Propagation of Ensete in vitro: a review

Copyright © NISC Pty Ltd

South African Journal of Botany 2004, 70(4): 497–501 Printed in South Africa — All rights reserved

SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299

Minireview

Propagation of Ensete in vitro: a review M Diro and J van Staden* Research Centre for Plant Growth and Development, School of Botany and Zoology, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa * Corresponding author, e-mail: [email protected] Received 12 March 2004, accepted in revised form 25 May 2004

The in vitro propagation of Ensete, an important food crop in parts of east Africa, has been investigated in a number of previous studies. These studies have reported on the culture of Ensete species using a variety of methods, including zygotic embryo germination, shoot tip culture, adventitious shoot regeneration and multiple shoot development, and somatic embryo cultures. Various problems, such as extensive blackening of

explants, necrosis and the formation of unwanted callus have been observed. E. ventricosum, in particular, is a very valuable food crop in Ethiopia and micropropagation techniques are important for clonal multiplication and germplasm conservation. This review brings together various aspects related to the in vitro micropropagation of Ensete.

Introduction The genera Ensete and Musa, belonging to the Musaceae, are monocotyledons. The genus Ensete consists of six species, E. gilletii, E. homblei, E. perrieri and E. ventricosum with an African distribution, and E. glaucum and E. superbum with an Asian distribution (Simmonds 1960). E. ventricosum is widespread in the wild in Africa from Cameroon to east Africa and is cultivated as a staple or co-staple food crop in Ethiopia (Bezuneh and Feleke 1966). In Ethiopia, E. ventricosum is known as ‘enset’ in the Amharic language. The use of tissue culture techniques to propagate plants in vitro is an extension of conventional propagation methods. Some aspects of in vitro propagation of E. ventricosum and E. superbum were previously investigated and can be used for the genetic improvement and conservation of Ensete. This review examines the available information on the propagation of Ensete in vitro. Importance of Ensete Enset (E. ventricosum) is a source of food, high in carbohydrates, which is grown and consumed by about 15 million Ethiopians (Spring 1996). A mixture of scraped leaf sheath and pulverised corms, after fermentation in a pit, results in the production of ‘kocho’, the main product of enset which is used to make a pancake-like food. ‘Bulla’ is another important food product from enset. It is produced from solidified liquid after dehydrating a fresh mixture of scraped leaf sheath and pulverised corms. ‘Bulla’ is consumed mainly as

porridge, in gruel and in a crumbled form. Corms of some clones are cooked and consumed in a similar way to roots and tubers of other food crops. Parts of some clones are also used in traditional medicine. A novel phenylphenalenone was detected and isolated from E. ventricosum (Holscher and Schneider 1998). A starch that can be used for paper, textile and adhesive industries is produced from enset (ESTC 2003). There is also a potential to use enset starch in binding and disintegrating of compressed tablets (Gebre and Nikolayev 1993). Fibre, a by-product of enset in food processing, is a versatile raw material for household usage and as a raw material for local fibre factories. Enset is a valuable security crop as it tolerates transient drought and can be harvested throughout the year at different growth stages to buffer seasonal food deficits. A large leaf area, closure of canopy and litter from enset contribute to the maintenance of soil fertility under enset cultivation. Therefore, enset contributes to sustainable agriculture and food security in Africa. Potential use of tissue culture of Ensete Enset is a subsistence crop that can easily be propagated using conventional vegetative propagation methods. The importance of micropropagation would therefore be to propagate new genotypes and/or specific clones tolerant to pathogens, for example, Xanthomonas campestris cv. musacearum, that causes enset wilt, a destructive disease

