In vitro propagation of Western Australian Rushes (Restionaceae and related families) by embryo culture. Part 1. In vitro embryo growth

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This study outlines the first method for initiating micropropagation of a wide range of wild and commercially significant species of various Southern Hemisphere rushes (Restionaceae) by embryo culture. Most species showed best embryo growth on
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  Plant Cell l~ssue and Organ Culture 41: 107-113, 1995. 107 @ 1995 KluwerAcademicPublishers. Printedin theNetherlands. n vitro propagation of Western Australian Rushes (Restionaceae and related families) by embryo culture. Part 1. n vitro embryo growth Kathy A. Meney Kingsley W. Dixon Kings Park and Botanic Garden, West Perth, Western Australia 6005 Received 9 November 1993; accepted n revised form 3 January 1995 Key words: germination, graminoids, micropropagation, rare and endangered species, restioids Abstract This study outlines the first method for initiating micropropagation of a wide range of wild and commercially significant species of various Southern Hemisphere rushes (Restionaceae) by embryo culture. Most species showed best embryo growth on half-strength Murashige and Skoog basal medium, with the addition of gibberellic acid (1-5 /~M) and/or zeatin (1-5 #M). Results showed there was a high level of variability in seed germinability as indicated by in vitro embryo growth (6-96%), with no apparent link to growth form, generic status or habitat preference. Embryo growth was achieved for several rare and/or restricted taxa (Loxocarya 'magna' ined Lepidobolus contorta Meney & Dixon Hopkinsia anoectocolea (E Muell.) Cutler, Lepidobolus contorta Meney & Dixon ined). The low germination of whole seeds of most species (average of 10%) indicates that whole seed germination under controlled conditions considerably understates the potential germinability of species of Restionaceae. In this context, embryo culture is useful for assessment of germinability and for initiating stock cultures for micropropagation. Abbreviations: ½MS -half-strength (minerals only) Murashige and Skoog (1962) basal medium; GA3 - gibberellic acid Introduction The Restionaceae is a predominantly Southern Hemi- sphere family of slow-growing perennial herbs. Species in this group comprise significant elements of the native vegetation of the south-west of Western Australia and represent a diversity of growth forms over a wide range of habitats (Pate et al. 1991). How- ever, these taxa have failed to satisfactorily re-establish after mining disturbance from either seed or vegetative material, which has consequently retarded the progress of post-mining rehabilitation. In addition, some species are in current use or have potential as major horticultur- al species for use as dried foliage for export or ~menity horticulture. All 18 species used for cut foliage are presently wild-picked. Successful propagation of many Restionaceae has been difficult to achieve by conventional methods using seed (Meney & Dixon 1988) and many demon- strate limited/slow response by rhizome division (Meney et al. 1990). No success has been obtained by using shoot or rhizome-bud material of Restionaceae as explants for in vitro propagation because of the scle- rifled nature of the tissues, difficulty of disinfestation from fungi and bacteria and lack of in vitro response (Meney et al. 1990). An earlier study outlined some preliminary results on the germination of three species of Restionaceae by in vitro extraction and culture of seed embryos (Meney & Dixon 1988). This paper presents research on the use of embryo culture as a means for initiating seedlings and providing a source of material for micropropagation and seed biology stud- ies. The taxonomy and phylogeny of the Restionaceae is currently under revision with the monograph soon due for publication in the Flora of Australia (Johnson and Briggs, pers. comm. Royal Botanic Gardens, Syd- ney). Where available, this paper uses the proposed new names and nomenclatural divisions by permission of the authors. The new treatment recognises Anarthria  108 Fig. 1. Typical members of the Ecdeiocoleaceae, Anarthriaceae and Restionaceae. (A) Ecdeiocolea monostachya (Ecdeiocoleaceae), (B) Anarthria scabra (Anarthriaceae), (C) Lepidobolus preissianus, (Restionaceae).  