line WIL-124C, which had a higher proportion of hexaploid cells at the earlier stage (Figure 2), had a lower regeneration rate. No plants were regenerated after treatment with 0.2% colchicine. Control cells of WIL-124C, however, had also a very poor regeneration capacity. This could be due to the different quality of cells between the two cell lines.

WIL-124T had a better regeneration capacity. Nevertheless, among the treated cells, the proportion of regenerated hexaploid plants (5.3%) still remained very low compared with triploid plants. Hence we speculate that triploid cells have an advantage over hexaploid cells during culture. Interestingly there were no mixoploids among the regenerated plants, which is consistent with a single cell origin of embryos. Thus embryogenic cell suspensions seem to be the material of choice for mutagenic treatments.

4. IRRADIATION OF ECS

4.1. Determination of the optimal timing for irradiation

4.1.1. Growth curve of ECS

The time at which the cells are irradiated is very critical. The growth of suspension cultures is usually divided into three phases: the lag phase, the exponential phase, and the stationary phase. Irradiation of the cells in the exponential (active growing) phase is usually recommended, to increase the chance of recovering higher numbers of mutants [20]. To determine the exponential phase, we measured the percentage of settled cell volume (% SCV) by regularly transferring the content of Erlenmeyer flasks to sterile centrifuge graduated tubes and using the following formula:

Five 100-ml Erlenmeyer flasks per cell line were inoculated with 15 ml of cell suspension (at an SCV of 3.3%). Growth curves were obtained by measuring the SCV twice per week (every 2-3 days) until day 29 (Figure 3). Cells were not subcultured until the exponential phase was reached, after 15 days or more. None of the five cell lines followed a sigmoid growth curve. Growth curves of cell lines of Three Hand Planty (THP1 and THP7) seemed to reach an exponential phase after 19 days. These two THP cell lines had a tendency to form clumps after some days after subculture, which could explain why their growth curves were different from the other three cell lines. The growth curves of the three other cell lines, Grande Naine (GN), Williams (WIL) and Bluggoe (BG), were linear rather than sigmoid. Additionally, cells in culture for periods longer than 2 weeks show a decreased quality. Thus, SCV did not seem suitable for the determination of the optimal timing for irradiation.

Figure 3 Growth curves of cell lines of cultivars Williams (WIL), Grande Naine (GN), Three Hand Planty (THP) and Bluggoe (BG). The curves were obtained by measuring the settled cell volume (SCV) every 2-3 days after subculture during 29 days without refreshing the medium. The SCV was set in the beginning at 3%. The SCVs of five 100-ml Erlenmeyer flasks per cell line were measured. Vertical bars represent standard deviations.

4.1.2. Cell-cycle analysis of ECS

Cell growth and division is a cyclical process. With flow cytometry, it is possible to study the proportion of cells in the G₁ and G₂ phases of the cell cycle, since the nuclear DNA content in G₂ is twice that in the G₁ phase. Cycling cells transit through the G₂ phase, whereas non-cycling cells usually remain in G₁ (G_0/1). Because the effect of a specific mutagen on chromosomes depends on the position of a cell in the cell cycle at the time of exposure, it is important to study the cell cycle of embryogenic cell suspensions [21]. We therefore analysed the proportion of G₂ and G₁ cells. Cells from four accessions (four 100-ml Erlenmeyer flasks per accession) were analysed every 2 or 3 days during 30 days of culture (Figure 4). Peaks indicated waves of mitotic activity, i.e. a certain degree of cell-cycle synchrony. A similar mitotic activity was described by Panis [22] by measuring the mitochondrial activity with the 2,3,5-triphenyl tetrazolium chloride reduction test during the culture of embryogenic banana cell suspensions. It seems that in cells transferred to a fresh medium, the cell cycle is stimulated relatively synchronously. The highest proportion of cells in G₂ phase was observed 8 days after subculture for Williams and the two THP accessions, and 6 days after subculture for Bluggoe. These are, however, preliminary results, and a more detailed study using synchronized cultures should be performed to confirm these findings.

Chromosome-type aberrations (involving both chromatids) are produced by ionising radiation in the G₁ phase of the cell cycle. During the S phase, the lesions are transmitted to both chromatids and are observed during metaphase or anaphase of the succeeding mitosis [21]. According to Figure 4, the majority of cells are in the G₁ phase during the first 3 days after subculture. Interestingly, the efficiency of biolistic transformation of ECS is highest during the first 3-4 days of culture (Swennen, personal communication). We thus consider 3 days after subculture the optimal moment for mutagenic treatment.

Figure 4 Changes in cell-cycle distribution during culture of embryogenic cell suspensions of four cell lines of cultivars Williams (WIL), Three Hand Planty (THP) and Bluggoe (BG). The curves were obtained by measuring the ratio of cells in the G₂ and G₁ phases of the cell cycle during 30 days without refreshing the medium. The G₂/G₁ ratio was measured for five 100-ml Erlenmeyer flasks per cell line. The lines represent the means.

4.2. Determination of the optimal mutagenic dose

4.2.1. Irradiation of ECS

Two cell lines of the varieties Williams (AAA) and Three Hand Planty (AAB) were sieved using a 1000-µm pore size mesh to obtain a fine suspension. The ECS were then subcultured in the maintenance medium (ZZ) in 100-ml flasks at a concentration of around 3% of settled cell volume (SCV). Three days after subculture, 0.5 ml of cells were transferred to a sterile Petri dish and the medium was removed. The cell aggregates were then irradiated at doses ranging from 0 to 250 Gy in steps of 25 Gy using a ⁶⁰Co gamma source at a dose rate of 30 Gy/min. After irradiation, the cells were resuspended in fresh medium in centrifuge tubes and transferred to 100 ml Erlenmeyer flasks at different quantities according the parameter to be analysed. To study the effect of gamma radiation on the growth of ECS, settled cell volume, fresh weight gain and regeneration capacity were determined. The results were expressed as percentages of the control (non-irradiated cells) at all doses.

