PiantCeli Reports
Plant Cell Reports (1996) 15:445-448
9 Springer-Verlag1996
Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds R. Afza, M. van Duren, and R. Morpurgo FAO/IAEA Agriculture and Biotechnology Laboratory, Plant Breeding Unit, A-2444 Seibersdorf, Austria Received 19 December 1994/Revised version received 20 June 1995 - Communicated by M. R. Davey
Abstract
In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with b e n z y l a m i n o p u r i n e (BAP) or 6 ~/-?dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on halfstrength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed. K e y words: Ensete ventricosum mutation breeding, regeneration, somatic embryo genesis Abbreviation: IAA = Indole-acetic acid, IBA =
Indole-butyric acid, BAP = Benzylaminopurine, 2iP = 6 ?-7-dimethylallylamino purine
Introduction Ensete is a herbaceous monocot belonging to the Musaceae family and for long time it was considered as aMusa. According to Cheesman (1947), all Ensete species, including E. ventricosum, studied so far are diploid (2n = 18). The genus Ensete contains eight different
species, but only two are of economic importance: E. ventricosum and E. edule. In Correspondence to: R. Afza
southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple-food crop supporting the diet of a quarter of the Ethiopian population. Furthermore, the leaves are used to produce fibre for rope making and used as feed for livestock (Benuzeh and Feleke, 1966). Ensete is part of a sustainable agricultural system d e v e l o p e d over many centuries. Its productivity in terms of calories/year/unit of land is comparable with that of Irish potatoes even without the application of fertilisers and pesticides. Thus, the potential of this crop is enormous. A major draw-back is its susceptibility to bacterial wilt caused by Xanthomonas musacearum. In the field germplasm bank conserved by the Institute of Agricultural Research OAR) in the substation of Areka Research Center, Ethiopia, preliminary investigations did not detect genetic variability for this trait. Thus, there is an urgent need for a breeding programme for resistance to bacterial wilt and fungal pathogens that attack leaves and pseudostem. An in vitro approach, combined with mutation breeding, was proposed by Novak (1991) in Musa to obtain resistant varieties. A major constraint in mutation breeding is the formation of chimeras, i.e. the development of an organ, tissue, or an individual with a heterogeneous genetic constitution (see TilneyBassett 1987 ). However, in mutation breeding, the formation of chimeras is not avoidable due to the fact that a mutation is always a single cell event. Various strategies have been developed in order to clean favourable mutation and obtain a stable genetic situation. One strategy, proposed by Roest et al. (1981)
446 is to induce de novo shoots through bud formation that have a single cell origin. Induction of adventitious buds is a normal event in vivo but time and space can often be limiting factors. Adventitious buds could be easily i n d u c e d in tissue culture by manipulation of the plant growth regulator balance; however, the success of an m vitro approach depends on the availability of an established tissue culture protocol, and, in turn, on the development of a well-balanced culture medium providing the explant with the optimal amount of micro and macro elements, vitamins and plant growth regulators. The present communication describes the regeneration from callus derived from corm tissues of E. ventricosum of plants and their establishment in vivo.
