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Phenotypic variation among in-vitro propagated plantain (Musa sp. cultivar ‘AAB’)
Scientia Horticulturae, 36 (1988) 79-88 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
79
Phenotypic Variation Among In-Vitro Propagated Plantain (Musa sp. cultivar 'AAB') DIRK VUYLSTEKE, RONY SWENNEN, GEORGE F. WILSON and EDMOND DE LANGHE x
International Institute of Tropical Agriculture, PMB 5320, Ibadan (Nigeria) 1International Network for the Improvement of Banana and Plantain, Avenue du Val de Mont[errand, BP 5035, 34032 Montpellier Cedex (France) (Accepted for publication 5 February 1988)
ABSTRACT Vuylsteke, D., Swennen, R., Wilson, G.F. and De Langhe, E., 1988. Phenotypic variation among in-vitro propagated plantain (Musa sp. cultivar 'AAb'). Scientia Hortic., 36: 79-88. In-vitro micropropagated plantain (Musa sp. cultivar 'AAB') plants were regenerated from meristem cultures and screened in the field for phenotypic variability. Observations of the plant crop and the successive ratoon indicated five different forms of phenotypic variation at a frequency of 6%. Inflorescence variants were of the "French" and 'Monganga' bunch type. Foliage variants were novel phenotypes and agronomically inferior. The latter off-types remained stable through conventional clonal propagation. Keywords: in-vitro micropropagation; Musa; plantain; shoot tip culture; somaclonal variation. Abbreviations: BA=n6-benzyladenine; IAA=indole-3-acetic acid; NAA=l-naphthaleneacetic acid.
INTRODUCTION
Plantain (Musa sp. 'AAB') is a major staple food in the humid lowlands of West and Central Africa. Increasing demand warrants increased production. However, expansion of production is limited by a shortage of suckers, the common planting material (Wilson, 1983). The transmission of harmful insects, nematodes and black Sigatoka disease (Raemaekers, 1975; Frossard, 1980) on field-grown suckers necessitates the production of clean planting material and impedes the international transfer of germplasm. Furthermore, the recent but rapid spread of the black Sigatoka disease on the African continent is emerging as a major threat to the cultivation of plantains because no resistant cultivars are known to exist (Wilson and Buddenhagen, 1986). These problems have 0304-4238/88/$03.50
© 1988 Elsevier Science Publishers B.V.
80 prompted an interest in the use of aseptic culture techniques, complementary to conventional approaches in the improvement ofMusa (Krikorian and Cronauer, 1984; Stover and Buddenhagen, 1986). Meristem culture has been suggested as a method for the rapid clonal propagation of selected {diseaseresistant) Musa cultivars and as a means of germplasm exchange (Jarret et al., 1985; Vuylsteke and De Langhe, 1985). The in-vitro micropropagation technique for cultivated Musa is well established (De Guzman et al., 1980; Cronauer and Krikorian, 1984; Jarret et al., 1985; Vuylsteke and De Langhe, 1985 ), but its use may be limited by the risk of somaclonal variation, a widespread occurrence in some in vitro cultures (Scowcroft, 1984 ). Off-types have been reported in dessert banana (Musa AAA group, 'Cavendish') (Reuveni et al., 1985; Hwang, 1986; Stover, 1987) and in plantain (Ramcharan et al., 1985). However, the report concerning plantain was a chance observation and not a systematic determination of the effect of the technique on phenotypic changes in the cultivar. Moreover, results on somaclonal variation in the AAA bananas might not be transposed to the genotypically different plantains. This paper presents findings of an attempt to determine the frequency and type of variants induced by meristem culture of plantain. MATERIALSAND METHODS The micropropagated plantain cultivar was 'Agbagba', the most popular in Nigeria. This cultivar produces bunches of 10-15 kg and 30-40 fingers within 12 months after planting. Its inflorescence is incomplete at maturity as there is no male bud. Hands consist of large fingers followed by a few hermaphrodite flowers (Fig. la). 'Agbagba' therefore belongs to the False Horn group of plantain (De Langhe, 1961; Tezenas du Montcel et al., 1983). To ascertain trueness-to-type, buds and suckers, used as source material, were collected from flowering plants. For culture initiation, cubical 1-2-cm :~ pieces containing the apices were excised from buds or suckers and surfacesterilized by washing with ethanol (95%) for 15 s, followed by soaking for 15 min in a 0.75% NaOC1 solution, to which one drop of Tween 80 was added per 50 ml, and then rinsed 3 times with sterile deionized water. Shoot tips of 1-2 mm in length, bearing 2-4 leaf primordia, were isolated aseptically and placed on a modified Murashige and Skoog medium (1962) with 0.4 g l-1 thiamine, 10 mg l-1 ascorbic acid, 4.5 g l - 1 0 x o i d agar, and without inositol (Vuylsteke and De Langhe, 1985). Cultures were maintained at 28-30 ° C on a 15 h light/ 9 h dark cycle. Shoot tips and meristems were induced to proliferate by adding 0.18 mg l-1 IAA and 4.5 mg l-1 BA to the basal medium. Proliferative growth in meristem cultures of plantains was characterized by the formation of a clump of small bulbous structures, which were covered with numerous minute meristems (Fig.
