8. Field evaluation of potential mutants obtained after gamma irradiation of banana and plantain (Musa spp.) Shoot-tip and embryogenic cell cultures - López*, J., H. Strosse**, J. de la C. Ventura*, R. Sánchez*, S. Rodríguez*, R. Swennen**, B. Panis**, R. Afza***

*Instituto de Investigaciones en Viandas Tropicales (INIVIT),
Apdo. 6, Santo Domingo,
Villa Clara, CP 53000,
Cuba

**Laboratory of Tropical Crop Improvement,
Catholic University Leuven (K.U. Leuven),
Kasteelpark Arenberg 13,
B-3001 Leuven,
Belgium

***Plant Breeding Unit,
FAO/IAEA Agriculture and Biotechnology Laboratory,
International Atomic Energy Agency Laboratories,
A-2444 Seibersdorf,
Austria

Abstract

Bananas and plantains provide an important carbohydrate source in the Cuban diet. Low yields and susceptibility to diseases (mainly black Sigatoka, caused by the fungus Mycosphaerella fijiensis) of current cultivars render the development of new varieties a high priority. Biotechnological and nuclear techniques have been successfully applied on shoot tips, resulting in the generation of improved cultivars. Potential induced mutants derived after gamma irradiation were tested in the field. A dwarf-like mutant of the cultivar 'Parecido al Rey' (clone V6-44) was obtained. Some mutants (Parecido al Rey variant 6-32 and 6-44 and Gran Enano variant 3-2) were selected for further field evaluation due to their tolerance to black Sigatoka disease, as well as their higher yield compared to non-irradiated control plants. These characters appeared to be unstable during following field cycles. To reduce the frequency of chimeras obtained after irradiation and to obtain a higher multiplication rate of irradiated samples, cell suspensions were established and used as starting material for mutation induction. The regeneration capacity of the established 'Navolean' cell culture was quantified. Homogenized samples of 500 µl cell suspension gave rise to between 1300 and 2650 embryos with a germination frequency of 20.7%. Percentages of survival ex vitro amounted to 95%. Field performance and genetic stability of plants regenerated from irradiated cell suspensions are currently under investigation.

1. INTRODUCTION

Bananas and plantains are among the most important food crops throughout the world, and constitute an important carbohydrate source in the Cuban diet. However, low yields and susceptibility to diseases of local cultivars (mainly black Sigatoka caused by the fungus Mycosphaerella fijiensis) necessitates the generation of improved clones [1-3]. Due to infection by black Sigatoka and the lack of resources to control this disease, the banana and plantain area in Cuba has been reduced, and banana cultivars replaced by black Sigatoka-resistant FHIA hybrids and cooking bananas (ABB group) despite the high preference for almost all plantain (AAB group) cultivars. Hence there is an urgent need to include plantains in the Cuban banana and plantain improvement program [3].

The genetic improvement of banana and plantain through conventional breeding has been hampered by the lack of a profound understanding of the genetics of these plants and their pathogens, the interaction and co-evolution of host and pathogens, and their high sterility, etc. [4]. Biotechnological and nuclear techniques have proved their merit for the induction of genetic variability and mutant selection. Gamma irradiation of in vitro shoot-tip cultures can induce changes in one or a few characteristics of the treated explants without altering their unique cultivar traits [4]. Irradiation of cell cultures followed by regeneration through somatic embryogenesis opens great possibilities, not only in relation to a high multiplication rate but also in the context of avoiding chimerism due to the presumed single cell origin of suspension-derived plants [5].

The present work was performed to obtain mutants characterized by reduced height, increased yield, and higher resistance or tolerance to black Sigatoka than the original clone. Furthermore, plant regeneration via somatic embryogenesis was optimised for its application in genetic improvement programs.

2. MATERIAL AND METHODS

2.1. In vitro establishment and gamma irradiation

2.1.1. Shoot-tips as starting material

Plant material selected for introduction in vitro consisted of vigorous and healthy suckers of commonly grown cultivars in Cuba. In vitro cultures of 'SH 3436-L9' (somaclonal variant of FHIA hybrid 'SH 3436', AAAA group), 'Parecido al Rey' (AAA group), 'Gran Enano' (AAA group) and 'Burro CEMSA' (ABB group) were established by placing their apical meristems onto MS-based culture initiation medium [6] (Table 1).