Material and methods Plants of E. ventricosum were obtained from Ethiopia. For micropropagation, a protocol used for banana was followed (Cronauer and Krikorian 1984) with minor modification in the plant growth regulator concentration: leaves were removed and a portion of corm with some of the youngest leaves surrounding the meristematic dome was surface sterilised with 70% ethanol for a few seconds, then for 30 min in 50% commercial bleach (5.25% active sodium hypochloride) containing some drops of Tween 20. The explants were washed three times with sterile distilled water and transferred onto a medium containing MS (Murashige and Skoog 1962) basal salts, supplemented with 12 laM B A P , 3 laM IAA, 1 mg 1-1 thiamine-HC1, 40 mg 1-1 cysteine, 30 g 1-1 sucrose and 7 g 1-1 agar. For somatic embryogenesis and adventitious regeneration a double step methodology was developed. Corm explants were cultured on a solid MS medium modified by the addition of 40 jaM BAP, 50 mg/1 cysteine, 2 g 1-1 activated charcoal, 30 g 1-1 sucrose, g 1-1 Gelrite and 5 g 1-1 agar. After 30 days, the callus-forming explants were transferred to fresh media containing different levels of either BAP or 2iP (5, 10, 20 ~tM). Regenerants were transferred to a rooting media as described elsewhere (Novak 1991). All media were autoclaved at 120~ for 20 min. Regenerants were hardened by washing off the residual agar from the roots, planting them in Jiffy-pots under constant humidity for two weeks. Histological analysis was performed using a protocol adapted from Johansen (1940). Explants were fixed in 1% CrO3, 6% acetic
acid, and 5% formaldehyde for 48 h, then washed for 4 h under running tap water and dehydrated over a series of water : ethanol : toluene for three days. Samples were saturated with paraffin oil with increasing density from highly liquid to viscous, and embedded in Paraplast (Monoject Scientifica, USA). Sections, 5-10 Jam, were stained with 1.5% basic fucsin and 1% indigocarmine in 0.33% picric acid, and mounted with Depex (Serva). The regenerated plants were analysed for phenotypical variation according to the following parameters: leaf blades shape, petiole and pseudostem color, i.e anthocyanin pigments and phyllotaxis. Results Our attempt to micro-propagate E. ventrieosum through axillary buds was only partially successful. In fact, when meristematic tips were transferred to the micropropagation medium only a few plants developed while extensive blackening was noted on the cut surface, creating an unsuitable environment for plant development. When corm tissue was plated and incubated at 3000 lux and 27 ~ onto a MS medium containing 40 ~tM BAP, caIlus induction was achieved after six weeks. Small green spots appeared on the callus (Fig. 1). The callus was transferred to the same basal medium containing either BAP or 2iP (5, 10, 20 ~tM). In all media tested, regeneration of plantlets was observed. However, the lower concentration of BAP or 2iP gave rise to a higher percentage (around 90%) of regenerating explants. No significant differences were recorded between the two cytokinins at low concentration (p = 0.05). However, BAP was more effective in promoting regeneration than 2iP. This difference in promoting regeneration was more evident at high concentration (Table 1). Regenerants were obtained either via direct regeneration, i.e. adventitious buds, as shown by histological section analysis (Fig 2 a,b), or through somatic embryogenesis (Fig. 3). Both somatic embryos and adventitious buds developed normally on the induction media. However, in the last case, i.e. adventitious buds, the use of a rooting medium containing 5 ~tM of IBA was necessary to obtain a well developed root system. The regenerated plants were maintained in a greenhouse for one year, and screened for variation induced by the in vitro passage. Two plants showed an altered leaf shape and eolour
447
Fig. 1 Callus growth on 40 ~tM BAP enriched medium. Arrows point to green spots from which regenerants originate.
Fig. 2 Longitudinal section through an Ensete adventitious bud.