81
Fig. 1. (a) The inflorescenceof a normalin-vitropropagatedplantain plant ('Agbagba') and (b) the inflorescencevariant with a "French"plantain bunchtype. 2a) (Vuylsteke and De Langhe, 1985; Banerjee et al., 1986). This pattern of meristem proliferation, which has been shown to be adventitious (Banerjee et al., 1986), resulted in a high multiplication rate, ranging from 10 to 30 per subculture every 2 months. Plant regeneration was accomplished on the same basal medium supplemented with 0.19 mg l- 1 NAA and 0.23 mg l - 1 BA. Within 2-3 months, rooted shoots 1-4 cm high had formed. These were transferred to the basal medium with macronutrients at half concentration and devoid of plant growth regulators. After 5-6 weeks, plantlets (Fig. 2b) were ready for transplanting to soil in 30 cm X 30 cmX 30 cm black polyethylene bags. Two months later, they were ready for field planting (Fig. 2c). In 1985, 965 regenerated plantlets were transplanted to the field and screened for phenotypic variability by monitoring growth parameters (Swennen and De Langhe, 1985) and phenotypic characteristics (De Langhe, 1961; Tezenas du Montcel et al., 1983 ) under standard agronomic practices. RESULTS AND DISCUSSION Phenotypic variations observed in the plant crop are presented in Table 1 and described below.
82
Inflorescence characteristics.-A typical "French" plantain bunch type was produced in 25 plants (2.6%) regenerated from three different non-successive multiplication cycles. A French inflorescence is complete at maturity, i.e. many hands consisting of many rather small fruits followed by many persistent her-
83
Fig. 2. In vitro micropropagationof the plantain 'Agbagba'by shoot tip/meristem culture. (a) Proliferative growthof bulbil-likestructures coveredwith meristems. (b) Regeneratedplantlets ready for soil transplantation. {c) Plants 2 months after transfer to soil, ready for fieldplanting. maphrodite flowers and male flowers. Its male bud is large and persistent (Fig. lb). Bunches of these variants had an average weight of 15.8 kg with 55-109 fingers. Precisely this sort of variation was reported earlier, but with a higher frequency of 21-38% depending on the cultivar used (Ramcharan et al., 1985 ). Two plants (0.2%), both regenerated from the second multiplication cycle, had a False Horn bunch type but with an extremely low finger number per hand. Bunches weighed only 3.3 kg and had 7-11 fingers. The whole inflorescence was very similar to what is produced by the 'Monganga' cultivar (De Langhe, 1961 ), which has a maximum of four strongly negative geotropic fingers per hand and a leafy appendage at the bract tips. These two off-types, together with the 'Agbagba'-type, actually represent typical steps of the so-called "plantain inflorescence degeneration line" (Tezenas du Montcel et al., 1983). Reversion in the field from one step to another has been reported (Cardenosa, 1953; Tezenas du Montcel et al., 1983). We can confirm the occurrence in situ of this phenomenon through observations on 2306 conventional propagules of 'Agbagba', among which 16 plants (0.7%) produced a French plantain inflorescence. Consequently, this type of variation should not be considered as being generated specifically by in vitro culture, although the latter appears to increase its normal frequency considerably. Inflorescence type variation may be expected to be ubiquitous (but not necessarily at high frequencies) when in vitro propagation of the plantain 'Agbagba', an intermediate type along the "plantain inflorescence degeneration line", is performed. This was illustrated by the regular re-occurrence of both off-types
84
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85 in later plantings which were not included in the present study (unpublished results).
Foliage characteristics.-A single plant {0.1% ) showed leaf variegation of a random pattern of normal green and pale green patches. This could result from a plastome mutation involving loss of chlorophyll in some cells (Vaughn, 1983 ). Leaves with the right half of the lamina distorted and narrow (Fig. 3a) were observed on 7 plants {0.7% ) that were regenerated from three different nonsuccessive multiplication cycles. It is noteworthy that one such variant was obtained directly from a regenerated shoot tip, hence without going through a multiplication cycle. The leaves were more erect than those of normal plants and the lamina distortion increased with leaf number. These variants flowered 1-2 months later t h a n normal plants. Their bunches were true-to-type, i.e. of the False Horn type, but smaller, with an average weight of 4.3 kg and 14-20 fingers. Drooping leaf habit, associated with slower growth, was observed in 22 plants (2.3%) (Fig. 3b). Flowering occurred 3-12 months later than normal in the 14 plants harvested to date. Bunches were of the False Horn type, but with an
Fig. 3. Foliage off-types of the in-vitro propagated plantain 'Agbagba' (a) Left, leaf No. 25 of a normal plant; right, leaf No. 25 of the variant with distorted lamina. (b } Variant with drooping leaf habit, 1 month after flowering.