498

(Ashagari 1985, Quimio and Tessera 1996). Enset root mealy bug, the root lesion nematode Pratylenchus goodeyi and the root knot nematode Meloidogyne sp., mosaic and chlorotic streaks, both of viral nature, are widely distributed in association with enset (Quimio and Tessera 1996). Tissue culture techniques can be used to produce pathogen-free plant material. A procedure for in vitro screening of Musa spp. for resistance to burrowing nematode (Radopholus similis) was developed (Elsen et al. 2002). Thus, in vitro techniques can also be used to screen enset genotypes against nematodes. Shoot tip culture can supplement a field genebank in conserving enset genetic resources using in vitro slow-growth conditions and/or cryopreservation (Negash et al. 2001). Genetic variability can be created using callus culture and in vitro mutation and important variants can be selected. Somatic hybridisation and recombinant DNA techniques can potentially be used in enset improvement. However, since enset is fertile and produces viable embryos, the use of existing variability through conventional breeding and selection would also be practically possible. In the breeding process the poor germination of intact seeds of enset can be overcome by germination of excised embryos in vitro. Therefore, tissue culture techniques can contribute to clonal multiplication, germplasm conservation and in developing enset genotypes with desirable traits. Propagation of Ensete in vitro Plant propagation in vitro can be achieved by zygotic embryo culture, organogenesis, or somatic embryogenesis. Organogenesis involves regeneration of organs from existing meristems or from de novo meristems. Various combinations of nutrients, plant growth regulators and environmental factors for different species or genotypes may stimulate the micropropagation of plants. Manipulation of these factors may enable plant breeders and propagators to control plant cell morphogenesis and to develop reliable cell to plant regeneration systems. Information on the type of explant, medium composition and culture environment used in the propagation of Ensete in vitro is summarised in Table 1. Different pathways for the regeneration of whole plants from excised enset plant parts are discussed. From the literature cited with respect to the micropropagation of Ensete it is quite clear that the voluminous literature on the in vitro culture of its fellow genus Musa has contributed positively to our present understanding of the biotechnology surrounding Ensete (Israeli et al. 1995, Zeweldu 1997, Diro Chimsa 2003) and will no doubt continue to do so in future. Zygotic embryo culture In this technique, mature or immature seed embryos are cultured in vitro to produce seedlings. Embryo cultures have been used to explore the nutritional and physical requirements for embryonic development to bypass seed dormancy which may shorten the breeding cycle, to test seed viability, to provide microcloning source material and to rescue immature hybrid embryos from incomparable crosses (Hu and Wang 1986, Pierik 1987). Mature embryos, in general,

Diro and Van Staden

require only inorganic salts supplemented with sucrose, whereas immature embryos have an additional requirement for vitamins, amino acids, growth regulators and sometimes endosperm extract. Germination of intact seeds of the Musaceae (Ensete and Musa) is very poor. In vitro germination of excised zygotic embryos was used as an alternative pathway to regenerate plants from seeds of Ensete (Bezuneh 1980, Negash et al. 2000, Diro and Van Staden 2003) and Musa (Cox et al. 1960, Afele and De Langhe 1991, Asif et al. 2001). Bezuneh (1980) regenerated plants from mature embryos of Ensete on a semi-solid medium modified from Murashige and Skoog (MS) (1962) and Cox et al. (1960), supplemented with 5g l–1 sucrose and agar. When the embryos were preincubated for 15–20min in 4mg l–1 gibberellic acid (GA3) additional swelling and additional elongation (10%) was observed. Negash et al. (2000) cultured mature enset embryos on benzyladenine (BA) and indole-3-acetic acid (IAA) containing MS medium. Recently the procedure for embryo culture of Ensete was further optimised (Diro and Van Staden 2003). Shoot tip culture Shoot tip culture is the use of a lateral or main shoot apex (apical dome plus a few subjacent leaf primordia), which may be up to 20mm in length, to produce multiple shoots (George and Sherrington 1984, Nehra and Kartha 1994). Murashige (1974) suggested three sequential stages of micropropagation; since then Stages 0 and 4 have been added (George and Sherrington 1984). Thus, the 5 Stages of micropropagation are: 1) preparation of the mother plants, 2) initiation of an aseptic culture, 3) multiplication of propagules, 4) regeneration of whole plants, and 5) hardening-off for subsequent field planting. In previous studies on the micropropagation of Ensete, mother plants were grown in greenhouses and shoot tips (corm tissues with some leaf primordia) were used (Afza et al. 1996, Morpurgo et al. 1996, Zeweldu 1997, Negash et al. 2000). In vitro germinated seedlings have also been used as a source of explants (Negash et al. 2000). Zeweldu (1997) used 2cm2 corm tissues of greenhouse-grown Ensete. These were decontaminated in 1.5% (w/v) sodium hypochlorite solution with some drops of Tween 20 for 10–15min, before rinsing three times with sterile distilled water. However, some endogenous contaminants were observed. Initiation of sterile shoot tips of E. ventricosum was difficult due to blackening of the explants. As a result swelling and callusing explants were obtained (Afza et al. 1996, Morpurgo et al. 1996, Zeweldu 1997). Shoot tips of E. superbum from a botanical garden were decontaminated with 0.1% mercuric chloride solution for 5min followed by three washings by sterile water (Mathew and Philip 1996). The shoot tips were incubated in liquid medium supplemented with 1.5mg l–1 BA and 1mg l–1 kinetin. In the culture of E. ventricosum, 2.25–5mg l–1 BA + 0.1–0.5mg l–1 IAA was used for the multiplication of propagules (Afza et al. 1996, Zeweldu 1997, Negash et al. 2000). In E. superbum, 1.5mg l–1 BA + 3mg l–1 IBA was used for multiplication (Mathew and Philip 1996). The plantlets from