Fig. 3 Somatic embryos of Ensete isolated at diffen developmental stage (40x)
448 but, after a few weeks in the greenhouse, they reverted to a normal phenotype. TABLE 1: Effects of different BAP and 2iP concentration on E. ventricosum shoot regeneration (numbers in column followed by the same number are not statistically significant accordin to the Duncan test at p=0.05)
gM
5 10 20 5 10 20
Number of explants BAP BAP BAP 2iP 2iP 2iP
35 41 40 48 34 29
Type of callus
Frequency of regeneration
Number of regenerants
Potted plants
Frequency of survival (%)
94 a 75 b 70b 88 a 50 c 35 d
132 a 80 b 86b 142 a 25 e 6d
79 31 31 76 15 0
60a 39b 36b 53a 38 b 0c
(%) 3 4 2 1 1
Type of callus: 1= browning, 2 = poor callus growth, 3 = moderate callus growth, 4 = good callus growth Discussion An in vitro system, as applied to mutation breeding, should respond to some preliminary requisites, i.e. to achieve a faster propagation rate, to be more efficient than in vivo systems in solving the chimera problem and to be free of somaelonal variation.The system developed in our laboratory seems to fulfil all these required criteria although a real micro-propagation system, i.e. induction of axillary bud formation has not been fully developed due to extensive blackening and tissue mortality. Ensete ventricosum is a vegetatively propagated plant. A high rate of propagation is achieved by cutting the pseudostem and removal of the meristem. Profuse callus formation is observed on the cut surface and 40-100 new shoots/plant develop from it. In our system, the propagation rate is comparable to that reported in the literature (Bezuneh and Feleke, 1966). Furthermore, regeneration of plants is achieved in limited space, while in the field Ensete ventricosum requires several square meters/plant as weel as complex labour intensive agronomic practices, and is dependent from the age of the plant, i.e only plants older than 4 years are used for the production of new planting material. The possibility of regenerating plants in a laboratory and to obtain more generation on a year round basis jointly with the formation of adventitious buds and somatic embryos can speed up
isolation of stable mutants. Our observation, at phenotypical level could not detect any influence of the in vitro passage on the stability of the genotype. However it is well known that the stability of the cultured banana, a crop closely related to the genus Ensete is genotype dependent (Smith 1988) and more studies should be carried out to clarify this aspect. References Bezuneh T Feleke A (1966) Soil and Crop Science Society of Florida 27:133-141 Cheesman EE (1947) Kew Bullettin 2:97-106 Cronauer SS Krikorian AD (1984) Ann. Bot. 53:321-328 Johansen DA (1940) Plant Microtechnique, McGraw-Hill, New York, Murashige T Skoog F (1962) Physiologia Plantarum 15:473-497 Novak FJ (1991) In: Plant Mutation Breeding for Crops Improvement. Vol. 2. IAEA, Vienna, pp 327-342 Roest S van Berkel AAE Bokelmann GS Broertij es C (1981) Euphytica 90:381-388 Smith M.K (1988) Fruits 43:219-223 Tilney-Basset RAE (1987) In: Improving Vegetatively Propagated Crop. Jabbot A.J.and Atkin RK Eds, Academic Press, London, pp.271-284
Plant Cell Reports (1996) 15:445-448
9 Springer-Verlag1996
Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds R. Afza, M. van Duren, and R. Morpurgo FAO/IAEA Agriculture and Biotechnology Laboratory, Plant Breeding Unit, A-2444 Seibersdorf, Austria Received 19 December 1994/Revised version received 20 June 1995 - Communicated by M. R. Davey
Abstract
In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with b e n z y l a m i n o p u r i n e (BAP) or 6 ~/-?dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on halfstrength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed. K e y words: Ensete ventricosum mutation breeding, regeneration, somatic embryo genesis Abbreviation: IAA = Indole-acetic acid, IBA =
Indole-butyric acid, BAP = Benzylaminopurine, 2iP = 6 ?-7-dimethylallylamino purine
Introduction Ensete is a herbaceous monocot belonging to the Musaceae family and for long time it was considered as aMusa. According to Cheesman (1947), all Ensete species, including E. ventricosum, studied so far are diploid (2n = 18). The genus Ensete contains eight different
species, but only two are of economic importance: E. ventricosum and E. edule. In Correspondence to: R. Afza
southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple-food crop supporting the diet of a quarter of the Ethiopian population. Furthermore, the leaves are used to produce fibre for rope making and used as feed for livestock (Benuzeh and Feleke, 1966). Ensete is part of a sustainable agricultural system d e v e l o p e d over many centuries. Its productivity in terms of calories/year/unit of land is comparable with that of Irish potatoes even without the application of fertilisers and pesticides. Thus, the potential of this crop is enormous. A major draw-back is its susceptibility to bacterial wilt caused by Xanthomonas musacearum. In the field germplasm bank conserved by the Institute of Agricultural Research OAR) in the substation of Areka Research Center, Ethiopia, preliminary investigations did not detect genetic variability for this trait. Thus, there is an urgent need for a breeding programme for resistance to bacterial wilt and fungal pathogens that attack leaves and pseudostem. An in vitro approach, combined with mutation breeding, was proposed by Novak (1991) in Musa to obtain resistant varieties. A major constraint in mutation breeding is the formation of chimeras, i.e. the development of an organ, tissue, or an individual with a heterogeneous genetic constitution (see TilneyBassett 1987 ). However, in mutation breeding, the formation of chimeras is not avoidable due to the fact that a mutation is always a single cell event. Various strategies have been developed in order to clean favourable mutation and obtain a stable genetic situation. One strategy, proposed by Roest et al. (1981)
446 is to induce de novo shoots through bud formation that have a single cell origin. Induction of adventitious buds is a normal event in vivo but time and space can often be limiting factors. Adventitious buds could be easily i n d u c e d in tissue culture by manipulation of the plant growth regulator balance; however, the success of an m vitro approach depends on the availability of an established tissue culture protocol, and, in turn, on the development of a well-balanced culture medium providing the explant with the optimal amount of micro and macro elements, vitamins and plant growth regulators. The present communication describes the regeneration from callus derived from corm tissues of E. ventricosum of plants and their establishment in vivo.