86 average weight of only 2.8 kg and 10-20 fingers. This phenotype suggests a change in ploidy, as drooping leaves and slow growth are typical vegetative features of plants with higher ploidy (Simmonds, 1948; Vakili, 1967). The fact that these plants were regenerated from the same multiplication cycle and that they were observed in the same row suggests that they originated from one single explant. Foliage variants with distorted laminae and with drooping leaves were agronomically inferior, since their aberrant phenotype resulted in later flowering and smaller bunches, i.e. lower productivity. Unlike the inflorescence type variants which occur naturally, foliage variants were novel and were generated during the in-vitro culture passage. Conclusions about the nature of this culture-induced variation remain speculative. Due to the extremely high sterility levels in the False Horn plantain (De Langhe, 1969), inheritance of variant traits by the sexual progeny of regenerants cannot be examined. As such, epigenetic and genetic variation cannot be distinguished (Chaleff, 1981 ). However, Scowcroft (1984) assumed that for asexually propagated crops, the transmission of off-type traits through at least two successive clonal generations is a reasonable criterion to determine a true genetic base. We have observed that the foliage malformations persisted in the first ratoon. Moreover, variant traits were consistent among sucker progeny, following a cycle of conventional clonal propagation. These two facts suggest that this variation is genetic in origin. CONCLUSION
Meristem tip cultures rank highest in phenotypic and genetic stability (Scowcroft, 1984 ), but considerable difference may exist between plants from axillary and adventitious meristems (Hussey, 1986 ). Jarret ( 1986 ) mentioned that both axillary and adventitious budding systems have been observed in Musa shoot tip cultures. Under conditions of intensive multiplication, the distinction between these two morphogenic pathways may become unclear (Hussey, 1986). However, Banerjee et al. (1986) demonstrated the adventitious origin of neoformed meristems in highly proliferative meristem cultures of Musa. These were characterized by the development of bulbil-like structures (Fig. 2a), a pattern similar to the in-vivo adventitious bud formation of true Horn plantain cultivars (Devos, 1985). A variation frequency of 6% expressed in five different phenotypes (two bunch and three foliage off-types) was encountered in our work. Ramcharan et al. (1985), also dealing with in-vitro propagated False Horn plantains, recorded a phenotypic variation ranging between 21 and 38%, all variants showing a French inflorescence. However, these in vitro mutation frequencies have no absolute value because unconscious selective multiplication of variants obviously enhances their incidence (e.g. the 22 plants with drooping leaf habit,
87
resulting in 14.6% of variation in the third multiplication cycle - Table 1). This is also illustrated by the large differences in variation frequency reported in Musa (AAA) 'Cavendish'. Frequencies varied between 2.4% (Hwang, 1986 ) and 25 % (Stover and Buddenhagen, 1986; Stover, 1987 ), with the single most common variant trait being dwarfism, accounting for 60-75% of the variability. This is not surprising in view of the various degrees of dwarf mutation which occur naturally in 'Cavendish' (Gross and Simmonds, 1954; Simmonds, 1966). Yet, it should be stressed that variants in plant stature, ubiquitous among 'Cavendish' plants, were not observed among 'Agbagba' plantains, indicating that in-vitro generated variability may be genotype-specific. Moreover, type and frequency of variation were not correlated to the number of sub-cultures (Table 1 ). Bearing in mind that the variants with foliage malformations can be detected during the nursery stage and can thus be rogued before field transplantation, in vitro micropropagation seems to be a reliable method for the rapid clonal propagation of the 'Agbagba' plantain.