and Ecdeiocolea as distinct from (and more primitive than) the Restionaceae and these have been moved into the families Anarthriaceae and Ecdeiocoleaceae, respectively. Unpublished names are indicated by use of inverted commas. Typical members of each family are indicated in Fig. 1. The genus Restio is considered incorrectly applied to Australian species and species currently treated under this genus will eventually be moved to at least two new genera yet to be deter- mined. Materials and methods Study species and site A total of 23 species were examined from open heath- land, seasonally wet, to forested habitats comprising 11 resprouters (fire-resistant) and 12 obligate seed- ers (fire-sensitive) (Pate et al. 1991). Previous stud- ies have shown that adult resprouter plants (indicated by (R) after species names) have significantly slow- er growth rates and reproductive capacity than seeder plants (indicated by (S) after species names) (Meney 1993; Pate et al. 1991), which may result in dif- ferent responses in terms of germination and growth response of in vitro-generated material. All species are rhizomatous ranging from strongly clonal with wide- ly spreading rhizomes to compact, tufted forms (Pate et al. 1991). Detailed media trials were undertaken on a selected number of representative or particularly recalcitrant species. Seeds of all species were collected at maturity in spring during 1987-1990. In most cases, seeds repre- senting a range of provenances for each species were collected randomly from habitats in the mediterranean zone of southwest Western Australia. Freshly collected seeds were stored dry for 2 to 8 months under ambient laboratory conditions until required for germination trials. Embryo extraction procedure and preparation of media After removal of the outer seed coats, seeds were dis- infested by first rinsing in a 1% sodium hypochlorite (NaOCI) solution containing 0.05% Tween 80 for 1 rain to remove surface contaminants. Testa-free seeds were then rinsed in distilled water and again placed in 1% NaOC1 under vacuum for 15 min (alternating 5 rain on, 5 min off, 5 min on). After double rinsing 109 in sterile distilled water, these seeds were transferred to a sterile laminar flow cabinet and left to imbibe for 2-12 h to soften the endosperm and embryos. Twenty to 45 embryos per replicate (2-3 replicates per species) were extracted using a dissecting micro- scope and placed singly in sterilised 30 ml polycarbon- ate tubes containing 10 ml of half-strength Murashige & Skoog (1962) basal salts media (½MS) containing supplementary vitamins (3.0/~M thiamine - HC1, 2.5 #M pyridoxine- HC1, 4.0 #M niacin), 500 #M myo- inositol and various concentrations of growth regula- tors (GA3 and zeatin). Sucrose (60mM) was added and for solid media 10 g 1-1 Difco Bacto-agar was added. Media were adjusted to pH 6.0. Basal medium was ster- ilised by autoclaving for 20 min at 120°C and 105 kPa. Growth regulators were filter-sterilised and added after media had cooled to 40°C. Embryos were incubated in the dark (see Meney & Dixon 1988) at 20°-22°C until growth had occurred. Tubes were inspected each 3 to 5 days up to 60 days and all growing embryos were recorded and moved to the light (cool white fluorescent tubes, 40/~mol m -z s -~ 16-h photoperiod). In vitro germination~growth of seeds and embryos Experiment 1 The whole seed germination versus embryo growth response of 10 common and two restricted (Loxocarya 'gigas', L. 'magna') species was compared on agar- gelled media as described above. For each species, 45 whole seeds (seed coat removed) (T1) and an equal number of embryos (T2) were tested (three replicates of 15 seeds each). In addition, embryo growth on solid versus liquid media was compared by placing a fur- ther 45 embryos of each species on filter paper domes suspended in liquid embryo media (T3). In each treat- ment, GA3 was added at 1/~M and zeatin at 0.5/~M. Seeds and embryos were incubated in the dark at 20- 22°C. Based on these results, the best medium was then used to test embryo growth response in 11 other species. In this trial, single batches of 30-50 embryos were used for each species. Statistical differences were tested using the Students t-test for large or small sam- pies as appropriate, where t = t-value, n = degrees of freedom. Experiment 2 Further embryo trials were conducted on a subset of six species (three replicates of 10 embryos each) that  110 demonstrated poor growth response of embryos (less than 50%) at low cytokinin levels. In this experiment, the same (1 #M) and higher (3 #M, 5 #M) levels of GA3 were initially tested in combination with 1 /zM zeatin (T1, T2, T3). Where there was no improved germination response, the level of zeatin was also increased to 5 #M, either in combination with 1 #M GA3 (T4), or used alone with no GA3 (T5). Statistical differences were tested by one-way ANOVA with least significant differences (Fishers PLSD) used to indicate differences between sample pairs when the F value was significant. Results Seed and embryo morphology Embryos of all species are of the broad basal type (Radford et al. 1974) and are normally located at the attachment end of the seed, which contains copi- ous endosperm. Embryos are characteristically small (approximately 0.1-0.3 mm). Seed size in the study species varies from 0.2 mm x 1 mm (e.g. Restio microcodon, R. 