Zygotic embryos

E. ventricosum (Genotypes Choro, Ketano and Nobo) E. ventricosum

Shoot tips 10–15mm in length from twothree-year-old plants grown in a botanical garden

About 10mm shoot MS tips from field grown plants Shoot tips from plants MS regenerated and maintained in vitro

Shoot tips from plants MS regenerated and maintained in vitro

E. superbum

E. superbum

E. superbum

E. superbum

E. ventricosum

Ensete sp. and E. ventricosum E. ventricosum (Genotypes Choro, Ketano and Nobo)

10 NAA + 2.5 Kinetin 2 NAA + 2.5 Kinetin for plant regeneration Embryogenic callus induction: 2 2,4D + 1.5 BA; Somatic embryo production: hormone free MS Embryogenic callus induction: 2 2,4D + 1.5 BA; Somatic embryo production: hormone free MS

Initiation: 1.5 BA + 1 Kinetin; Multiplication: 1.5 BA + 3 IBA; Rooting: 3 IBA + 0.1 BA

2.25 BA

–

Somatic embryos were compared with zygotic embryos and similarities were found between their ontogenetic stages

Initiation: 24 ± 2°C 84 shoots per corm in three months in the dark; Multiplication: 12h photo-period with an irradiance of 30µmol m–2 s–1 – A proliferating callus in 120 days after first inoculation; shoots with roots were regenerated after 60 days of subculture – Embryogenic callus in 90 days and somatic embryos after 40 days

At 15°C or 18°C for Shoots were stored for six months and slow growth regenerated

26°C

Rooting: 1.02 IBA + 0.225 BA + 1 000 AC MS with 1 000 or 2 000mM mannitol Initiation: MS Rooting: Halfstrength MS gelled with agar

25°C, 16h photo- About 30 shoots per explant in four period 3 000µmol months m–2 s–1

Initiation: 2.25 BA + 0.17 IAA Multiplication: 2.25 or 4.5 BA + 0.17 IAA;

MS

Adventitious shoots

27°C and 3 000µmol Callus was induced in six weeks; adventim–2 s–1 tious shoots regenerated from callus and plants from somatic embryos 27°C and 16h pho- Swelling and callusing explants after 3–4 toperiod months

A few plants were regenerated; extensive explant blackening occurred

Plant regeneration 20–60% germinated, callusing and swelling embryos in 8 weeks 14–71% in vitro germiation of the embryos in 10–12 days