Table 1 Medium composition for in vitro culture initiation, multiplication (used at IAEA or INIVIT) and rooting

^a Multiplication medium used for 'SH 3436-L9'and 'Paracido al Rey'
^b Multiplication medium used for 'Gran Enano' and 'Burro CEMSA'

Once successfully initiated, explants were cultured on multiplication medium (Table 1) until the required amount of starting material for irradiation was obtained. Shoot tips consisting of the apical dome and 2-4 leaf primordia were excised from rooted plants or multiple shoot-tip cultures and submitted to gamma irradiation in Cuba ('SH 3436-L9', 'Gran Enano' and 'Burro CEMSA') or at IAEA ('Parecido al Rey') according to [4-5].

To determine the optimal irradiation dose for 'SH 3436-L9', a dose-response curve was generated by irradiating 40 shoot-tips to doses of 0, 25, 35, 45 and 60 Gy respectively. Treated explants were then cultured on multiplication medium (Table 1) for 21 days, and the surviving shoot-tips and the number of proliferating buds counted. From these data, the mean lethal doses (LD₅₀) for irradiation of 'SH 3436-L9' shoot-tips could be determined by Probit analysis [7]. The optimal irradiation doses to induce mutants of 'Paracido al Rey' (60 Gy), 'Gran Enano' (35 Gy) and 'Burro CEMSA' (35 Gy) had already been obtained from earlier experiments. After mutagenic treatment, shoot-tips were cultured for three cycles on fresh multiplication medium (Table 1). The M₁V₄ multiple shoot cultures obtained were separated into individual shoots and transferred onto rooting medium (Table 1) for 2-3 weeks. Rooted plants were hardened in the greenhouse for approximately 60 days.

If the desired number of plants for field evaluation (>1000) was not reached, plants were multiplied by corm sectioning so that each section contained at least one bud. This material was incubated in chambers under high humidity conditions. This technique is called SPC (Seed Production Chamber) and was developed at INIVIT. For each explant, 45 days of culture resulted in 10-15 additional 'seeds' for field evaluation.

2.1.2. Embryogenic cell suspensions as starting material

Scalp-derived embryogenic cell suspension of 'Navolean' (AAB group) were developed according to Schoofs [8]. This methodology requires a material preparation phase followed by induction of embryogenesis, and initiation and maintenance of the embryogenic cell suspension (see also Chapter 1 in this book). After inoculation of apical meristems onto culture initiation medium, in vitro meristem cultures were optimised by subsequent cycles on BAP-rich multiplication medium (Table 2).

Table 2 Medium composition for culture initiation and multiplication of meristem cultures, embryogenesis induction and maintenance (ZZ), regeneration (RD1) and germination of somatic embryos and rooting of embryogenic cell suspension derived plants

Optimal explants ('scalps') for induction of embryogenesis were excised from the uppermost part of the meristem culture and cultured on semi-solid ZZ medium (Table 2). Embryogenic calli formed on embryogenesis-induced explants were transferred to liquid ZZ medium (semi-solid ZZ medium without gelling agent) in Erlenmeyer flasks with a volume of either 10 or 25 ml. If the quantity of embryogenic material was so low that the minimal inoculum density required for a successful establishment of an embryogenic cell suspension was not reached, two or three embryogenic calli (derived from different induced explants) were cultured together in one Erlenmeyer flask of 25 ml. Liquid cultures were kept on a rotary shaker (70 r.p.m.) under a light regime of 16:8 light:dark cycle. During the early initiation phase of the embryogenic cell suspension, the culture medium was refreshed every third day. Four weeks after initiation, renewal of liquid ZZ medium was done at bi-weekly intervals. As soon as the cell cultures reached the phase of mass multiplication, the settled cell volume (SCV) was adjusted to 3% at the onset of the subculture period.

To determine the regeneration capacity of the established cell cultures, a homogenized sample of 500 µl suspension (3% SCV) was plated onto RD1 (Table 2). The weight of a representative sample of a 45-day-old regenerated cell culture was measured, and the embryos counted under a stereomicroscope. In this way, the relationship between the weight and the number of embryos could be determined [9]. After embryo differentiation, 12 samples each containing 0.1 g globular embryos were incubated on four different but similar germination media (Table 2). Percentages of embryo germination were recorded 30 days later according to [10]. Once explants with leafs and/or small roots were formed, shoots were transferred to rooting medium (Table 2). Twenty-day-old rooted plantlets that had reached a length of 5 cm were grown ex vitro, and surviving percentages were evaluated after 45 days.

Before irradiation, the SCV of the Navolean embryogenic suspension was increased from 3 to 25%. Samples of 500 µl of this concentrated suspension were irradiated at 10, 20 and 30 Gy respectively. After irradiation, the SCV of the suspension was again adjusted to 3%.

