In Vitro Cell.Dev.Biol.Plant (2007) 43:1620 DOI 10.

1007/s11627-006-9010-9

DEVELOPMENTAL BIOLOGY/MORPHOGENESIS

Somatic embryogenesis and adventitious shoot formation in Burma reed (Neyraudia arundinacea Henr.)
Guohua Ma & Guojiang Wu & Eric Bunn

Received: 15 October 2006 / Accepted: 17 October 2006 / Published online: 9 February 2007 / Editor: D.D. Songstad # The Society for In Vitro Biology 2007

Abstract Burma reed (Neyraudia arundinacea Henr.) is a C4 grass native to Southeast Asia and Indomalaya that grows quickly, exhibits strong resistance to environmental stresses, and is extremely adaptable. It can be widely utilized as a bioenergy crop for biomass conversion. In vitro multiple shoots were first established from axillary buds and then subcultured on propagation medium containing 10 M 6benzylaminopurine (BA) and 2.0 M naphthaleneacetic acid (NAA). Multishoot clumps were used as explants to induce somatic embryogenesis and adventitious shoot formation. The results showed that auxin 2,4-dichlorophenoxyacetic acid or NAA play a key role for the induction of somatic embryogenesis and adventitious shoot formation, whereas cytokinin BA or kineatin enhance shoot proliferation and plant regeneration from callus and somatic embryos. Efficient somatic embryogenesis, mass propagation, and plant regeneration systems in Burma reed were established. Keywords Neyraudia arundinacea Henr. . Somatic embryogenesis . Adventitious shoot formation . Mass propagation . Plant regeneration

Introduction Continuous use of fossil fuels at present rates risks exhausting known energy resources prematurely. Additionally, climbing prices and serious pollution problems (especially
G. Ma (*) : G. Wu South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, China e-mail: magh@scib.ac.cn E. Bunn Kings Park and Botanic Garden, West Perth, Western Australia 6005, Australia

CO2 emissions) signal a time to develop new and green energy resources. The need for the development of biomass energy has therefore become more urgent (Overend and Chornet, 1999; Ramamurthi et al., 2001; Yuan, 2002). Some quick-growing plant species, such as the grass Miscanthus sinensis, Burma reed, have been identified as ideal biomass energy plants (El Bassam, 1998; Tsao, 1999). Miscanthus sinensis has been widely utilized as an energy crop in temperate regions of the world (Othar et al., 1993; Clifton and Lewandowski, 2002). Neyraudia arundinacea, known as Burma reed, silk reed, or cane grass, is a large caespitose perennial C4 plant that is distributed widely in tropical and subtropical areas of Southeast Asia and Indomalaya (Bor, 1960; Piao et al., 2004). In South China, Burma reed grows quickly and plays important roles as a native pioneer plant in controlling soil erosion and renewing mine castoff areas (Lin, 2004; Sun, 2004). Burma reeds drought tolerance and environmental adaptability exceeds that of Vetiveria zizanioides, M. sinensis, and Paspalum notatum (Deng, 2004; Lin, 2005). Burma reed is regarded as an optimal biomass energy crop in tropical and subtropical regions. However, Burma reed can reproduce seeds and spread around easily, so it may cause ecological invasion disaster. For better utilization of this quickgrowing and high-biomass plant species, efficient propagation and regeneration systems are necessary for genetic improvement (especially triploidy breeding and somaclonal variation). However, tissue culture in Burma reed has not been reported before. Here we report somatic embryogenesis and adventitious shoot formation in Burma reed.

Materials and Methods Axillary shoots of N. arundinacea were harvested from the plants growing in the South China Botanical Garden. The

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explants were sterilized in 70% alcohol for 10 s and 0.1% mercuric chloride for 8 min, rinsed in three changes of sterile distilled water, then inoculated on (Murashige and Skoog, 1962) basal medium supplemented with 10 M 6benzylaminopurine (BA) and 2.0 M naphthaleneacetic acid (NAA) for shoot propagation. The culture jars were placed in an air-conditioned culture room at 262C with 14/10 h photoperiod providing 80 mol m2 s1 fluorescent light and subcultured every 2 mo. After 6 mo. of subcultures for shoot mass propagation, the multiple shoots were divided into single shoots. Shoot base sections were cut into 5-mm-long explants, then transferred to different induction media for dark culture. All the media contained 30 g l1 sucrose and were adjusted to pH 5.7 and solidified with 0.6% agar (Shantou, China).
Figure 1. Somatic embryogenesis and adventitious shoot formation in Burma reed (bar = 2 mm). Globular somatic embryos (a) and green adventitious shoots (b) were induced on the induction medium containing 5 M 2,4-D after culturing for 14 and 21 d, respectively. Adventitious shoots were developed from the callus after transferring to the medium containing 5.0 M BA and 1.0 M NAA for 7 (c) and 21 d (d), respectively. Shoot mass propagation (e). Root formation within 7 d after transferred to the regeneration medium ( f ).

