Plant Cell Tiss Organ Cult (2009) 97:197–202 DOI 10.1007/s11240-009-9515-0 ORIGINAL PAPER Incubation temperature critical to successful stimulation of in vitro zygotic embryo growth in four Australian native Cyperaceae species M. Panaia Æ E. Bunn Æ S. R. Turner Æ J. McComb Received: 3 December 2008 / Accepted: 9 February 2009 / Published online: 3 March 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Many species of Western Australian Cyperaceae (sedges) are vital components of the indigenous flora but commonly display low seed set, poor seed quality and intractable seed dormancy. We report the effects of incubation temperature and in vitro growth media on whole seed germination compared with extracted zygotic embryo growth in Tetraria capillaris, T. octandra, Lepidosperma drummondii and L. tenue. No germination was observed from intact whole seeds of all test species regardless of the treatment evaluated. In contrast, excised zygotic embryos of all study species exhibited significant increases in growth when cultured at 15°C compared to embryos incubated at 25°C; however, optimal media for embryo growth were genera specific. Extracted embryos of T. capillaris and T. octandra exhibited maximum percentage growth (30 and 40%, respectively) at 15°C on ‘ MS medium with no plant growth regulators required. In the case of L. drummondii and L. tenue 1 lM thidiazuron was a necessary addition to the ‘ MS medium resulting in 40 and 77% growth of embryos (at 15°C), respectively. Incubation of extracted embryos at 25°C (regardless of M. Panaia
J. McComb Biological Sciences, Murdoch University, South Street, Murdoch, WA 6150, Australia M. Panaia
E. Bunn (&)
S. R. Turner Botanic Gardens and Parks Authority, Fraser Avenue, West Perth, WA 6005, Australia e-mail: ebunn@bgpa.wa.gov.au S. R. Turner School of Pharmacy, Faculty of Health Sciences, The University of Queensland, Brisbane, QLD 4072, Australia E. Bunn University of Western Australia, Crawley, WA 6009, Australia medium treatment) resulted in \10% embryo growth for T. octandra and L. tenue, while the remaining two species (L. drummondii, T. capillaris) showed no embryo growth at 25°C on any medium treatment. Keywords Lepidosperma drummondii
Lepidosperma tenue
Temperature
Tetraria capillaris
Tetraria octandra
Seed germination Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid. TDZ Thidiazuron K Kinetin (6-furfurylaminopurine) BA 6-Benzylaminopurine MES 2-(N-morpholino) ethanesulfonic acid TTC Triphenyl tetrazolium chloride Ca(OCl)2 Calcium hypochlorite Introduction The Cyperaceae (sedges) are grass-like plants found in most habitats throughout the world with an estimated 100 genera and *5,000 species worldwide (Goetghebeur 1998). Out of this worldwide total, Australia hosts about 12% of these species (650) and almost 50% of the genera (Johnson and Briggs 1983). In south west Western Australia, particularly the biodiverse Kwongan (Pate et al. 1984), the Cyperaceae are highly diverse and prominent understorey species, filling ecological niches typically occupied by grasses (Poaceae) in other ecosystems (Meney and Dixon 1988). Given their relative dominance in many south west environments where bauxite and sand mining occurs, their return to the post mining environment during 123 198 rehabilitation work is viewed as a high priority (Willyams 2005). There is also evidence that many species are Phytophthora resistant (and can even reduce the spread of dieback disease) making their return to susceptible plant communities all the more important (Sieler 1996). However, unlike grasses which can be relatively easy to propagate from seeds (Clarke and French 2005; Read and Bellairs 1999) seed-based propagation for most West Australian dry-land Cyperaceae is very difficult, particularly with species of Lepidosperma, Mesomelaena Tetraria, and Schoenus (Sieler 1996; Willyams 2005). This lack of germination success for Cyperaceae as a whole is due to a number of known factors such as poor seed set, low seed quality, poorly understood seed collection strategies and most importantly deep seed dormancy (Willyams 2005; Leck and Schutz 2005; Merritt et al. 2007) so that direct seeding or seedling propagation of many Cyperaceae is currently highly restricted or impossible. Direct transplanting from natural bushland to