Plant Cell Rep (2002) 20:1107–1111 DOI 10.1007/s00299-002-0454-z
CELL BIOLOGY AND MORPHOGENESIS
V. Sarasan · M.M. Ramsay · A.V. Roberts
In vitro germination and induction of direct somatic embryogenesis in ‘Bottle Palm’ [Hyophorbe lagenicaulis (L. Bailey) H.E. Moore], a critically endangered Mauritian palm Received: 18 July 2001 / Revised: 24 January 2002 / Accepted: 28 January 2002 / Published online: 10 April 2002 © Springer-Verlag 2002
Abstract Hyophorbe lagenicaulis is a critically endangered palm of Mauritius. Zygotic embryos were isolated from seeds and germinated in vitro on MS salts and vitamins containing activated charcoal. When seedlings were pre-treated in vitro for 2 weeks in liquid medium containing 0.05 mg l–1 paclobutrazol, 80% survived the transfer to soil. Three-week-old seedlings were sectioned longitudinally and partially embedded in medium that contained MS salts and vitamins, 30 g l–1sucrose and 3 mg l–1 2,4-dichlorophenoxyacetic acid. Somatic embryos were formed, in profusion, directly (without callus) from the haustorium, plumule and radicle. Direct regeneration is important for the conservation of endangered species, as fewer somaclonal variants are likely to arise than from indirect regeneration. When the haustorium, plumule and radicle of the longitudinally sectioned seedlings were separated, they formed more callus but fewer embryos. Plantlets derived from somatic embryos have not yet been successfully established in soil. Keywords In vitro germination · Somatic embryogenesis · Critically endangered species · Bottle palm Abbreviations BA: 6-Benzyladenine · 2,4-D: 2,4-Dichlorophenoxyacetic acid · GA3: Gibberellic acid · NAA: β-Naphthaleneacetic acid · PBZ: Paclobutrazol · TDZ: Thidiazuron
Communicated by M.R. Davey V. Sarasan (✉) · M.M. Ramsay Micropropagation Unit, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK e-mail:
[email protected] Tel.: +44-208-3325559 A.V. Roberts School of Biosciences, University of East London, Romford Road, London E15 4LZ, UK
Introduction The ‘Bottle Palm’, Hyophorbe lagenicaulis (L. Bailey) H.E. Moore, is endemic to Mauritius, where it is confined to lowland palm savannah on Round Island. It is critically endangered (Oldfield et al. 1998), and a survey for the International Union for Conservation of Nature and Natural Resources conducted in 1996 reported fewer than ten wild plants. In the wild, germination of seeds takes 5–6 months and is sporadic. Palms are recalcitrant in culture, and the development of in vitro strategies are complicated by the presence of only a single shoot meristem per plant and the genetic diversity of the seed (El Hadrami et al. 1995). In vitro germination has been used to improve the germination rates of palms (Zaid and Tisserat 1984). Douglas (1987) investigated embryo culture in H. amaricaulis, a species closely related to H. lagenicaulis, but only one embryo from 51 seeds germinated to produce a true leaf. Propagation by somatic embryogenesis has been reported in Cocos nucifera (Chan et al. 1998), Elaeis guineensis (Teixeira et al. 1993), Phoenix dactylifera (Veramendi and Navarro 1996) and P. canariensis (Huong et al. 1999). As the conservation of palm seeds through storage at ambient temperature or in liquid nitrogen presents difficulties because of the high moisture content of the seeds, the storage of somatic embryos might provide an alternative approach. The aim of the investigation reported here was to use in vitro methods to improve the germination rates of seeds of bottle palm and develop a protocol for in vitro propagation by somatic embryogenesis. Zygotic embryos were chosen as explants because of the ease with which they can be transported from their place of origin and because zygotic embryos are the best source of somatic embryos in many species of angiosperms.