Effect of plant growth regulators on induction of somatic embryogenesis. The induction media contained 20 M 2,4dichlorophenoxyacetic acid (2,4-D), 5.0 M 2,4-D, 5.0 M NAA, 5.0 M BA, and 5.0 M kineatin (KIN). After the shoot base explants were cultured for 14 d in darkness, the culture jars were then transferred to light culture. The induction of callus, somatic embryos, or adventitious shoot formation were investigated. Effect of plant growth regulators on regeneration from the callus. After the shoot base section explants were cultured on the induction medium containing 20 M 2,4-D for 21 d in darkness, the induced callus clumps were transferred to different media for further development. The media contained 20 M 2,4-D, 5.0 M 2,4-D, 5.0 M

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MA ET AL. Table 3. Shoot mass propagation of Burma reed in different media Plant growth regulator combination in the propagation media (M) BA BA BA BA 20+NAA 2.0 10+NAA 2.0 5.0+NAA 1.0 1.0+NAA 1.0 Propagation index per 45 d 6.5 6.9 4.8 3.1 c c b a

Table 1. Effects of plant growth regulators on the induction of somatic embryogenesis and adventitious shoot formation from shoot base sections of Burma reed after culturing for 21 d Plant growth regulator in the media (M) 2,4-D 20 2,4-D 5.0 Observation results from the explants Mean number of adventitious shoot per explant 0a 37.1 d

NAA 5.0

BA 5.0 KIN 5.0

Callus Callus, somatic embryo and adventitious shoot buds Callus, somatic embryo and adventitious shoot buds No callus, shoots No callus, shoots

The same letter in the same column denotes no significant difference by least significant difference (0.05) test. 13.6 c

ric acid (IBA) for root formation. After 1 mo. of culture, the plantlets were removed from jars and rinsed of agar, then directly transplanted to a sandy propagation bed.

3.1 b 2.6 b

The same letter in the same column denotes no significant difference by least significant difference (0.05) test.

Results Effect of plant growth regulators on induction of somatic embryogenesis. When the shoot base section explants were cultured on the induction medium containing 20 M 2,4-D for dark culture, some yellow-white callus was induced at the cut surface of the explant within 1 wk. With culture time prolongation, the friable yellow-white callus proliferated normally; however, no somatic embryo formation was observed. On the induction medium containing 5.0 M 2,4-D, callus was also induced within 1 wk, and some white globular somatic embryos generally occurred on the surface of the callus (Fig. 1a). With the culture time prolongation to 3 wk, it was found that some somatic embryos germinated to develop into green adventitious shoots (Fig. 1b). Induction medium containing 5.0 M NAA could also induce callus. However, the induction of callus was slower and less callus formed compared with the callus rate and amount of 2,4-D. Somatic embryos and adventitious shoots were formed on the surface of callus within 23 wk. However, the quantity of somatic embryos or adventitious shoots was less than that induced by 5.0 M 2,4-D induced (Table 1). When the induction media contained BA or KIN alone, no callus was produced (Table 1). However, with the culture time prolongation to 1 mo., few shoots grew from the shoot base sections. These shoots might have originated and proliferated from the shoot bud primordia (Table 1). Effects of plant growth regulators on regeneration from the callus. When the 20 M 2,4-D-induced callus was transferred to the same medium for subculture, the callus proliferated normally to form yellow-white friable callus. However, no somatic embryo was visible on the callus (Table 2). As the callus was transferred to the medium

NAA, 5.0 M BA, 5.0 M KIN, and 5.0 M BA +1.0 M NAA. After 2 wk of dark culture, the formation of callus, somatic embryo, and adventitious shoot was investigated based on the appearance of developmental characters. Explant numbers per experiment were 70100. Experiments were repeated twice over a 2-month period. All experimental data were statistically analyzed by one-way ANOVA using the protected least significant difference test (p =0.05) to separate treatment means. Mass propagation and plant regeneration. The multiple shoots were transferred to different propagation media containing different concentrations of BA and NAA. After 45 d of light culture, their propagation indexes were investigated. For plant regeneration, the multiple shoots were divided into single or few shoots and then transferred to the generation medium containing 1.0 M 3-indolebutyTable 2. Proliferation and regeneration from the callus of Burma reed after culturing for 45 d Plant growth regulators in the media (M) 2,4-D 20 2,4-D 5.0 NAA 5.0 BA 5.0 KIN 5.0 BA 5.0+NAA 1.0 Observation results from the culture of callus Callus Callus, somatic embryo and adventitious shoots Callus, somatic embryo and adventitious shoots Adventitious shoots Adventitious shoots Adventitious shoots Mean number of shoot per explant

0a 45.3 c 42.1 c 31.5 bc 27.6 b 33.2 bc

The same letter in the same column denotes no significant difference by least significant difference (0.05) test.