4.5 BA + 0.017 IAA or 0.19 NAA

Initiation: 5 BA + 0.1 IAA and transferred to 5 BA + 1 TDZ

9 BA

–

25°C in the dark

Culture conditions –

MS

MS

Shoot tips with 2–3 leaf primordia with small portion of corm from six-month-old green house-grown plants Swelling and callusing explants 5mm long corm and 2cm long leaf explants from 5-month-old green-house-grown plants or from in vitro germinated zygotic seedlings Shoot tips

Ensete sp. and E. ventricosum

MS

Corm explants

E. ventricosum

2.7 BA + 0.5 IAA

Culture medium Plant growth regulators (mg l–1) Modified MS, Pre-incubation in 4 GA3 for 15–20min Cox MS Germination: 0.45 BA + 0.17 IAA Rooting: 1.02 IBA

Shoot tips of green- MS house-grown suckers

Initial explant Zygotic embryos

Genus/species Ensete sp.

Table 1: Summary of in vitro propagation studies of Ensete. Cox = Cox et al. 1960, MS = Murashige and Skoog 1962

et

et

al.

al.

Mathew and Philip 2003

Mathew et al. 2000

Mathew and Philip 1996

Negash 2001

Negash 2000

Zeweldu 1997

Afza et al. 1996, Morpurgo et al. 1996 Afza et al. 1996, Morpurgo et al. 1996 Zeweldu 1997

Negash et al. 2000

References Bezuneh 1980

South African Journal of Botany 2004, 70: 497–501 499

500

Ensete showed high rooting capacity on hormone-free MS medium (Zeweldu 1997), while 1.02mg l–1 IBA + 0.22mg l–1 BA + 1g l–1 activated charcoal for rooting of E. ventricosum (Negash et al. 2000) and 3mg l–1 IBA + 0.1mg l–1 BA for E. superbum (Mathew and Philip 1996) were used. Rooted in vitro plants of E. superbum (Mathew and Philip 1996) were planted in plastic cups containing compost and acclimatised in a mist chamber. Bezuneh (1980) observed that blackening of explants, formation of unwanted callus and a low rate of multiplication in vitro were associated with shoot tip culture. The blackening of cultures and callusing of explants inhibited formation of roots from zygotic embryos of Ensete in vitro. Shoot tips of E. ventricosum at the initiation stages turned brown/black and became necrotic, which resulted in failure of initiation of the explants in vitro (Afza et al. 1996, Morpurgo et al. 1996, Zeweldu 1997). Maintenance of initiating shoot tips of E. superbum in the same medium for more than four weeks resulted in blackening and necrosis of the explants (Mathew and Philip 1996). Activated charcoal and frequent subculturing were used to reduce the problem of explant blackening. Swelling and callusing of shoot tip explants of Ensete at the initiation stage was also a problem (Afza et al. 1996, Zeweldu 1997). The indirect production of adventitious shoots from explants via a callus stage may increase the problem of genetic instability of the progeny. It is suggested that the more organised the explant, the less the variation in progeny; and the less organised the starting material, the more the variation (Krikorian 1989). Using this view, shoot tips would vary least; callus, cell suspension or protoplast procedures would progressively generate the most varied plantlets. The occurrence of somaclonal variation has not been investigated in Ensete cultures. The rate of in vitro multiplication for E. ventricosum, a species of economic importance in Ethiopia, was low and erratic from the shoot tips while the rate of adventitious shoot regeneration from callus was comparable with that from conventional vegetative propagation, about 200 shoots per explant per year (Afza et al. 1996, Morpurgo et al. 1996). The regeneration of about 30 shoots per explant in four months (Negash et al. 2000) is better than that from conventional propagation but it is a low rate of multiplication when compared with 84 shoots per corm explant in three months for E. superbum (Mathew and Philip 1996) and for bananas that can be over four million shoots per shoot tip per year, depending on the stooling properties of the individual clone (Cronauer and Krikorian 1988). Callus culture and somatic embryogenesis Callus is a coherent and amorphous tissue formed when plant cells multiply in a disorganised way (George and Sherrington 1984). Root formation generally takes place in a medium with a relatively high auxin and low cytokinin concentration while formation of adventitious shoots takes place if there is a low auxin and high cytokinin concentration (Pierik 1987). Mostly, whole plant regeneration from cultured cells may occur either through shoot-bud differentiation (organogenesis) or somatic embryogenesis. Organogenic events of Ensete callus are influenced by the ratios of aux-