At present, efforts are concentrated on the establishment of embryogenic cell suspensions and plant regeneration via somatic embryogenesis for cultivars varying in their resistance to Fusarium and Sigatoka diseases (Zanzibar, CEMSA 3/4, Burro CEMSA, FHIA 21, Calcutta 4, Niyarma Yik, Pisang Berlin, Pisang Lilin, Rose, Bluggoe, Manzano, Gros Michel, Pisang Ceylan, Pisang Rastali, Yangambi Km5, Pisang Tugi and Pisang Mas). Cell cultures are developed according to the scalp methodology [8] and/or the male flower technique [11].

2.2. Field evaluation of potential mutants obtained by gamma irradiation

After hardening in the greenhouse, control plants (non-irradiated) and irradiated plants derived from shoot tips (at a dose corresponding to LD₅₀) were grown in the field. At flowering and harvest each of the cultivars involved (SH 3436-L9, Parecido al Rey, Gran Enano and Burro CEMSA) was evaluated for plant height (cm), pseudostem circumference (cm), weeks until flowering, number of hands per bunch, number of fingers per bunch, bunch weight (kg) and incidence of black Sigatoka disease at flowering, according to internationally accepted criteria [12]. Statistical analysis of the mean values obtained was done by Duncan's Test.

Based on desirable agronomic traits such as low height, early flowering, high yield and/or increased tolerance towards black Sigatoka, potential improved variants were selected for further field evaluation. Prior to the following field cycle, selected clones were multiplied by SPC until the required number of hardened plants was obtained. The genetic stability of potential variants was deduced from their behaviour during subsequent field cycles. Similarly, plants derived from shoot tips, from suspension culture, and control plants of Navolean were evaluated for their field performance six months after planting.

3. RESULTS AND DISCUSSION

3.1. Shoot-tip derived plants as starting material

As illustrated in Figure 1, increasing irradiation of shoot tips of SH 3436-L9 resulted in fewer surviving explants. Simultaneously, a linear decrease in the proliferation rate of treated explants was noted (112,68,49,29 and 4 buds counted on explants irradiated at 0,25,35,45 and 60 Gy respectively).

Figure 1 Dose-response curve of 'SH 3436-L9' shoot-tips established 21 days after exposure of explants to different irradiation doses (40 shoot tips were treated per irradiation dose).

According to Probit analysis [7] the mean lethal irradiation dose (LD₅₀) of SH 3436-L9 ranged between 39.6 and 47 Gy. Hence, 45 Gy was assumed as most promising for successful mutation induction of SH 3436-L9 shoot tips. Consequently, only plants derived from explants submitted to this irradiation dose (45 Gy) were evaluated in the field.

Results on mean field performance values of potentially induced mutants and control plants of SH 3436-L9, Parecido al Rey, Gran Enano and Burro CEMSA are summarized in Table 3. In a first harvesting cycle, nine irradiated clones of SH 3436-L9 were selected for further evaluation due to their lower height (280-300 cm compared to a mean height of 306 cm for the control), earlier flowering (41-42 weeks compared to a mean value of 43.2 weeks for the control), number of hands per bunch (11-13 compared to a mean number of 11 for the control), number of fingers per bunch (177-225 compared with a mean number of 170 for the control) and bunch weight (32.8-36.2 kg compared to 32 kg for the control). However, these advantageous agronomic traits appeared to be unstable during the second field cycle (data not shown).

For Parecido al Rey, a total of 900 irradiated plants and a similar number of control plants were evaluated for their field performance. In the first cycle, 58% of potentially induced mutants showed phenotypic variations. In particular, bunches were deformed (about 50%). Dwarf and extra-dwarf plants were found, as well as deformed leaves and split fingers. Probably irradiation doses were too high, as each genotype has its optimal LD₅₀ [13-15]. Variants could also be associated with chimerism and the number of subculture cycles on proliferation medium [16]. Because of this very high frequency of variation, no selection was carried out in the first cycle, but material was propagated for further evaluation. In the second cycle eight irradiated clones of Parecido al Rey seemed to perform better in height and yield than the control plants. Two of these better performing clones (V6-32 and V6-44) showed also some tolerance to black Sigatoka (Table 3) and were thus evaluated in a third cycle. In the third evaluation cycle, clones V6-32 and V6-44 were significantly smaller than the control (176.5 and 160.1 cm versus 214.2 cm) and flowered earlier (40.6 and 40.1 weeks versus 42.1 weeks for the control) (Table 3). Since, in addition, a significantly higher mean bunch weight was obtained for variant V6-44 (23.8 kg compared to 20.0 kg for the control), this clone was further evaluated for its field performance. In a fourth cycle, the yield of mutants of V6-44 was substantially higher but not different from the control (data not shown).