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containing lower concentration of 2,4-D (5.0 M), some yellow-white callus proliferated normally and then globular somatic embryos generally developed within 1 wk. The somatic embryos germinated and some adventitious shoots developed and grew successively on the surface of the callus. With the culture time prolongation to 3 wk, it was found that more than 40 adventitious shoots developed on the medium (Table 2). On the medium containing 5.0 M NAA, somatic embryos and adventitious shoots developed on the surface of the callus within 1 wk. When the callus was transferred to the media containing 5.0 M BA, 5.0 M KIN, or 5.0 M BA + 1.0 M NAA, green adventitious shoots were developed within 7 d (Fig. 1c). With the culture time prolongation to 3 wk, the adventitious shoots developed into a multiple-shoot clump (Fig. 1d). Mass propagation and plant regeneration. When the multiple shoots were cultured on the different propagation media for light culture, the shoots could proliferate 37 times every 1.5 mo. (Table 3). Among the media, the combination of 10 M BA and 2.0 M NAA in the propagation medium could proliferate the highest multipleshoot propagation index (Fig. 1e). When the shoots were transferred to the medium containing 0.5 M IBA, root formation was observed within 1 wk (Fig. 1f). One mo. later, when the plantlets were transplanted to a sandy bed, 96% of the plantlets could survive in 2 wk.

Ma et al., 1998, 2002). Cytokinin (BA or KIN) did not play any role in the earlier induction period in Burma reed. However, as the callus or somatic embryo was induced, the cytokinin could enhance plant regeneration and recovery from somatic embryos. Optimal combinations of BA and NAA in the propagation media could also efficiently improve shoot proliferation in Burma reed. Through this study on tissue culture in Burma reed, the induction of callus, somatic embryogenesis, shoot proliferation, and plant regeneration were well established. This will provide a substantial base and an efficient protocol for future biotechnology research.
Acknowledgements Support from the Biology Special Program in Energy Plant Biotechnology & Biomass Conversion (KSCX2-SW130) of the Chinese Academy of Sciences, the Program of Hundred Talents of the Chinese Academy of Sciences, and the Program of Tropical and Subtropical Plant Germplasm Construction in Guangdong Province (2005B20801009) are greatly acknowledged.

References
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Discussion It is obvious that auxin (including 2,4-D and NAA) plays a key role in the induction of somatic embryogenesis from the shoot base section explants of Burma reed. However, high concentrations of auxin (e.g., 20 M 2,4-D) could not induce somatic embryogenesis; these concentrations could only induce friable callus. With the optimal concentrations of 2,4-D (5.0 M) or NAA (5.0 M) in the induction media, somatic embryos were induced on the surface of the callus, and some adventitious shoots also developed from the somatic embryos. The effects of 2,4-D on the tissue culture of Burma reed seem to reveal two key effects. Firstly, 2,4-D can induce callus and somatic embryogene-sis, and secondly, it restrains further development of the somatic embryo. Our studies concur with previous studies on the induction of somatic embryogenesis in some other plants such as vetiver (Marco et al., 1993; Ma et al., 2000;) sugarcane (Ho and Vasil, 1983; Fitch and Moore, 1993), P. notatum (Marousky and West, 1990) and cassava (Raemakers et al., 1993;

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MA ET AL. Raemakers, C. J. J. M.; Schavemake, C. M.; Jacobsen, E.; Visser, R. G. F. Improvements of cyclic somatic embryogenesis of cassava (Manihot esculenta Crantz). Plant Cell Rep. 12: 226229; 1993. Ramamurthi, R.; Kastury, S.; Smith, W. H. (ed). Bioenergy: Vision for the New Millennium. Science Publishers Inc., Enfield. 2001. Sun, F. Z. Valuation of Burma reed in water and soil conservation and slope stabilization. Grassland and Turf 104: 6669; 2004. Tsao, G. T. Recent progress in bioconversion of lignocelluloses. Springer-Verlag, Berlin Heidelberg New York. 1999. Yuan, Z. Research and development on biomass energy in China. Int. J. Energy Tech. Policy 1: 108; 2002.

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