restoration sites is considered unhygienic due to risks associated with spreading soil-borne pathogens (e.g. Phytophthora) that may be lethal to other species (even if not directly affecting sedges themselves). Vegetative propagation by division or cuttings has not (to our knowledge) been widely successful as most species are very slow growing and extremely difficult to establish (A. Shade and D. Willyams, personal communication). Some success has been achieved with tissue culture propagation methods for species of the related family Restionaceae utilising extracted zygotic embryos as the preferred starting point for micropropagation due to lack of success with vegetative material (Meney and Dixon 1995a, b). The success of this work has enabled the return of Restionaceae plants to post mined restoration landscapes in south Western Australia that were previously extremely difficult or impossible to propagate (Willyams 2005). Species such as Loxocarya ‘magna’, Loxocarya ‘gigas’, Lyginia barbata and Restio microcodon exhibited no germination for whole seed (seed coat removed), however these and Restio tremulus, Lepidobolus prissianus and Alexgeorgea subterranean showed some growth of extracted embryos on ‘ MS basal medium when incubated in the dark at 20–22°C (Meney and Dixon 1995a). However, Meney and Dixon (1995a), Sieler (1996) and Willyams (2005) did not investigate the effect of different incubation temperatures on zygotic embryo growth of Restionaceae species and development in vitro primarily because adequate growth of embryos was obtained in most cases at 20– 25°C to enable successful in vitro propagation. In contrast, many factors remain to be adequately investigated to optimise in vitro protocols for propagation of Cyperaceae. One is the impact of incubation temperature which is known to be a critical factor for germination 123 Plant Cell Tiss Organ Cult (2009) 97:197–202 success with a range of native south west Western Australian species (Bellairs and Bell 1990). The aim of this study was to investigate the effect of incubation temperature on germination of whole seed and growth of excised zygotic embryos (as a pre-requisite for in vitro mass propagation by shoot culture and/or somatic embryogenesis) of four important but difficult to propagate sedge species (Lepidosperma drummondii, L. tenue, Tetraria capillaris and Tetraria octandra) required for large-scale land restoration following bauxite mining. Materials and methods Seed was obtained from several sources (Table 1) and screened for seed fill—initially a simple flotation test with seeds placed in *250 ml water with a drop of Tween-80 surfactant in a 500 ml glass beaker and stirred slowly. After 5–10 min floating seeds were separated from sunken seeds and both classes of seed were immediately dried on filter paper. A FaxitronÒ X-ray specimen radiography system was subsequently used to more accurately identify fully-filled plump endosperms for whole seed germination and excision of zygotic embryos. Due to difficulties inherent in harvesting seeds from wild populations of the four study species the amounts and potential viability of seeds available proved to be highly variable. Therefore it was not possible to test all treatments equally on all study species and consequently for growth/germination the four species were treated as individual experiments with some necessary variation of treatments as applied to seed of each species. When not used for experiments seed was stored (dry) at 5°C. The ages of seed in months from time of harvest to time of use is stated in Figs. 1, 2, 3, and 4. The following materials and methods are common to all experiments unless otherwise stated: Basal medium (BM) consisted of 1/2 strength Murashige and Skoog (1962; MS) medium with 100 lM NaFeEDTA, 512 lM MES, 20 g l-1 (58.4 mM) sucrose, 6 g l-1 agar, pH 6.0 (adjusted prior to autoclaving) with the following additional components: 500 lM myo-Inositol, 4 lM niacin, 3 lM thiamine HCl and 2.5 lM pyridoxine HCl. All non-heat labile plant growth regulators (PGR) such as 2, 4-D, K and BA (see footnote, Table 3) were added to the media prior to autoclaving (20 min at 121°C), whilst TDZ was filter sterilised and added after autoclaving. Explants were cultured in Petri dishes containing 25 ml of treatment medium and