4.2.2. Radiosensitivity of ECS based on the percentage of settled cell volume

After several preliminary experiments, we observed that the variation of SCV between flasks was too high. Therefore we divided each Erlenmeyer flask into two identical subsamples each of 3% SCV. For each dose (from 0 to 250 Gy), one subsample was irradiated and the other served as control (non-irradiated). The SCV was calculated every week according to the formula given above (Section 4.1.1). Clearest results were obtained after 21 days (Figure 5A). Based on SCV, the radiosensitivity curves were quite similar for Three Hand Planty and Williams. This would indicate that the response was not genotype-dependent. Surprisingly, Figure 5 shows that even at a relatively high dose of 250 Gy (SCV₂₅₀ = 7.8%), cells have a settled cell volume corresponding to nearly 50% of the control (SCV₀ = 14.3%). At doses over 100 Gy cells looked brownish but were still able to divide.

4.2.3. Radiosensitivity of ECS based on fresh weight gain

To study the fresh weight gain after irradiation, the SCV was adjusted to 10% and 0.5 ml of the homogenized suspension was distributed over a moistened filter paper saturated with the regeneration medium R₁. Four Petri dishes were inoculated per dose. For each Petri dish, the saturated filter paper was weighed before and after spreading the cells. The fresh weight of the cells was calculated using the following formula:

Fresh weight of cells = total weight (filter paper + cells) - filter paper weight

Figure 5 Effect of increasing doses of gamma radiation on three parameters: (A) settled cell volume (SCV) 21 days after irradiation; (B) fresh weight gain 28 days after irradiation; and (C) regeneration capacity at 60-90 days after irradiation. All parameters were expressed as percentages of the control (non-irradiated ECS). Cell suspension cultures of the cultivars Williams (WIL) and Three Hand Planty (THP) were used. For each treatment the equivalent of four Petri dishes were measured. 50% of control corresponds to around 100 Gy for the SCV but corresponds to 50-75 Gy for the fresh weight gain and regeneration capacity.

Each week, during a 56-day period, the cell fresh weight was compared to the control. The optimal time to observe the effect of gamma radiation on fresh weight was 28 days after irradiation (Figure 5B). The radiosensitivity curves for the varieties Williams and Three Hand Planty are quite similar. After 28 days, the cell-weight gain at 75 Gy (CWG₇₅ = 0.84 g) was 50% of the control (CWG₀ = 1.68 g). The effect of gamma radiation on fresh weight gain thus seems to be greater than on the SCV. This difference could also be due to the different medium constitution (semi-solid versus liquid medium).

4.2.4. Radiosensitivity of ECS based on regeneration capacity

Approximately 40 days after irradiation and transfer onto semi-solid regeneration medium R₁, the pro-embryos (Figure 1B) with filter paper were transferred to the second semi-solid regeneration medium R₂ for about 15-20 days until embryo formation. Because of the high number of embryos (Figure 1C), the experiment was limited to one Petri dish per dose. The content of a Petri dish was transferred to a TIS vessel (Figure 1D) containing the third regeneration medium R3 (liquid), except for the non-irradiated cells where the content of a Petri dish was transferred to two TIS vessels because of the higher number of embryos. Later, green plantlets were counted and transferred to Magenta GA7 boxes containing semi-solid R₃ regeneration medium for further growth before acclimatization. The radiosensitivity curve was obtained by comparing the number of regenerated plantlets for each dose with the control plants (from non-irradiated ECS) (Figure 5C).

Radiation at a low level seems to stimulate regeneration, especially in Williams. We must, however, take into account that in control plants, the density of embryos in the TIS vessels was too high and hence a considerable number of small plantlets could not develop. In both genotypes no plants regenerated above 200 Gy. The number of regenerated plants drops drastically above 50 Gy for the variety Williams, whereas for Three Hand Planty the number of regenerated plantlets decreases less dramatically. The regenerated plants have recently been transferred to the greenhouse and the field for further phenotypic and agronomical evaluation.

5. CONCLUSIONS

Our observations clearly demonstrate that a combination of colchicine treatment and DNA flow cytometry is useful for the verification of the unicellular origin of somatic embryos. The results demonstrated that embryogenic cell suspensions (ECS) are probably the material of choice for in vitro mutation induction, as their use overcomes the problem of chimerism. This will speed up the in vitro mutagenesis process since there is no need to dissociate chimeras after mutagenic treatment. DNA flow cytometric analysis is also very powerful to study cell-cycle kinetics of ECS and to determine the optimal timing for irradiation. Three days after subculture seems to be the optimal moment for mutagenic treatment. Nevertheless, additional studies should be carried out, preferably with synchronized cells, to obtain more precise results. To study the effect of gamma irradiation, regeneration capacity seemed to be more appropriate than settled cell volume or fresh weight gain. The optimal dose for the irradiation of suspensions was determined as 50-75 Gy for Williams (AAA) and Three Hand Planty (AAB). Hence embryogenic cell suspensions from Musa can tolerate higher doses than shoot tips.

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