Diro and Van Staden

ins and cytokinins as well as the light regime (Zeweldu 1997). Under the same hormonal treatment, light promoted differentiation of callus with subsequent organ formation, whereas the callus that was maintained in darkness usually grew without further differentiation. The greatest number of adventitious shoots was observed with 4.5mg l–1 BA in combination with either 0.017mg l–1 IAA or 0.19mg l–1 NAA (Zeweldu 1997). Morpurgo et al. (1996) established callus cultures from corm explants and adventitious buds of E. ventricosum. Somatic embryos of E. ventricosum could be obtained by lowering the level of cytokinins in the medium (Afza et al. 1996, Morpurgo et al. 1996). In E. superbum, the shoot tips from in vivo conditions cultured on MS medium supplemented with 10mg l–1 NAA, 2.5mg l–1 kinetin and 1g l–1 L-glutamine produced a proliferating callus within 120 days of culture from which adventitious shoots were regenerated but somatic embryos were not formed (Mathew et al. 2000). The presence of 2mg l–1 2,4-D and 1.5mg l–1 BA was important for induction of a proliferating embryogenic callus of E. superbum from the corm tissue at the base of in vitro generated plants. However, the differentiation and maturation of somatic embryos happened only in hormone-free medium (Mathew and Philip 2003). Conclusions The agricultural importance of Ensete is not widely known. As a result, only limited in vitro investigations have been carried out in the past. Studies using shoot tip explants of E. ventricosum have shown low levels of multiple shoot formation. This is possibly due to a monopodial corm morphology and complete apical dominance. The use of cytokinins does not result in a large amount of multiple shoot formation from the shoot tip explants in E. ventricosum. Moreover, extensive blackening, as a result of phenolic oxidation creates unfavourable culture environments and causes high rates of tissue mortality. Further in vitro investigation on the propagation of Ensete species is important, given the value of this crop as a staple food in many African countries. Acknowledgements — The financial support of the Agricultural Research and Training Project of Ethiopian Agricultural Research Organization and University of KwaZulu-Natal is appreciated.

References Afele JC, De Langhe E (1991) Increasing in vitro germination of Musa balbisiana seed. Plant Cell, Tissue and Organ Culture 27: 33–36 Afza R, Van Duren M, Morpurgo R (1996) Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds. Plant Cell Reports 15: 445–448 Ashagari D (1985) Studies on bacterial wilt of enset (Ensete ventricosum) and prospects for its control. Ethiopian Journal of Agricultural Science 7: 1–14 Asif MJ, Mak C, Othman RY (2001) In vitro zygotic culture of wild Musa acuminata ssp. malaccensis and factors affecting germination and seedling growth. Plant Cell, Tissue and Organ Culture 67: 267–270 Bezuneh T (1980) The morphology of Ensete embryo in vitro culture. Ethiopian Journal of Agricultural Science 2: 23–28