Table 3 Summary results on field performance at flowering and harvest of promising variants (V) (irradiated at LD₅₀) and control plants (non-irradiated) of four genotypes (SH 3436-L9, Parecido al Rey, Gran Enano and Burro CEMSA) during different field cycles

Incidence to black Sigatoka is characterized by Incubation Time (IT), Disease Development Time (DDT), Symptom Evolution Time (SET), Youngest leaf with streaks (YLWS), Youngest Leaf in Stage 6 (YLS-6) and Total Number of Leaves (NL) and compared to four reference cultivars (Niyarma Yik, Pisang Berlin, Pisang Lilin and Calcutta 4).

For each experiment, means in the same column followed by same letter are not different (P £ 0.05) based on the Duncan test.

* Highly susceptible

** Susceptible

*** Highly resistant to black Sigatoka

Among the initial population of 3000 Gran Enano plants evaluated for field performance, two mutants (V2-9 and V3-2) were selected because of their shorter height (data only shown for variant V3-2). These mutants were multiplied by SPC until 70-80 plants per mutant were obtained. In the second cycle, mutant V3-2 remained significantly smaller than its control (184 cm versus 192 cm) and seemed to be less susceptible to black Sigatoka than its control. Other agronomic characteristics remained unaffected (Table 3). These two important characteristics were, however, unstable in the third cycle.

A total of 1730 (irradiated and non-irradiated plants) of Burro CEMSA were grown in the field. Based on desirable agronomic traits such as early flowering and lower plant height, two lines (V2-16-1 and V4-20-2) were better performing during a first field evaluation cycle (Table 3). Selected lines were multiplied via SPC and followed for their field performance during a second growing season. During the second field cycle, these two selections lost their superiority over the control (Table 3). All these results obtained in different cultivars indicate that selected materials were unstable during subsequent field cycles.

3.2. Establishment of embryogenic cell suspensions and field evaluation of gamma-irradiated suspension-derived plants

Optimised in vitro meristem cultures of Navolean were obtained after three subsequent cycles on BAP-rich multiplication medium (Table 2). From these highly proliferating meristem cultures, a total of 150 scalps were excised and induced for embryogenesis. One week after induction of embryogenesis, explants showed no evident changes. However, 4-5 weeks after placement of the explants on ZZ medium, the original explant had doubled in size. From the fourth to the sixth week, yellowish-white nodular tissue appeared at the surface of the induced culture. White meristematic globules (nodular callus) gave rise to the first somatic embryos and the formation of embryogenic complexes similar to those previously described [8, 10, 11, 17]. In seven out of 150 cases, embryogenic calli suitable for transfer to liquid medium were observed. The embryogenic frequency obtained (5%) is considered to be high for bananas and plantains, since these crops are rather recalcitrant towards embryogenesis [15, 18, 19]. Transfer of embryogenic calli to either 10 or 25 ml Erlenmeyer flasks revealed a higher proliferation rate of embryogenic cell clusters and subsequently more rapid establishment of the cell culture if the smallest container was used for initiation. Since (a) the quantity of available embryogenic cell material is often low, and (b) a minimal inoculum density is required for successful initiation in liquid medium, the use of 10 ml Erlenmeyer flasks is an important and advanced step in the establishment of embryogenic cell suspensions. Cell cultures which had reached the phase of mass multiplication consisted of a great number of actively dividing spherical cells and heterogeneous translucent and non-translucent aggregates similar to those previously described [8,20]. Forty-five days after transfer of suspension samples (500 µl, 3% SCV) onto regeneration medium RD1, 0.1 g embryogenic tissue contained between 325 and 663 globular embryos. Transfer of regenerated cultures (0.1 g per sample) onto germination medium 1 (containing 0.3 mg/l BAP and 1.0 mg/l IAA as plant growth regulators) was found to be significantly better (Duncan's test, P £ 0.05) than the other germination media tested (Table 4).

Table 4 Mean number of embryos counted 30 days after transfer of 0.1 g of embryogenic tissue onto germination media differing in BAP concentration (see Table 2)

Number of repetitions is 12
Statistics according to Duncan's test (P £ 0.05).