Material and methods Plants of E. ventricosum were obtained from Ethiopia. For micropropagation, a protocol used for banana was followed (Cronauer and Krikorian 1984) with minor modification in the plant growth regulator concentration: leaves were removed and a portion of corm with some of the youngest leaves surrounding the meristematic dome was surface sterilised with 70% ethanol for a few seconds, then for 30 min in 50% commercial bleach (5.25% active sodium hypochloride) containing some drops of Tween 20. The explants were washed three times with sterile distilled water and transferred onto a medium containing MS (Murashige and Skoog 1962) basal salts, supplemented with 12 laM B A P , 3 laM IAA, 1 mg 1-1 thiamine-HC1, 40 mg 1-1 cysteine, 30 g 1-1 sucrose and 7 g 1-1 agar. For somatic embryogenesis and adventitious regeneration a double step methodology was developed. Corm explants were cultured on a solid MS medium modified by the addition of 40 jaM BAP, 50 mg/1 cysteine, 2 g 1-1 activated charcoal, 30 g 1-1 sucrose, g 1-1 Gelrite and 5 g 1-1 agar. After 30 days, the callus-forming explants were transferred to fresh media containing different levels of either BAP or 2iP (5, 10, 20 ~tM). Regenerants were transferred to a rooting media as described elsewhere (Novak 1991). All media were autoclaved at 120~ for 20 min. Regenerants were hardened by washing off the residual agar from the roots, planting them in Jiffy-pots under constant humidity for two weeks. Histological analysis was performed using a protocol adapted from Johansen (1940). Explants were fixed in 1% CrO3, 6% acetic
acid, and 5% formaldehyde for 48 h, then washed for 4 h under running tap water and dehydrated over a series of water : ethanol : toluene for three days. Samples were saturated with paraffin oil with increasing density from highly liquid to viscous, and embedded in Paraplast (Monoject Scientifica, USA). Sections, 5-10 Jam, were stained with 1.5% basic fucsin and 1% indigocarmine in 0.33% picric acid, and mounted with Depex (Serva). The regenerated plants were analysed for phenotypical variation according to the following parameters: leaf blades shape, petiole and pseudostem color, i.e anthocyanin pigments and phyllotaxis. Results Our attempt to micro-propagate E. ventrieosum through axillary buds was only partially successful. In fact, when meristematic tips were transferred to the micropropagation medium only a few plants developed while extensive blackening was noted on the cut surface, creating an unsuitable environment for plant development. When corm tissue was plated and incubated at 3000 lux and 27 ~ onto a MS medium containing 40 ~tM BAP, caIlus induction was achieved after six weeks. Small green spots appeared on the callus (Fig. 1). The callus was transferred to the same basal medium containing either BAP or 2iP (5, 10, 20 ~tM). In all media tested, regeneration of plantlets was observed. However, the lower concentration of BAP or 2iP gave rise to a higher percentage (around 90%) of regenerating explants. No significant differences were recorded between the two cytokinins at low concentration (p = 0.05). However, BAP was more effective in promoting regeneration than 2iP. This difference in promoting regeneration was more evident at high concentration (Table 1). Regenerants were obtained either via direct regeneration, i.e. adventitious buds, as shown by histological section analysis (Fig 2 a,b), or through somatic embryogenesis (Fig. 3). Both somatic embryos and adventitious buds developed normally on the induction media. However, in the last case, i.e. adventitious buds, the use of a rooting medium containing 5 ~tM of IBA was necessary to obtain a well developed root system. The regenerated plants were maintained in a greenhouse for one year, and screened for variation induced by the in vitro passage. Two plants showed an altered leaf shape and eolour
447
Fig. 1 Callus growth on 40 ~tM BAP enriched medium. Arrows point to green spots from which regenerants originate.
