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In Vitro Cell.Dev.Biol.—Plant (2007) 43:16–20 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 μM 6benzylaminopurine (BA) and 2.0 μM 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 reed’s 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

the multiple shoots were divided into single shoots. The culture jars were placed in an air-conditioned culture room at 26±2°C with 14/10 h photoperiod providing 80 μmol m−2 s−1 fluorescent light and subcultured every 2 mo. then inoculated on (Murashige and Skoog.0 μM kineatin (KIN).4-D. 5. Effect of plant growth regulators on regeneration from the callus. The media contained 20 μM 2. 5. 5. Shoot mass propagation (e).4-D. the induced callus clumps were transferred to different media for further development. 5. rinsed in three changes of sterile distilled water.4-D.4dichlorophenoxyacetic acid (2. All the media contained 30 g l−1 sucrose and were adjusted to pH 5. Figure 1.0 μM 2. 5.0 μM naphthaleneacetic acid (NAA) for shoot propagation. the culture jars were then transferred to light culture. then transferred to different induction media for dark culture.0 μM NAA for 7 (c) and 21 d (d). respectively. After 6 mo. Effect of plant growth regulators on induction of somatic embryogenesis. The induction media contained 20 μM 2.0 μM NAA. or adventitious shoot formation were investigated. China).1% mercuric chloride for 8 min. The induction of callus. 1962) basal medium supplemented with 10 μM 6benzylaminopurine (BA) and 2.7 and solidified with 0.SOMATIC EMBRYOGENESIS AND SHOOT FORMATION IN BURMA REED 17 explants were sterilized in 70% alcohol for 10 s and 0.0 μM BA. of subcultures for shoot mass propagation. After the shoot base explants were cultured for 14 d in darkness.6% agar (Shantou.0 μM BA and 1. Adventitious shoots were developed from the callus after transferring to the medium containing 5. somatic embryos. Somatic embryogenesis and adventitious shoot formation in Burma reed (bar = 2 mm). and 5.4-D).4-D for 21 d in darkness. Globular somatic embryos (a) and green adventitious shoots (b) were induced on the induction medium containing 5 μM 2. Shoot base sections were cut into 5-mm-long explants.4-D after culturing for 14 and 21 d. After the shoot base section explants were cultured on the induction medium containing 20 μM 2. Root formation within 7 d after transferred to the regeneration medium ( f ). respectively.0 μM .0 μM 2.

0 μM KIN. the quantity of somatic embryos or adventitious shoots was less than that induced by 5. When the 20 μM 2. Results Effect of plant growth regulators on induction of somatic embryogenesis. For plant regeneration.0 1.8 3. callus was also induced within 1 wk.6 b 33.0+NAA 1. then directly transplanted to a sandy propagation bed.0 Observation results from the culture of callus Callus Callus. the friable yellow-white callus proliferated normally. As the callus was transferred to the medium NAA. All experimental data were statistically analyzed by one-way ANOVA using the protected least significant difference test (p =0. After 45 d of light culture. no callus was produced (Table 1). few shoots grew from the shoot base sections. Table 3. 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 (μM) 2. When the induction media contained BA or KIN alone.5 bc 27.4-D induced (Table 1).05) to separate treatment means. 3. 5. However.5 6. .0 μM BA.18 MA ET AL. After 2 wk of dark culture.0 μM 2. Explant numbers per experiment were 70–100.4-D. it was found that some somatic embryos germinated to develop into green adventitious shoots (Fig. Experiments were repeated twice over a 2-month period.05) test.0 Propagation index per 45 d 6. somatic embryo and adventitious shoots Adventitious shoots Adventitious shoots Adventitious shoots Mean number of shoot per explant 0a 45.1 c 31.0 BA 5. shoots No callus. On the induction medium containing 5.0 KIN 5. 