The use of germination medium 1 resulted in a germination frequency of 21% and is similar to reports on the diploid cultivar Musa acuminata (20-36%) but higher than for the clone 'French Sombre' (10-14%) [20]. An increase in BAP concentration from 0.3 to 0.6 mg/l resulted in a decrease of embryo germination from 21 to 7.6% for the cultivar Navolean (AAB group). Six weeks after hardening ex vitro 95% of the plants had survived.

Compared to the control, a growth increase was remarked for Navolean suspensions irradiated with a dose of 10 Gy. Since the suspension irradiated at 30 Gy was still regenerating, it was not possible to determine the mean lethal and subsequent optimal irradiation dose for Navolean cell cultures. Field evaluation of suspension-derived irradiated (0-30 Gy) and control plants are currently under investigation. Besides the highly regenerable Navolean cell suspension, cell cultures were also successfully established for Zanzibar and CEMSA3/4. The development of embryogenic cell suspensions for other cultivars is in progress.

REFERENCES

[1] MORPURGO, R., et al., Enigma of banana breeding a challenge for biotechnology, Agro-Food-Industry Hi-Tech, July/August (1997) 16-21.

[2] LÓPEZ, J., et al., Establecimiento de suspensiones celulares en plátanos vianda del grupo (AAB), Biotecnología Vegetal 1 (2000) 59-61.

[3] LÓPEZ, J., et al., Suspensiones celulares y formación de embriones somáticos en plátano vianda (Grupo AAB). Taller Internacional de Biotecnología Vegetal, Reportes Cortos, Centro de Bioplantas, Ciego de Avila. (2001).

[4] NOVAK, F.J., et al., Mutation induction by gamma irradiation of in vitro culture shoot-tips of banana and plantain (Musa spp.), Trop. Agr. (Trinidad) 67 (1990) 21-28.

[5] NOVAK, F.J., Plant tissue culture techniques for mutation, training manual, Program IAEA, Laboratories-Seibersdorf, FAO/IAEA (1991) 245.

[6] MURASHIGE, T., SKOOG, F., A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant. 15 (1962) 473-497.

[7] FINNEY D.J., Probit Analysis, Cambridge University Press, London, 3rd edition.

[8] SCHOOFS, H., The origin of embryogenic cells in Musa. Ph.D. thesis, K.U.Leuven, Belgium (1997).

[9] GEORGET, F., et al., Morphohistological study of the different constituents of a banana (Musa AAA, cv Grande naine) embryogenic cell suspension, Plant Cell Rep. 19 (2000) 748-754.

[10] DHED'A, D., et al., Plant regeneration in cell suspension cultures of the cooking banana cv. Bluggoe (Musa spp. ABB group), Fruits 46 (1991) 125-135.

[11] ESCALANT, J.V., et al., Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.), In Vitro Plant Cell. Dev. Biol. 30 (1994) 181-186.

[12] FOURE, E., Leaf sport diseases of banana and plantain caused by Mycosphaerella musicola and M. fijiensis, (1994) 37-46, 126-278.

[13] BASILIO, D., "Mutagenesis applied for the improvement of vegetatively propagated plants", Plant Breeding, Seibersdorf, Austria (1992) 40.

[14] AFZA, R., ROUX, N., "Biotechnological approaches which will be applied for Musa improvement in the near future", Banana and Plantain Breeding, Priorities and Strategies, INIBAP, Honduras (1994) 35-36.

[15] MAK CHAI, KW, L.M., et al., Banana improvement by using induced mutation and related biotechnology, (Report of the first FAO/IAEA mutation techniques for creation of new useful Banana Genotypes, 20-24 November), Vienna (1995) 27-38.

[16] ROUX, N., et al., Effectiveness of three micropropagation techniques to dissociate cytochimeras in Musa spp., Plant Cell, Tissue Organ Culture 66 (2001) 189-197.

[17] DHED'A, D., Culture de suspensions cellulaires embryogéniques et régénération en plantules par embryogénèse somatique chez le bananier et le bananier plantain (Musa spp.), Ph.D. Thesis, K.U.Leuven, Belgium, (1992).

[18] SCHOOFS, H., et al., Cuellos de botella en la generación y mantenimiento de las suspensiones celulares morfogénicas de banano y la regeneración de las plantas via embriogénesis somática a partir de ellas, InfoMusa 9 (1999) 3-6.

[19] COTE, F.X., et al., Variations in micropropagated bananas and plantains: literature survey, Fruits 48, Special Bananas: Genetic Improvement (1993) 11-18.

[20] GRAPIN, A., et al., Establishment of embryogenic callus and initiation and regeneration of embryogenic cell suspensions from female and male immature flowers of Musa spp., InfoMusa 7 (1998) 13-15.