observations were recorded weekly. Cultures were maintained in the dark at 15 or 25°C for 6 weeks. Each treatment comprised 10 whole seeds or 10 excised zygotic embryos per Petri dish with a minimum of three replications per treatment. Plant Cell Tiss Organ Cult (2009) 97:197–202 199 Table 1 Sources of various Cyperaceae seed Species namea Supplier Identification no. Location Date coll Lepidosperma drummondii Benth South west native seed supplyb SWS-11231 collected 24/5/2006d Boddington marradong Dec/2005 Lepidosperma tenue Benth South west native seed supply SWS-11230 collected 22/5/2006d Boddington bauxite mine Dec/2005 Tetraria capillaris (F.Muell) J.M.Black Nindethana seed servicec NS-28363 collected 11/1/2005d Red hill reserve Jan/2005 Tetraria octandra (Nees) Kuk Nindethana seed service NS-28644 collected 12/1/2005d Boddington marradong zone 7 Jan/2004 a Plant name authority from FloraBase (http://florabase.calm.wa.gov.au) South west native seed supply (www.swnativeseeds.com.au) and c Nindethana seed service (www.nindethana.iinet.net.au) are registered Australian seed collecting and exporting enterprises d Fresh seeds dried at room temperature (22–24°C) and stored at 5°C Lepidosperma drummondii 100 Tetraria capillaris 25°C 60 40 20 0 zygotic embryo zygotic embryo zygotic embryo 1/2 MS 1/2 MS +1 µM 2,4-D 1/2 MS +1 µM TDZ whole seed whole seed whole seed 1/2 MS 1/2 MS +1 µM 2,4-D 40 30 20 10 0 1/2 MS +1 µM TDZ Fig. 1 Growth of excised zygotic embryos and germination of whole seed for Lepidosperma drummondii on various media and incubated at 15 and 25°C. Bars on columns indicate SE of means but in most cases are too small to show. The difference in excised embryo versus whole seed growth at different incubation temperatures was significant (p value \ 0.05). (Seed age = 14 months) zygotic embryo zygotic embryo zygotic embryo 1/2 MS 1/2 MS + 1 µM 2,4-D Water agar whole seed whole seed whole seed 1/2 MS + 1 µM 2,4-D Water agar 1/2 MS Fig. 3 Growth of excised zygotic embryos and germination of whole seed for T. capillaris on various media and incubated at 15 and 25°C. Bars on columns indicate SE of means but in most cases are too small to show. Excised embryo growth was significantly greater than whole seed germination at 15°C (p value \ 0.05). (Seed age = 31 months) Tetraria octandra Lepidosperma tenue 100 Growth/Germination (%) Growth/Germination (%) 80 15°C 25°C 50 15°C 25°C 80 60 40 20 zygotic embryo zygotic embryo whole seed whole seed 1/2 MS 1/2 MS + 1 µM TDZ 1/2 MS 1/2 MS + 1 µM TDZ Fig. 2 Growth of excised zygotic embryos and germination of whole seed for Lepidosperma tenue on various media and incubated at 15 and 25°C. Bars on columns indicate SE of means but in most cases are too small to show. Excised embryo growth was significantly enhanced at 15°C compared to whole seed germination (p value \ 0.05). (Seed age = 15 months) 25°C 40 30 20 10 0 0 15°C 50 15°C Growth/Germination (%) Growth/Germination (%) b zygotic embryo zygotic embryo zygotic embryo whole seed 1/2 MS 1/2 MS + 2.5 µM K + 0.25 µM BA Water agar 1/2 MS whole seed whole seed 1/2 MS + 2.5 Water agar µM K + 0.25 µM BA Fig. 4 Growth of excised zygotic embryos and germination of whole seed for Tetraria octandra on various media and incubated at 15 and 25°C. Bars on columns indicate SE of means but in most cases are too small to show. Excised embryo growth was significantly greater than whole seed germination at 15°C (p value \ 0.05). (Seed age = 49 months) 123 200 Plant Cell Tiss Organ Cult (2009) 97:197–202 All seed batches of the study species were surface sterilised for a total of 30 min in 4.0% (w/v) Ca(OCl)2 with intermittent vacuum (10 min on/10 min off/10 min on) then rinsed three times in sterile deionised water (sdw) before imbibing overnight in sdw. The following day sterilized seed were either moved intact to Petri dishes (containing various treatment media) or zygotic embryos were aseptically excised and transferred to duplicate media treatments in Petri plates (Table 2) and incubated in darkness at either 15 or 25°C. Following 6 weeks incubation, non-germinated whole seeds were tested for viability using a 1% w/v TTC stain cut test method (Conacher et al. 1994). Embryos staining pink or red were considered viable. Growth was assessed based on radicle and shoot development