5.2 bc The same letter in the same column denotes no significant difference by least significant difference (0.0 NAA 5. Proliferation and regeneration from the callus of Burma reed after culturing for 45 d Plant growth regulators in the media (μM) 2.0+NAA 1. the induction of callus was slower and less callus formed compared with the callus rate and amount of 2. These shoots might have originated and proliferated from the shoot bud primordia (Table 1).1 c c b a Table 1.05) test. With culture time prolongation.1 d NAA 5. however. the formation of callus. with the culture time prolongation to 1 mo. somatic embryo and adventitious shoot buds Callus. their propagation indexes were investigated. Shoot mass propagation of Burma reed in different media Plant growth regulator combination in the propagation media (μM) BA BA BA BA 20+NAA 2. 13. When the shoot base section explants were cultured on the induction medium containing 20 μM 2.3 c 42. The multiple shoots were transferred to different propagation media containing different concentrations of BA and NAA. However.9 4.1 b 2.0 KIN 5.0 5. no somatic embryo formation was observed.0 μM BA +1. shoots The same letter in the same column denotes no significant difference by least significant difference (0.4-D 5. 1b). and adventitious shoot was investigated based on the appearance of developmental characters.0 BA 5.4-D 20 2. the callus proliferated normally to form yellow-white friable callus. However. Effects of plant growth regulators on regeneration from the callus. 1a). and 5.0 Observation results from the explants Mean number of adventitious shoot per explant 0a 37.05) test.4-D.0+NAA 1. no somatic embryo was visible on the callus (Table 2).4-D for dark culture.6 c ric acid (IBA) for root formation. Somatic embryos and adventitious shoots were formed on the surface of callus within 2–3 wk..0 Callus Callus. of culture. Induction medium containing 5.6 b The same letter in the same column denotes no significant difference by least significant difference (0. After 1 mo.4-D 20 2.0 μM 3-indolebutyTable 2. the multiple shoots were divided into single or few shoots and then transferred to the generation medium containing 1. and some white globular somatic embryos generally occurred on the surface of the callus (Fig.0 μM NAA could also induce callus. With the culture time prolongation to 3 wk.0 μM NAA. the plantlets were removed from jars and rinsed of agar.0 10+NAA 2. Mass propagation and plant regeneration. somatic embryo and adventitious shoot buds No callus.4-D 5.0 μM 2. somatic embryo and adventitious shoots Callus. somatic embryo.0 BA 5.4-D-induced callus was transferred to the same medium for subculture. some yellow-white callus was induced at the cut surface of the explant within 1 wk. However.

L. M. later.) I. Discussion on utilization and benefit of Burma reed.) sugarcane (Ho and Vasil. Induction of somatic embryogenesis and adventitious shoot formation from immature leaves of cassava. 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. the cytokinin could enhance plant regeneration and recovery from somatic embryos. K.0 μM BA + 1. With the culture time prolongation to 3 wk. Q. 96% of the plantlets could survive in 2 wk. Direct primary somatic embryogenesis and shoot formation in Manihot esculenta. Plant Cell Tiss. Silvano. Cult. Cult. G. Burma reed. H. I. 1983. Xian. Xian. as the callus or somatic embryo was induced. 16: 67–70. Y. J. Somatic embryogenesis in sugarcane (Saccharum officianarum L. 1990) and cassava (Raemakers et al. Studies on the Utilization of Burma reed. Pergamon Press. W. N. G. Org. 2000. 2004. H.0 μM NAA.0 μM). 35: 267–271. 1960. 1993. Xu. Acknowledgements Support from the Biology Special Program in Energy Plant Biotechnology & Biomass Conversion (KSCX2-SW130) of the Chinese Academy of Sciences. these concentrations could only induce friable callus. 1990. somatic embryogenesis.g. root formation was observed within 1 wk (Fig. On the medium containing 5. Mass propagation and plant regeneration. Agronomy 16: 97–110. 