South African Journal of Botany 2004, 70: 497–501

Bezuneh T, Feleke A (1966) The production and utilization of genus Ensete in Ethiopia. Economic Botany 20: 65–70 Cox EA, Stotzky G, Goos RD (1960) In vitro culture of Musa balbisiana Colla embryos. Nature 185: 403–404 Cronauer SS, Krikorian AD (1988) Temporal, spatial and morphological aspects of multiplication in aseptically cultured Musa clones. In: Valentine FA (ed) Forest and Crop Biotechnology Progress and Prospects. Springer Verlag, New York, pp 45–57 Diro Chimsa M (2003) In vitro Propagation of Enset (Ensete ventricosum (Welw.) Cheeseman). PhD Thesis, University of Natal, Pietermaritzburg, South Africa Diro M, Van Staden J (2003) In vitro regeneration of Ensete ventricosum from zygotic embryos of stored seeds. South African Journal of Botany 69: 364–369 Elsen A, Stoffelen R, Tuyet NT, Baimey H, De Boulois HD, De Waele D (2002) In vitro screening for resistance to Radopholus similes in Musa spp. Plant Science 163: 407–416 ESTC (Ethiopian Science and Technology Commission) (2003) Commission awards individuals for outstanding achievements. Available at: http://www.capitalethiopia.com/archive/2003/july/ week3/index.htm Gebre MT, Nikolayev AS (1993) Evaluation of starch obtained from Ensete ventricosum as a binder and disintegrant for compressed tablets. Journal of Pharmacy and Pharmacology 45: 317–320 George EF, Sherrington PD (1984) Plant Propagation by Tissue Culture. Exegetics Ltd, Basingstoke Hu C, Wang P (1986) Embryo culture: Technique and applications. In: Evans DA, Sharp WR, Ammirato PV (eds) Handbook of Plant Cell Culture: Techniques and Applications. MacMillan Publishing Company, New York, pp 43–96 Israeli Y, Lahav E, Reuveni O (1995) In vitro culture of bananas. In: Gowen S (ed) Bananas and Plantains. Chapman and Hall, London, pp 147–178 Krikorian AD (1989) In vitro culture of bananas and plantains, back ground, update, and call for information. Tropical Agriculture (Trinidad) 66: 194–200 Mathew MM, Manuel R, Philip VJ (2000) Callus regeneration and somatic embryogenesis in Ensete superbum (ROXB: CHEESMAN). Indian Journal of Plant Physiology 5: 392–396 Mathew MM, Philip VJ (1996) Clonal propagation of enset (Ensete superbum (ROXB.)) through shoot tips cultures. Plant Cell Reports 16: 232–234

Edited by CH Bornman

501

Mathew MM, Philip VJ (2003) Somatic embryogenesis versus zygotic embryogenesis in Ensete superbum. Plant Cell, Tissue and Organ Culture 72: 267–275 Morpurgo R, Afza R, Novak FJ (1996) Biotechnology and enset achievements and perspectives. In: Abate T, Hiebsch C, Brandt SA, Gebremariam S (eds) Proceedings from the International Workshop on Enset. 13–20 December 1993, Addis Ababa, Ethiopia, pp 256–270 Murashige T (1974) Plant propagation through tissue culture. Annual Review of Plant Physiology 25: 135–166 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473–479 Negash A, Krens F, Schaart J, Visser B (2001) In vitro conservation of enset under slow-growth conditions. Plant Cell, Tissue and Organ Culture 66: 107–111 Negash A, Puite K, Schaart J, Visser B, Krens F (2000) In vitro regeneration and micropropagation of enset from Southwestern Ethiopia. Plant Cell, Tissue and Organ Culture 62: 153–158 Nehra NS, Kartha KK (1994) Meristem and shoot tip culture: Requirements and applications. In: Vasil IK, Thorpe A (eds) Plant Cell and Tissue Culture. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 37–70 Pierik RLM (1987) In Vitro Culture of Higher Plants. Martinus Nijhoff Publishers, Dordrecht, The Netherlands Quimio AJ, Tessera M (1996) Diseases of Enset. In: Abate T, Hiebsch C, Brandt SA, Gebremariam S (eds) Enset-based Sustainable Agriculture in Ethiopia. Proceedings from the International Workshop on Enset, 13–20 December 1993, Institute of Agricultural Research, Addis Ababa, Ethiopia, pp 188–203 Simmonds NW (1960) Notes on banana taxonomy. Kew Bulletin 14: 198–212 Spring A (1996) Enset farming system in Southern Region, Ethiopia. Report on a rapid rural appraisal in Gurage, Hadiya and Sidama Zones. Enset Need Assessment Project Phase I, Awasa, Ethiopia Zeweldu T (1997) Comparative Tissue Culture Study on Banana and Plantain (Musa spp.) and Development of in vitro Methods for Propagation of Enset (Ensete spp.). PhD Thesis, Humbolt University of Berlin, Verlag Dr. Koster, Berlin, Germany