of excised zygotic embryos (i.e. development of a seedling) and expressed as a percentage of the total number of excised embryos averaged across all replicates for all treatments. Data were statistically analysed via GenstatÒ using ANOVA where appropriate. Mean comparisons between treatments were made using Fisher’s protected LSD with 95% confidence intervals. Percentage data was arcsin transformed for analysis but non-transformed data are presented in Figs. 1, 2, 3, and 4. Results The percentage of ‘sinker’ seeds shown to have a full endosperm ranged from 92% for L. tenue to 46% for T. capillaris. However, some ‘floater’ seeds also contained full endosperms (from 46% for L. drummondii to 16% in L. tenue and T. capillaris (Table 3). Latter investigations of seed of L. drummondii, L. tenue, T. capillaris and T. octandra (data not presented) using a Faxitron X-ray analysis system to distinguish filled from unfilled seed was found to be far more sensitive and accurate compared to the flotation/sinking Table 2 Murashige & Skoog basal media (half strength) with plant growth regulator additives to assess effects on growth of excised zygotic embryos or germination of whole seed at 15 or 25°C for method. There was no germination of intact seeds of L. drummondii, L. tenue, T. capillaris or T. octandra across all temperature and growth media treatments (Figs. 1, 2, 3, and 4.). This was despite high seed viability (after 6 weeks incubation) of 76, 78, 83 and 90%, respectively, (as determined by TTC staining). However, enhanced growth of excised zygotic embryos was observed at 15°C for L. drummondii on ‘ MS ? 1 lM TDZ (40%), on ‘ MS with no PGR (27%) and on ‘ MS ? 1 lM 2,4-D (10%), but no growth of embryos at 25°C on any media treatments (Fig. 1). Growth of excised embryos of L. tenue occurred on ‘ MS ? 1 lM TDZ (77%) and ‘ MS minus PGR (47%) at 15°C, while 10% of excised zygotic embryos of L. tenue also grew on ‘ MS ? 1 lM TDZ at 25°C (Fig. 2). Excised zygotic embryos of T. capillaris grew best at 15°C on ‘ MS minus PGR (30%) and ‘ MS ? 1 lM 2,4-D (27%), however, growth of zygotic embryos at both 15 and 25°C on water agar treatment was much lower at 3% (Fig. 3). Growth of excised zygotic embryos of T. octandra was different on ‘ MS minus PGR with 40% growth of embryos at 15°C compared to 3% at 25°C, while 15% of embryos grew on ‘ MS with 2.5 lM kinetin ? 0.25 lM BA and 10% on water agar (Fig. 4). However, it was noted in an earlier experiment (data not presented) that higher embryo growth (*35%) occurred at 25°C on ‘ MS basal medium (minus PGR) with T. octandra seeds 28 months after collection (and storage at 5°C) compared to subsequent experiments with 49 month old seed (Fig. 4). Discussion The four species assessed in this study (L. drummondii, L. tenue, T. capillaris and T. octandra) have very low seed set and even lower seed fill percentages making the implementation of large-scale experiments extremely Lepidosperma drummondii, Lepidosperma tenue, Tetraria capillaris and Tetraria octandra Species name Explant type Media Lepidosperma drummondii Zygotic embryo & whole seed ‘ MS Lepidosperma drummondii Zygotic embryo & whole seed ‘ MS ? 1 lM 2,4-D Lepidosperma drummondii Zygotic embryo & whole seed ‘ MS ? 1 lM TDZ Lepidosperma tenue Zygotic embryo & whole seed ‘ MS Lepidosperma tenue Zygotic embryo & whole seed ‘ MS ? 1 lM TDZ Tetraria capillaris Zygotic embryo & whole seed ‘ MS Tetraria capillaris Tetraria capillaris Zygotic embryo & whole seed Zygotic embryo & whole seed ‘ MS ? 1 lM 2,4-D Water agar Tetraria octandra Zygotic embryo & whole seed ‘ MS Tetraria octandra Zygotic embryo & whole seed ‘ MS ? 2.5 lM K ? 0.25 lM BA Tetraria octandra Zygotic embryo & whole seed Water agar (0.6% w/v) 123 Plant Cell Tiss Organ Cult (2009) 97:197–202 201 Table 3 Seed of Cyperaceae spp.: percentages of potentially viable seed (as determined via flotation testing) Species/age of seed (months) Total number of seeds assessed Floaters (%) Sinkers (%) Sinkers with endosperm (%) Floaters with endosperm (%) Lepidosperma drummondii (5) 1,323 21 79 87 46 Lepidosperma tenue (6) 4,132 43 57 92 16 Tetraria capillaris (16) Tetraria octandra (28) 1,430 1,344 90 77 10 23 46 62 16 23 difficult and only a limited number of experiments could be conducted. This also necessitated the