Fig. 2 Longitudinal section through an Ensete adventitious bud.
Fig. 3 Somatic embryos of Ensete isolated at diffen developmental stage (40x)
448 but, after a few weeks in the greenhouse, they reverted to a normal phenotype. TABLE 1: Effects of different BAP and 2iP concentration on E. ventricosum shoot regeneration (numbers in column followed by the same number are not statistically significant accordin to the Duncan test at p=0.05)
gM
5 10 20 5 10 20
Number of explants BAP BAP BAP 2iP 2iP 2iP
35 41 40 48 34 29
Type of callus
Frequency of regeneration
Number of regenerants
Potted plants
Frequency of survival (%)
94 a 75 b 70b 88 a 50 c 35 d
132 a 80 b 86b 142 a 25 e 6d
79 31 31 76 15 0
60a 39b 36b 53a 38 b 0c
(%) 3 4 2 1 1
Type of callus: 1= browning, 2 = poor callus growth, 3 = moderate callus growth, 4 = good callus growth Discussion An in vitro system, as applied to mutation breeding, should respond to some preliminary requisites, i.e. to achieve a faster propagation rate, to be more efficient than in vivo systems in solving the chimera problem and to be free of somaelonal variation.The system developed in our laboratory seems to fulfil all these required criteria although a real micro-propagation system, i.e. induction of axillary bud formation has not been fully developed due to extensive blackening and tissue mortality. Ensete ventricosum is a vegetatively propagated plant. A high rate of propagation is achieved by cutting the pseudostem and removal of the meristem. Profuse callus formation is observed on the cut surface and 40-100 new shoots/plant develop from it. In our system, the propagation rate is comparable to that reported in the literature (Bezuneh and Feleke, 1966). Furthermore, regeneration of plants is achieved in limited space, while in the field Ensete ventricosum requires several square meters/plant as weel as complex labour intensive agronomic practices, and is dependent from the age of the plant, i.e only plants older than 4 years are used for the production of new planting material. The possibility of regenerating plants in a laboratory and to obtain more generation on a year round basis jointly with the formation of adventitious buds and somatic embryos can speed up
isolation of stable mutants. Our observation, at phenotypical level could not detect any influence of the in vitro passage on the stability of the genotype. However it is well known that the stability of the cultured banana, a crop closely related to the genus Ensete is genotype dependent (Smith 1988) and more studies should be carried out to clarify this aspect. References Bezuneh T Feleke A (1966) Soil and Crop Science Society of Florida 27:133-141 Cheesman EE (1947) Kew Bullettin 2:97-106 Cronauer SS Krikorian AD (1984) Ann. Bot. 53:321-328 Johansen DA (1940) Plant Microtechnique, McGraw-Hill, New York, Murashige T Skoog F (1962) Physiologia Plantarum 15:473-497 Novak FJ (1991) In: Plant Mutation Breeding for Crops Improvement. Vol. 2. IAEA, Vienna, pp 327-342 Roest S van Berkel AAE Bokelmann GS Broertij es C (1981) Euphytica 90:381-388 Smith M.K (1988) Fruits 43:219-223 Tilney-Basset RAE (1987) In: Improving Vegetatively Propagated Crop. Jabbot A.J.and Atkin RK Eds, Academic Press, London, pp.271-284