'isomorphus') to 5 mm x 3 mm in Loxocarya 'gigas' and 12 mm x 7 mm in Alexge- orgea subterranea. Extraction of embryos was rela- tively simple after seeds had fully imbibed revealing distinct embryos. In most cases, the most effective extraction technique was to remove the seed coat and inner integuments thereby exposing the embryo and facilitating extraction. Germination~embryo growth trials Experiment 1 Using solid media, germination of extracted embryos (T2) was significantly better than whole seeds for all taxa (T1, tn ffi -3.008~2, p < 0.003) (Table 1). Whole seeds of four species failed to germinate. Six species demonstrated significantly better embryo germination on liquid (T3) than on solid (T2) medium and in particular the following significant dif- ferences were observed, Ecdeiocolea eorgei', Restio tremulus, (p < 0.001), Loxocarya 'magna', Restio 'sin- uosus', (/9 < 0.01), Lepidobolus chaetocephalus, Restio microcodon, (p < 0.05) (Table 1). However, consider- ing all the species combined the differences of embryo germination in liquid and solid media was not statis- tically significant (tn ffi -1.56772, p < 0.061) (Table 1). The average germination of extracted embryos on liq- uid media with low cytokinins varied from 6.2% in Lyginia barbata var. barbata (resprouter) to 96.0% in the seeder species, Lepidobolus chaetocephalus. Results of a further 11 species also tested on the same liquid embryo medium (n -~ 30 embryos per species) were as follows: -Seeders: 'Desmocladus castaneus' (20%), 'D. elongatus' (56%), 'D. flexuosus' (75%), Lyginia barbara var. imberbis (33%), Restio 'sinuosus var. erectus' (32%), R. ' stenandra' (60%a). - Resprouters: Anarthria prolifera (45%), A. scabra (34%), Hopkinsia anoectocolea (32%) Hypolaena 'pubescens' (11%), Lepidobolus ' contorta' (83%). Combining both sets of data, mean embryo growth was 47.6% 4- 8.9% for resprouters compared to 53.1% 4- 8.0% for seeders, indicating there was no significant difference between regeneration mode and embryo growth (tn ffi -0.4512~). Experiment 2 A subset of species that showed less than 50% germi- nation response in Experiment 1 was exposed to higher levels of cytokinins (Table 2). For all but two species, 1 /zM GA3 in combination with 1/zM zeatin (T1) result- ed in no significant improvement in embryo growth over control plants (½MS only). The first exception was Lyginia barbata var. barbata, which showed poor embryo growth (6.1%) at 1 #M GA3, but no embryo response in either the control or if2 (3/~M GA3, 1/zM zeatin). The second exception was Loxocarya 'magna' in which T1 and T2 resulted in significantly better embryo growth than the control (PLSD = 17.617, p < 0.01), but T3 (5 /zM GA3, 1 #M zeatin) showed a slightly depressed response compared with T2 (not significant). T2 resulted in significantly poorer embryo growth than both the control and T1 for Anarthria prolifera (PLSD ffi 23.044, p < 0.001) and the seed- er, Restio 'sinuosus var. erectus' (PLSD ffi 18.447, p < 0.001). Embryo growth in the resprouter, Restio 'sinuosus', was also significantly better in the control without added growth regulators (PLSD ffi 12.299, p < 0.01). One species, Hopkinsia anoectocolea, showed no significant difference among T1, T2 and the con- trol. Two treatments in which 5/zM zeatin was added either in combination with 1 #M GA3 (T4) or singly (T5) were tested on Hopkinsia anoectocolea and Lox- ocarya 'magna' (Table 2). In the first species, zeatin alone resulted in the best overall response, but this  111 Table 1. In vitro growth of whole seeds and embryos on ½MS basal medium. Species Fire response Treatments a T1 T2 T3 Alexgeorgea subterranea Carlquist S 44.3 4- 2.3 80.7 -I- 1.8 81.3 4- 1.8 Ecdeiocolea monostachya R 19.3 4- 1.8 E Muell. 79.0 4- 1.5 85.0 + 1.7 Ecdeiocolea ' georgei' L. Johnson & B. Briggs (ined) R 16.7 4- 1.8 17.3 4- 1.3 53.0 4- 1.7 Hypolaena exsulca R. Br. R 10.0 4- 5.8 51.3 4- 4.0 48.7 4- 4.0 Lepidobolus chaetocephalus S E Muell. ex Benth. 6.7 4- 1.3 81.3 4- 4.8 96.0 4- 3.0 Lepidobolus preissianus R Loxocarya 'magna S Meney & Dixon (ined) 11.3 4- 1,8 77.3 4- 2.4 78.3 4- 3.0 0 7.3 4- 0.7 26.7 4- 12.0 Loxocarya ' gigas ' L. Johnson & B. Briggs (ined) S 0 59.3 4- 11.8 68.0 4- 7.2 Lyginia barbata var. barbata (Labill.) R. Br. R 0 4.7 4- 0.7 6.2 q- 2.4 Restio microcodon L. Johnson & B. Briggs (ined) S 0 4.0 4- 2.3 12.0 4- 2.3 Res6o ~inuosus var, erectus' L. Johnson & B. Bfiggs (ined) S 8.0 4- 1.2 12.0 ± 2.3 32.3 4- 2.3 Restio tremulus R. Br. S 10.0 4- 1.0 20.7 4- 4.4 43.3 4- 11.7 MEAN + SE 10.5 q- 3.6 41.2 4- 9.6 52.6 4- 8.6 aMean % 4- standard error Treatments (T1-T3) as follows: T1 = whole seeds on solid medium with 1 #M GA3 and 0.5/zM zeatin; T2 = embryos on liquid medium with 1/zM GA3 and 0.5 #M zeatin; T3 = embryos on liquid medium with 3/zM GA3 and 0.5 #M zeatin. R = resprouter, S = seeder. was not significantly different from the control or oth- er treatments (except T3, PLSD = 24.397, p < 0.01). In contrast, increased zeatin levels resulted in higher ger- mination of embryos for Loxocarya 'magna ', with both T4 and T5 significantly better than both the control and T1 (PLSD = 24.696, p < 0.001). T4 was significantly different from all other treatments (PLSD = 17.617, p < 0.01).
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