storage of seed for varying lengths of time (at 5°C in this study) as reliable supplies of fresh seed of all species were unobtainable. The simple flotation test utilized initially did not adequately select all filled seeds while the subsequent availability of a Faxitron X-ray specimen radiography system resolved this problem by allowing more accurate selection of filled seeds. Careful attention to harvesting and selection of seed has been shown to be extremely important for in vitro culture of species of Cyperaceae (Panaia et al. 2008). The fact that these species appear to set so few viable seeds means that the seeds collected are all the more valuable and their use must be maximised, making in vitro propagation of this material crucial for large-scale revegetation programs. The observation that T. octandra seeds exhibited higher embryo growth (*35%) at 25°C on ‘ MS basal medium (minus PGR) 28 months after collection (and storage at 5°C) compared to subsequent experiments with 49 month old seed (same storage conditions) was unusual. As seed was randomly selected (within a single seed batch) for experimental use this discrepancy may suggest a deepening of seed dormancy over time (Baskin and Baskin 2004) as seed viability remained at similar levels over the different trials. A similar but far less obvious trend was also evident with T. capillaris and possibly with the two Lepidosperma species, however, this apparent change in seed embryo response to in vitro conditions over time requires further investigation to resolve. In contrast, growth from excised embryos was high at 15°C but not at 25°C, however, time of storage may also impact on embryo response (as evidenced by the higher embryo growth response of 2-yearold seed of T. octandra compared to 4-year-old seed). Thus in all four species the key driver of growth of embryos removed from the endosperm appears to be temperature, with exogenously applied plant growth regulators having a lesser effect. It would appear that temperature sensitivity resides within the embryo rather than in the surrounding endosperm tissues, even though the embryos in all (4) study species are only a small fraction of the total seed volume. For several other native species germination of whole seeds has been shown to be better at temperatures lower than 20°C (Bellairs and Bell 1990). Daytime temperatures lower than 20°C occurs during the winter season in south west Western Australia where all four studied species occur. This part of Australia experiences a ‘‘classic’’ Mediterranean climate with hot dry summers followed by cool wet winters. For most south west native species the winter wet season, (May–September) is the only time of year when seeds can germinate and grow sufficiently large enough and/or attain a level of hardiness sufficient to withstand the often extremely hot dry conditions of summer (Merritt et al. 2007, Turner et al. 2006). Embryo culture has been previously shown to be an effective means for micropropagation of some Cyperaceae species but the effect of incubation temperature has not been reported for these species, which appear to give satisfactory (in vitro) germination at 20–25°C. For Caustis dioica zygotic embryos incubated on ‘ MS medium at 22–25°C there was 50% germination (Rossetto et al. 1992). Extracted zygotic embryos of C. dioica, Gahnia trifida, Lepidosperma gladiatum and L. angustatum all grew at 22°C (Sieler 1996) while Meney and Dixon (1995a) reported variation in growth of embryos from Restionaceae species (from 4% embryo growth of Restio microcodon to 81% with Lepidobolus chaetocephalus embryos), when incubated at 20–22°C on ‘ MS medium. None of the above studies alluded to either significant seed viability issues or restrictions on seed availability therefore seed dormancy may have been the reason for low seed embryo propagation performance for at least some of the species. This study has shown that excision of zygotic embryos and incubation at 15°C (for L. drummondii, L. tenue, T. capillaris and T. octandra) significantly increases growth of excised zygotic embryos and development into seedlings which can then be utilised for initiation of stock cultures necessary for micropropagation (via shoot culture and/or somatic embryogenesis). 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