REFERENCES Banerjee, N., Vuylsteke, D. and De Langhe, E., 1986. Meristem tip culture of Musa: histomorphological studies of shoot bud proliferation. In: L.A. Withers and P.G. Alderson (Editors), Plant Tissue Culture and its Agricultural Applications. Butterworths, London, pp. 139-147. Cardenosa, R., 1953. E1 genero Musa en Colombia. Notas Agron., 6: 373. Chaleff, R.S., 1981. Genetics of Higher Plants. Applications of Cell Culture. Developmental and Cell Biology Series, 9. Cambridge University Press, Cambridge, London, New York, 184 pp. Cronauer, S.S. and Krikorian, A.D., 1984. Multiplication of Musa from excised stem tips. Ann. Bot., 53: 321-328. De Guzman, E.V., Decena, A.C. and Ubalde, E.M., 1980. Plantlet regeneration from unirradiated and irradiated banana shoot tip tissues cultured in vitro. Philipp. Agric., 63: 140-146. De Langhe, E., 1961. La taxonomie du bananier plantain en Afrique ~quatoriale. J. Agric. Trop. Bot. Appl., T. VIII, No. 10-11: 417-449. De Langhe, E., 1969. Bananas (Musa spp.). In: F.P. Ferwarda and F. Wit (Editors), Outlines of Perennial Crop Breeding in the Tropics. H. Veenman en Zonen N.V., Wageningen, pp. 53-78. Devos, P., 1985. The location of lateral buds of banana (Musa sp.) clarified by the discovery of two new types of adventitious buds within the AAB plantain group. Fruits, 40: 471-474. Frossard, P., 1980. Apparition d'une nouvelle et grave maladie foliaire des bananiers et plantains au Gabon: la maladie des raies noires, Mycosphaerella fijiensis Morelet. Fruits, 35: 519-527. Gross, R.A. and Simmonds, N.W., 1954. Mutations in the Cavendish banana group. Trop. Agric. (Trinidad), 31: 131-132. Hussey, G., 1986. Problems and prospects in the in vitro propagation of herbaceous plants. In: L.A. Withers and P.G. Alderson (Editors), Plant Tissue Culture and its Agricultural Applications. Butterworths, London, pp. 69-84. Hwang, S.C., 1986. Variation in banana plants propagated through tissue culture, J. Chin. Soc. Hortic. Sci., 32:117-125 (in Chinese, with English summary). Jarret, R.L., 1986. Bananas and plantains. A report presented to the IBPGR. AGPG: IBPGR/86/ 105 (unpublished). International Board for Plant Genetic Resources, Rome, 51 pp.
88 Jarret, R.J., Rodriguez, W. and Fernandez, R., 1985. Evaluation, tissue culture propagation, and dissemination of 'Saba' and 'Pelipita' plantains in Costa Rica. Scientia Hortic., 25: 137-147. Krikorian, A.D. and Cronauer, S.S., 1984. Aseptic culture techniques for banana and plantain improvement. Econ. Bot., 38: 322-331. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Raemaekers, R., 1975. Black leaf streak like disease in Zambia. PANS, 21: 396-400. Ramcharan, C., Gonzalez, A. and Knausenberger, W.I., 1985. Performance of plantains produced from tissue culture plantlets in St. Croix, U.S. Virgin Islands. Proc. 3rd Meet. Int. Assoc. Research on Plantain and Banana, 27-31 May 1985, Abidjan, CSte d'Ivoire, pp. 36-39. Reuveni, O., Israeli, Y., Degani, H. and Eshdat, Y., 1985. Genetic variability in banana plants multiplied via in vitro techniques. Research Report AGPG: IBPGR/85/216 (unpublished). International Board for Plant Genetic Resources, Rome, 23 pp. Scowcroft, W.R., 1984. Genetic Variability in Tissue Culture: Impact on Germplasm Conservation and Utilization. IBPGR Report AGPG: IBPGR/84/152. International Board for Plant Genetic Resources, Rome, 41 pp. Simmonds, N.W., 1948. The effects of ploidy upon the leaf of Musa. Ann. Bot., 12: 441-453. Simmonds, N.W., 1966. Bananas. 2nd edn. Longman, London, New York, 512 pp. Stover, R.H., 1987. Somaclonal variation in Grand Naine and Saba bananas in the nursery and field. In: G.J. Persley and E.A. De Langhe (Editors), Banana and Plantain Breeding Strategies. Proc. Int. Workshop, 13-17 October 1986, Cairns, Australia, ACIAR Proc. No. 21, pp. 136-139. Stover, R.H. and Buddenhagen, I.W., 1986. Banana breeding: polyploidy, disease resistance and productivity. Fruits, 41:175-191. Swennen, R. and De Langhe, E., 1985. Growth parameters of yield of plantain (Musa cv.AAB ). Ann. Bot., 56: 197-204. Tezenas du Montcel, H., De Langhe, E. and Swennen, R., 1983. Essai de classification des bananiers plantains (AAB). Fruits, 38: 461-474. Vakili, N.G., 1967. The experimental formation of polyploidy and its effect in the genus Musa. Am. J. Bot., 34: 24-36. Vaughn, K.C., 1983. Chimeras and variegation: problems in propagation. Hort Science, 18: 845848. Vuylsteke, D. and De Langhe, E., 1985. Feasibility of in vitro propagation of bananas and plantains. Trop. Agric. (Trinidad), 62: 323-328. Wilson, G.F., 1983. Production de plantains: Perspectives pour am~liorer~a situation alimentaire sous les tropiques. Fruits, 38: 229-239. Wilson, G.F. and Buddenhagen, I., 1986. The black Sigatoka threat to plantain and banana in West Africa. IITA Res. Briefs, 7 (3):3.