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Materials and methods
Table 1 Classification of seeds
Introduction into culture
Class
Fruit-wall colour
Endosperm
Number tested
M1 M2 M3
Green Green Dark brown
Soft Semi-hard Hard
30 71 47
Seeds, 2 cm in length, from the National Parks and Conservation Service, were classified by characters of the fruit wall and endosperm (Table 1) into categories of increasing maturity (M1–M3). Between 3 days and 14 days after collection, the outer flesh and inner fibrous layers of the fruit were removed to expose the seeds, which were cleaned and left overnight in distilled water. The seeds were then surface-sterilised by first being dipped (40 s) in 70% alcohol, then immersed for 40 min in an aqueous solution of 0.5% (w/v) sodium dichloroisocyanurate (Sigma-Aldrich, UK) containing a drop of Tween-20 (Sigma-Aldrich) on an orbital shaker (100 rpm). Sterilised seeds were then washed in three changes of sterile distilled water, each of 5 min, and the embryos were excised. This was achieved with M1 seeds by making an incision in the testa with a scalpel blade and squeezing the embryo out. The endosperm hardened with age, and M3 embryos were released by chipping off the endosperm with a scalpel blade. Tissue culture methods Culture media consisted of MS salts and vitamins (Murashige and Skoog 1962) at full strength or half strength and other ingredients as specified. The pH was adjusted to 5.8 and, if a semi-solid medium was required, 0.8 g l–1 agar (A-7002; Sigma Chemicals, UK) was added. Media were autoclaved at 121°C for 15 min. Cultures were maintained at 26°±2°C and illuminated with cool-white fluorescent lights (PPFD: 25 µmol m–2 s–1) under a 12/12-h (day/night) photoperiod or, where specified, in darkness. Germination of zygotic embryos and transfer to soil Intact embryos were inoculated onto agar-solidified medium containing MS salts and vitamins at full strength, sucrose (30 g l–1) and activated charcoal (2 g l–1). Aliquots of 50 ml were dispensed into honey jars with plastic lids (355-ml capacity; Miele, Belgium) and autoclaved. Haustoria, if present, were removed after 4 weeks, and seedlings with at least two roots were transferred to liquid medium containing MS salts and vitamins at full strength, sucrose (20 g l–1) and activated charcoal (2 g l–1) either with or without PBZ (0.05 mg l–1). The sterilised medium was dispensed in 50-ml aliquots into Phytatray II pots (114 mm×86 mm×102 mm high; Sigma-Aldrich). In the lid of each pot, a circular hole (5 mm in diameter) had been drilled and sealed with sterile Micropore tape (Merck, UK) to allow gaseous exchange. After 2 weeks, seedlings were transferred to sucrose-free liquid medium containing halfstrength MS medium and activated charcoal (2 g l–1) for a further 2 weeks. Seedlings were transferred to a potting mixture that consisted of equal proportions (v/v/v) of sand, coir and charcoal grit in plastic pots (21.5 cm high × 25.5 cm in diameter) and maintained in a glasshouse. A high humidity was maintained by covering the pots with polythene bags that were removed after 8 weeks. Initiation of somatic embryos from seed The culture medium that was adopted for somatic embryo induction (SEI medium) consisted of full-strength MS salts and vitamins, sucrose (30 g l–1) and 2,4-D (3 mg l–1) and was solidified with agar. Other components that were tested included maltose (30 g l–1) in place of sucrose, charcoal (2 g l–1), alternative concentrations of 2,4-D, and BA and TDZ, as specified. The culture medium was autoclaved and poured in 25-ml aliquots into singlevented petri dishes (90 mm in diameter) that were sealed with Parafilm. Two- to 3-week-old seedlings with haustoria (Fig. 1A) were sectioned longitudinally into two halves through the haustorium, plumule and root. In treatment 1 (T1), the half seedlings (one per petri dish) were not further divided, whereas in treat-
ment 2 (T2), the haustorium, plumule and the root were separated. The sectioned seedlings were then partially embedded in the culture medium. All cultures were incubated in darkness for 12 weeks. Germination of somatic embryos Somatic embryos that had started to elongate were cultured on agar-solidified germination media containing full-strength MS salts and vitamins, BA (0.1, 0.5, 1 and 2 mg l–1) or 0.5 mg l–1 BA with either NAA (0.1, 0.5, 1 and 2 mg l–1) or GA3 (0.1, 0.5, and 1 mg l–1). Four replicates of five embryos per petri dish were cultured in these media for 6–10 weeks. Discoloured embryos were transferred to the same medium at intervals of 2 weeks.
Results and discussion Introduction into culture Each of the 148 zygotic embryos tested were introduced into culture without loss through death or contamination. This result supports the view (Parkinson et al. 1996) that sodium dichloroisocyanurate is an effective bleaching agent that causes little damage to plant material. Germination of zygotic embryos and transfer to soil Zygotic embryos enlarged rapidly after their transfer to culture medium and doubled in volume within 3 days. Browning of tissues, with consequentially slow growth, was prevented by the inclusion of activated charcoal in the culture medium. Charcoal was similarly used by Tisserat (1979) to overcome browning in cultures of P. dactylifera. Signs of germination, including the appearance of haustorial, plumular and radicular nodules (Fig. 1A), occurred in M1 embryos within 7–11 days and in both M2 and M3 embryos within 7 days. Embryos that did not germinate within first 2 weeks did not develop further. Four weeks after initiation into culture, seedlings were classified according to whether the haustorium was: (1) absent, (2) small (5 mm). The proportion in each class varied amongst M1, M2 and M3 embryos (Fig. 2), and 2×2 contingency χ2 tests showed that a haustorium was present in significantly (P