2005. New York. Energy plant species. Ma G. When the shoots were transferred to the medium containing 0. However. H.. Deng. Our studies concur with previous studies on the induction of somatic embryogenesis in some other plants such as vetiver (Marco et al. London. Q. J. some yellow-white callus proliferated normally and then globular somatic embryos generally developed within 1 wk. Bot. 2002). Discussion It is obvious that auxin (including 2.4-D on the tissue culture of Burma reed seem to reveal two key effects.4-D and NAA) plays a key role in the induction of somatic embryogenesis from the shoot base section explants of Burma reed. and plant regeneration were well established. shoot proliferation. Firstly. 1993. India and Pakistan. Q.. H. 1983. J. P. it was found that more than 40 adventitious shoots developed on the medium (Table 2). P. 2004. With the optimal concentrations of 2. M. M. .0 μM) in the induction media. When the multiple shoots were cultured on the different propagation media for light culture. However.. Acta Bot. Moore. H. Among the media. and some adventitious shoots also developed from the somatic embryos.. (Table 3). 1f). somatic embryos and adventitious shoots developed on the surface of the callus within 1 wk. B. notatum (Marousky and West. Org. Plant Cell Tiss. Xu. Somatic embryogenesis and plant regeneration from cultured mature caryopses of bahiagrass (Paspalum notatum Flugge). 16: 63–67. L. P. H.SOMATIC EMBRYOGENESIS AND SHOOT FORMATION IN BURMA REED 19 containing lower concentration of 2. Somatic embryogenesis and shoot formation from explants of Vetiveria zizanioides. or 5.4-D (5. H. Ma. Optimal combinations of BA and NAA in the propagation media could also efficiently improve shoot proliferation in Burma reed.. Lin.5 μM IBA. This will provide a substantial base and an efficient protocol for future biotechnology research. L. James & James Science Publishers. The Grasses of Burma. J. Vasil. Ceylon. 1998. and secondly. With the culture time prolongation to 3 wk. I. Y. Marousky. 1998. 8: 55–59.0 μM BA.. M. J. 1993. 40: 503–507. Clifton.. Cytokinin (BA or KIN) did not play any role in the earlier induction period in Burma reed. when the plantlets were transplanted to a sandy bed.0 μM) or NAA (5. 5. it restrains further development of the somatic embryo. Through this study on tissue culture in Burma reed. S. Lewandowski. Callus induction and plant regeneration in Vetiveria zizanioides. Protoplasma 118: 169–180. the induction of callus. 70: 281–288. 1e). 1993. Long-term culture of embryogenic sugarcane callus. their use and impact on environment and development.. the adventitious shoots developed into a multiple-shoot clump (Fig. J. 1993). Plant Cell Tiss. N. S. Marisa. Fujian Soil and Water Conserv. green adventitious shoots were developed within 7 d (Fig.. The morphology and physiology of callus formation and the ontogeny of somatic embryos. Org.4-D can induce callus and somatic embryogene-sis. an excellent grass for soil and water conservation... The effects of 2.. 2002. Trop.4-D (5. Europ. 2000. 2. 1998.. 32: 335–343.. 1c). Ma. F. X.0 μM NAA. El Bassam. H. C. Fujian Soil and Water Conserv. high concentrations of auxin (e. Fitch. the combination of 10 μM BA and 2. Fitch and Moore. Plant Cell Tiss. Ma et al. Q. Subtrop.. Xia.. G. The somatic embryos germinated and some adventitious shoots developed and grew successively on the surface of the callus. Sin. 1d). Marco.0 μM KIN. Org. Ma et al. S. Ho. References Bor. When the callus was transferred to the media containing 5.4-D) could not induce somatic embryogenesis. Screening Miscanthus genotypes in field trials to optimise biomass yield and quality in Southern Germany. Cult. West. Lin.0 μM NAA in the propagation medium could proliferate the highest multipleshoot propagation index (Fig. 20: 125–129. 2002. the shoots could proliferate 3–7 times every 1. somatic embryos were induced on the surface of the callus.5 mo. S. 20 μM 2. One mo. Longyan Univ. Cult. 23: 106–107.

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