79
Phenotypic Variation Among In-Vitro Propagated Plantain (Musa sp. cultivar 'AAB') DIRK VUYLSTEKE, RONY SWENNEN, GEORGE F. WILSON and EDMOND DE LANGHE x
International Institute of Tropical Agriculture, PMB 5320, Ibadan (Nigeria) 1International Network for the Improvement of Banana and Plantain, Avenue du Val de Mont[errand, BP 5035, 34032 Montpellier Cedex (France) (Accepted for publication 5 February 1988)
ABSTRACT Vuylsteke, D., Swennen, R., Wilson, G.F. and De Langhe, E., 1988. Phenotypic variation among in-vitro propagated plantain (Musa sp. cultivar 'AAb'). Scientia Hortic., 36: 79-88. In-vitro micropropagated plantain (Musa sp. cultivar 'AAB') plants were regenerated from meristem cultures and screened in the field for phenotypic variability. Observations of the plant crop and the successive ratoon indicated five different forms of phenotypic variation at a frequency of 6%. Inflorescence variants were of the "French" and 'Monganga' bunch type. Foliage variants were novel phenotypes and agronomically inferior. The latter off-types remained stable through conventional clonal propagation. Keywords: in-vitro micropropagation; Musa; plantain; shoot tip culture; somaclonal variation. Abbreviations: BA=n6-benzyladenine; IAA=indole-3-acetic acid; NAA=l-naphthaleneacetic acid.
INTRODUCTION
Plantain (Musa sp. 'AAB') is a major staple food in the humid lowlands of West and Central Africa. Increasing demand warrants increased production. However, expansion of production is limited by a shortage of suckers, the common planting material (Wilson, 1983). The transmission of harmful insects, nematodes and black Sigatoka disease (Raemaekers, 1975; Frossard, 1980) on field-grown suckers necessitates the production of clean planting material and impedes the international transfer of germplasm. Furthermore, the recent but rapid spread of the black Sigatoka disease on the African continent is emerging as a major threat to the cultivation of plantains because no resistant cultivars are known to exist (Wilson and Buddenhagen, 1986). These problems have 0304-4238/88/$03.50
© 1988 Elsevier Science Publishers B.V.
80 prompted an interest in the use of aseptic culture techniques, complementary to conventional approaches in the improvement ofMusa (Krikorian and Cronauer, 1984; Stover and Buddenhagen, 1986). Meristem culture has been suggested as a method for the rapid clonal propagation of selected {diseaseresistant) Musa cultivars and as a means of germplasm exchange (Jarret et al., 1985; Vuylsteke and De Langhe, 1985). The in-vitro micropropagation technique for cultivated Musa is well established (De Guzman et al., 1980; Cronauer and Krikorian, 1984; Jarret et al., 1985; Vuylsteke and De Langhe, 1985 ), but its use may be limited by the risk of somaclonal variation, a widespread occurrence in some in vitro cultures (Scowcroft, 1984 ). Off-types have been reported in dessert banana (Musa AAA group, 'Cavendish') (Reuveni et al., 1985; Hwang, 1986; Stover, 1987) and in plantain (Ramcharan et al., 1985). However, the report concerning plantain was a chance observation and not a systematic determination of the effect of the technique on phenotypic changes in the cultivar. Moreover, results on somaclonal variation in the AAA bananas might not be transposed to the genotypically different plantains. This paper presents findings of an attempt to determine the frequency and type of variants induced by meristem culture of plantain. MATERIALSAND METHODS The micropropagated plantain cultivar was 'Agbagba', the most popular in Nigeria. This cultivar produces bunches of 10-15 kg and 30-40 fingers within 12 months after planting. Its inflorescence is incomplete at maturity as there is no male bud. Hands consist of large fingers followed by a few hermaphrodite flowers (Fig. la). 'Agbagba' therefore belongs to the False Horn group of plantain (De Langhe, 1961; Tezenas du Montcel et al., 1983). To ascertain trueness-to-type, buds and suckers, used as source material, were collected from flowering plants. For culture initiation, cubical 1-2-cm :~ pieces containing the apices were excised from buds or suckers and surfacesterilized by washing with ethanol (95%) for 15 s, followed by soaking for 15 min in a 0.75% NaOC1 solution, to which one drop of Tween 80 was added per 50 ml, and then rinsed 3 times with sterile deionized water. Shoot tips of 1-2 mm in length, bearing 2-4 leaf primordia, were isolated aseptically and placed on a modified Murashige and Skoog medium (1962) with 0.4 g l-1 thiamine, 10 mg l-1 ascorbic acid, 4.5 g l - 1 0 x o i d agar, and without inositol (Vuylsteke and De Langhe, 1985). Cultures were maintained at 28-30 ° C on a 15 h light/ 9 h dark cycle. Shoot tips and meristems were induced to proliferate by adding 0.18 mg l-1 IAA and 4.5 mg l-1 BA to the basal medium. Proliferative growth in meristem cultures of plantains was characterized by the formation of a clump of small bulbous structures, which were covered with numerous minute meristems (Fig.
81
Fig. 1. (a) The inflorescenceof a normalin-vitropropagatedplantain plant ('Agbagba') and (b) the inflorescencevariant with a "French"plantain bunchtype. 2a) (Vuylsteke and De Langhe, 1985; Banerjee et al., 1986). This pattern of meristem proliferation, which has been shown to be adventitious (Banerjee et al., 1986), resulted in a high multiplication rate, ranging from 10 to 30 per subculture every 2 months. Plant regeneration was accomplished on the same basal medium supplemented with 0.19 mg l- 1 NAA and 0.23 mg l - 1 BA. Within 2-3 months, rooted shoots 1-4 cm high had formed. These were transferred to the basal medium with macronutrients at half concentration and devoid of plant growth regulators. After 5-6 weeks, plantlets (Fig. 2b) were ready for transplanting to soil in 30 cm X 30 cmX 30 cm black polyethylene bags. Two months later, they were ready for field planting (Fig. 2c). In 1985, 965 regenerated plantlets were transplanted to the field and screened for phenotypic variability by monitoring growth parameters (Swennen and De Langhe, 1985) and phenotypic characteristics (De Langhe, 1961; Tezenas du Montcel et al., 1983 ) under standard agronomic practices. RESULTS AND DISCUSSION Phenotypic variations observed in the plant crop are presented in Table 1 and described below.
82
Inflorescence characteristics.-A typical "French" plantain bunch type was produced in 25 plants (2.6%) regenerated from three different non-successive multiplication cycles. A French inflorescence is complete at maturity, i.e. many hands consisting of many rather small fruits followed by many persistent her-
83
Fig. 2. In vitro micropropagationof the plantain 'Agbagba'by shoot tip/meristem culture. (a) Proliferative growthof bulbil-likestructures coveredwith meristems. (b) Regeneratedplantlets ready for soil transplantation. {c) Plants 2 months after transfer to soil, ready for fieldplanting. maphrodite flowers and male flowers. Its male bud is large and persistent (Fig. lb). Bunches of these variants had an average weight of 15.8 kg with 55-109 fingers. Precisely this sort of variation was reported earlier, but with a higher frequency of 21-38% depending on the cultivar used (Ramcharan et al., 1985 ). Two plants (0.2%), both regenerated from the second multiplication cycle, had a False Horn bunch type but with an extremely low finger number per hand. Bunches weighed only 3.3 kg and had 7-11 fingers. The whole inflorescence was very similar to what is produced by the 'Monganga' cultivar (De Langhe, 1961 ), which has a maximum of four strongly negative geotropic fingers per hand and a leafy appendage at the bract tips. These two off-types, together with the 'Agbagba'-type, actually represent typical steps of the so-called "plantain inflorescence degeneration line" (Tezenas du Montcel et al., 1983). Reversion in the field from one step to another has been reported (Cardenosa, 1953; Tezenas du Montcel et al., 1983). We can confirm the occurrence in situ of this phenomenon through observations on 2306 conventional propagules of 'Agbagba', among which 16 plants (0.7%) produced a French plantain inflorescence. Consequently, this type of variation should not be considered as being generated specifically by in vitro culture, although the latter appears to increase its normal frequency considerably. Inflorescence type variation may be expected to be ubiquitous (but not necessarily at high frequencies) when in vitro propagation of the plantain 'Agbagba', an intermediate type along the "plantain inflorescence degeneration line", is performed. This was illustrated by the regular re-occurrence of both off-types
84
o
o
0
¢q
0~
0~
0
t,
¢q
"7.
~
-~ r.4
85 in later plantings which were not included in the present study (unpublished results).
Foliage characteristics.-A single plant {0.1% ) showed leaf variegation of a random pattern of normal green and pale green patches. This could result from a plastome mutation involving loss of chlorophyll in some cells (Vaughn, 1983 ). Leaves with the right half of the lamina distorted and narrow (Fig. 3a) were observed on 7 plants {0.7% ) that were regenerated from three different nonsuccessive multiplication cycles. It is noteworthy that one such variant was obtained directly from a regenerated shoot tip, hence without going through a multiplication cycle. The leaves were more erect than those of normal plants and the lamina distortion increased with leaf number. These variants flowered 1-2 months later t h a n normal plants. Their bunches were true-to-type, i.e. of the False Horn type, but smaller, with an average weight of 4.3 kg and 14-20 fingers. Drooping leaf habit, associated with slower growth, was observed in 22 plants (2.3%) (Fig. 3b). Flowering occurred 3-12 months later than normal in the 14 plants harvested to date. Bunches were of the False Horn type, but with an
Fig. 3. Foliage off-types of the in-vitro propagated plantain 'Agbagba' (a) Left, leaf No. 25 of a normal plant; right, leaf No. 25 of the variant with distorted lamina. (b } Variant with drooping leaf habit, 1 month after flowering.
86 average weight of only 2.8 kg and 10-20 fingers. This phenotype suggests a change in ploidy, as drooping leaves and slow growth are typical vegetative features of plants with higher ploidy (Simmonds, 1948; Vakili, 1967). The fact that these plants were regenerated from the same multiplication cycle and that they were observed in the same row suggests that they originated from one single explant. Foliage variants with distorted laminae and with drooping leaves were agronomically inferior, since their aberrant phenotype resulted in later flowering and smaller bunches, i.e. lower productivity. Unlike the inflorescence type variants which occur naturally, foliage variants were novel and were generated during the in-vitro culture passage. Conclusions about the nature of this culture-induced variation remain speculative. Due to the extremely high sterility levels in the False Horn plantain (De Langhe, 1969), inheritance of variant traits by the sexual progeny of regenerants cannot be examined. As such, epigenetic and genetic variation cannot be distinguished (Chaleff, 1981 ). However, Scowcroft (1984) assumed that for asexually propagated crops, the transmission of off-type traits through at least two successive clonal generations is a reasonable criterion to determine a true genetic base. We have observed that the foliage malformations persisted in the first ratoon. Moreover, variant traits were consistent among sucker progeny, following a cycle of conventional clonal propagation. These two facts suggest that this variation is genetic in origin. CONCLUSION
Meristem tip cultures rank highest in phenotypic and genetic stability (Scowcroft, 1984 ), but considerable difference may exist between plants from axillary and adventitious meristems (Hussey, 1986 ). Jarret ( 1986 ) mentioned that both axillary and adventitious budding systems have been observed in Musa shoot tip cultures. Under conditions of intensive multiplication, the distinction between these two morphogenic pathways may become unclear (Hussey, 1986). However, Banerjee et al. (1986) demonstrated the adventitious origin of neoformed meristems in highly proliferative meristem cultures of Musa. These were characterized by the development of bulbil-like structures (Fig. 2a), a pattern similar to the in-vivo adventitious bud formation of true Horn plantain cultivars (Devos, 1985). A variation frequency of 6% expressed in five different phenotypes (two bunch and three foliage off-types) was encountered in our work. Ramcharan et al. (1985), also dealing with in-vitro propagated False Horn plantains, recorded a phenotypic variation ranging between 21 and 38%, all variants showing a French inflorescence. However, these in vitro mutation frequencies have no absolute value because unconscious selective multiplication of variants obviously enhances their incidence (e.g. the 22 plants with drooping leaf habit,
87
resulting in 14.6% of variation in the third multiplication cycle - Table 1). This is also illustrated by the large differences in variation frequency reported in Musa (AAA) 'Cavendish'. Frequencies varied between 2.4% (Hwang, 1986 ) and 25 % (Stover and Buddenhagen, 1986; Stover, 1987 ), with the single most common variant trait being dwarfism, accounting for 60-75% of the variability. This is not surprising in view of the various degrees of dwarf mutation which occur naturally in 'Cavendish' (Gross and Simmonds, 1954; Simmonds, 1966). Yet, it should be stressed that variants in plant stature, ubiquitous among 'Cavendish' plants, were not observed among 'Agbagba' plantains, indicating that in-vitro generated variability may be genotype-specific. Moreover, type and frequency of variation were not correlated to the number of sub-cultures (Table 1 ). Bearing in mind that the variants with foliage malformations can be detected during the nursery stage and can thus be rogued before field transplantation, in vitro micropropagation seems to be a reliable method for the rapid clonal propagation of the 'Agbagba' plantain.
REFERENCES Banerjee, N., Vuylsteke, D. and De Langhe, E., 1986. Meristem tip culture of Musa: histomorphological studies of shoot bud proliferation. In: L.A. Withers and P.G. Alderson (Editors), Plant Tissue Culture and its Agricultural Applications. Butterworths, London, pp. 139-147. Cardenosa, R., 1953. E1 genero Musa en Colombia. Notas Agron., 6: 373. Chaleff, R.S., 1981. Genetics of Higher Plants. Applications of Cell Culture. Developmental and Cell Biology Series, 9. Cambridge University Press, Cambridge, London, New York, 184 pp. Cronauer, S.S. and Krikorian, A.D., 1984. Multiplication of Musa from excised stem tips. Ann. Bot., 53: 321-328. De Guzman, E.V., Decena, A.C. and Ubalde, E.M., 1980. Plantlet regeneration from unirradiated and irradiated banana shoot tip tissues cultured in vitro. Philipp. Agric., 63: 140-146. De Langhe, E., 1961. La taxonomie du bananier plantain en Afrique ~quatoriale. J. Agric. Trop. Bot. Appl., T. VIII, No. 10-11: 417-449. De Langhe, E., 1969. Bananas (Musa spp.). In: F.P. Ferwarda and F. Wit (Editors), Outlines of Perennial Crop Breeding in the Tropics. H. Veenman en Zonen N.V., Wageningen, pp. 53-78. Devos, P., 1985. The location of lateral buds of banana (Musa sp.) clarified by the discovery of two new types of adventitious buds within the AAB plantain group. Fruits, 40: 471-474. Frossard, P., 1980. Apparition d'une nouvelle et grave maladie foliaire des bananiers et plantains au Gabon: la maladie des raies noires, Mycosphaerella fijiensis Morelet. Fruits, 35: 519-527. Gross, R.A. and Simmonds, N.W., 1954. Mutations in the Cavendish banana group. Trop. Agric. (Trinidad), 31: 131-132. Hussey, G., 1986. Problems and prospects in the in vitro propagation of herbaceous plants. In: L.A. Withers and P.G. Alderson (Editors), Plant Tissue Culture and its Agricultural Applications. Butterworths, London, pp. 69-84. Hwang, S.C., 1986. Variation in banana plants propagated through tissue culture, J. Chin. Soc. Hortic. Sci., 32:117-125 (in Chinese, with English summary). Jarret, R.L., 1986. Bananas and plantains. A report presented to the IBPGR. AGPG: IBPGR/86/ 105 (unpublished). International Board for Plant Genetic Resources, Rome, 51 pp.
88 Jarret, R.J., Rodriguez, W. and Fernandez, R., 1985. Evaluation, tissue culture propagation, and dissemination of 'Saba' and 'Pelipita' plantains in Costa Rica. Scientia Hortic., 25: 137-147. Krikorian, A.D. and Cronauer, S.S., 1984. Aseptic culture techniques for banana and plantain improvement. Econ. Bot., 38: 322-331. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Raemaekers, R., 1975. Black leaf streak like disease in Zambia. PANS, 21: 396-400. Ramcharan, C., Gonzalez, A. and Knausenberger, W.I., 1985. Performance of plantains produced from tissue culture plantlets in St. Croix, U.S. Virgin Islands. Proc. 3rd Meet. Int. Assoc. Research on Plantain and Banana, 27-31 May 1985, Abidjan, CSte d'Ivoire, pp. 36-39. Reuveni, O., Israeli, Y., Degani, H. and Eshdat, Y., 1985. Genetic variability in banana plants multiplied via in vitro techniques. Research Report AGPG: IBPGR/85/216 (unpublished). International Board for Plant Genetic Resources, Rome, 23 pp. Scowcroft, W.R., 1984. Genetic Variability in Tissue Culture: Impact on Germplasm Conservation and Utilization. IBPGR Report AGPG: IBPGR/84/152. International Board for Plant Genetic Resources, Rome, 41 pp. Simmonds, N.W., 1948. The effects of ploidy upon the leaf of Musa. Ann. Bot., 12: 441-453. Simmonds, N.W., 1966. Bananas. 2nd edn. Longman, London, New York, 512 pp. Stover, R.H., 1987. Somaclonal variation in Grand Naine and Saba bananas in the nursery and field. In: G.J. Persley and E.A. De Langhe (Editors), Banana and Plantain Breeding Strategies. Proc. Int. Workshop, 13-17 October 1986, Cairns, Australia, ACIAR Proc. No. 21, pp. 136-139. Stover, R.H. and Buddenhagen, I.W., 1986. Banana breeding: polyploidy, disease resistance and productivity. Fruits, 41:175-191. Swennen, R. and De Langhe, E., 1985. Growth parameters of yield of plantain (Musa cv.AAB ). Ann. Bot., 56: 197-204. Tezenas du Montcel, H., De Langhe, E. and Swennen, R., 1983. Essai de classification des bananiers plantains (AAB). Fruits, 38: 461-474. Vakili, N.G., 1967. The experimental formation of polyploidy and its effect in the genus Musa. Am. J. Bot., 34: 24-36. Vaughn, K.C., 1983. Chimeras and variegation: problems in propagation. Hort Science, 18: 845848. Vuylsteke, D. and De Langhe, E., 1985. Feasibility of in vitro propagation of bananas and plantains. Trop. Agric. (Trinidad), 62: 323-328. Wilson, G.F., 1983. Production de plantains: Perspectives pour am~liorer~a situation alimentaire sous les tropiques. Fruits, 38: 229-239. Wilson, G.F. and Buddenhagen, I., 1986. The black Sigatoka threat to plantain and banana in West Africa. IITA Res. Briefs, 7 (3):3.