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Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.)
Scientia Horticulturae 105 (2005) 411–420 www.elsevier.com/locate/scihorti
Short communication
Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.) Sumay Arunyanart a,*, Montira Chaitrayagun b a
Department of Horticulture, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology, Ladkrabang, Bangkok 10520, Thailand b Programme Agriculture, Faculty of Agricultural Technology, Rajabhat Institute Phuket, Phuket 83000, Thailand Received 5 July 2004; received in revised form 6 January 2005; accepted 31 January 2005
Abstract Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 mM 2,4dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 mM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 mM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 mM 2,4-D and 1 mM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 mM 2,4-D and 1 mM BA and subsequently transferred to media containing 2–4 mM 2,4-D and 0 or 0.5 mM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 mM 2,4-D and 1 mM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 mM 2,4-D and 0.5 mM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks. # 2005 Elsevier B.V. All rights reserved. Keywords: Somatic embryogenesis; 2,4-Dichlorophenoxy acetic acid (2,4-D); Benzyladenine (BA); 6-Furfuryl aminopurine (kinetin); Lotus
* Corresponding author. Tel.: +66 2 3264318; fax: +66 2 3264318. E-mail address: [email protected] (S. Arunyanart). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.01.034
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S. Arunyanart, M. Chaitrayagun / Scientia Horticulturae 105 (2005) 411–420
1. Introduction Lotus (Nelumbo nucifera Gaertn.) is a south-east Asian aquatic edible plant known for the delicate beauty of its water flowers. Tubers, nuts, young leaves, embryos and stamen are all edible and produced for local market in Thailand and flowers and rhizomes are used for medicinal purposes (Suvatabandhu, 1958). The production of edible parts in Thailand is low and seed and rhizome products are imported from China (Dusadeemaetha, 2003). The flowers are considered sacred by Buddhists and are used for decorative purposes but they exhibit a narrow range of colours and shapes. They are produced for local and export markets. There are four commercial varieties of lotus available in Thailand which have had limited development through breeding programmes. These varieties are vegetatively propagated through the use of rhizomes. There is considerable potential for improvement of these plants through the use of tissue culture and preliminary work on micropropagation (Arunyanart, 1998) and mutagenesis (Arunyanart and Soontronyatara, 2002) has been useful. The use of somatic embryogenesis in developmental studies, crop improvement and genetic transformation is well recognized (Dandekar, 1995; Jain et al., 1995; Neumann, 2000). This process allows not only clonal plant propagation but also specific changes that provide desirable, elite individuals through genetic engineering. The development of a somatic embryogenesis protocol for lotus will provide useful tool for the further development of this plant. So far, there are no reports on an efficient culture system for somatic embryo induction in this species. This work was undertaken to study the effect of different explant types and plant growth regulators on the induction of somatic embryogenesis in lotus.
2. Materials and methods 2.1. Explant sources and surface sterilization Seeds of lotus were rinsed in 70% ethanol for 1 min, then surface sterilized in 5% (v/v) NaOCl (plus two drops of tween20) for 20 min and finally rinsed three times in sterile distilled water. Two-week-old seedlings were used as a source of cotyledons and young leaves. The aquatic habit of lotus makes obtaining aseptic cultures from field-growing plants very difficult, therefore, surface sterilization was carried out using a sequence of rinses in sterilizing agents. Buds from rhizomes of field-grown plants of lotus cv. Satabankacha were initially rinsed in 70% ethanol for 1 min. Explants were then transferred to 0.1% (w/ v) mercuric chloride (plus two drops of tween20) for 10 min followed by 30 min in 5% (w/ v) calcium hypochlorite (plus two drops of tween20) and then 10 min in 1% calcium hypochlorite (plus two drops of tween20). Finally, the explants were rinsed three times in sterile distilled water. 2.2. Culture medium Murashige and Skoog (1962) (MS) medium supplemented with 2 g l 1 phytagel was used as the basal medium for all experiments. Media were adjusted to pH 5.5 before
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autoclaving at 121 8C for 20 min. Cultures were maintained in the dark at 25 8C for 16 weeks and sub-cultured every 4 weeks. 2.3. Callus induction Callus was initiated by culturing buds from rhizome, cotyledon and young leaf explants on media containing combinations of various plant growth regulators (PGR) using two independent experiments. In the first experiment, the effect of MS medium containing a combination of 0, 4, 6, 8 and 10 mM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 mM 6-furfuryl aminopurine (kinetin) was examined. In the second experiment, MS medium supplemented with a combination of 0, 4, 6, 8 and 10 mM 2,4-D and 0, 0.5 and 1.0 mM benzyl adenine (BA) was examined. A two factorial (PGR explant), randomized complete block design with five blocks (four explants per block) was used in each experiment (i.e. 20 explants per treatment). The growth of callus was recorded as the percent explants forming callus and callus weight. 2.4. Induction of somatic embryogenesis To induce somatic embryos, friable calli derived from buds explants on media with 4, 6, 8 or 10 mM 2,4-D and 1 mM BA (callus induction experiment 2) were transferred to MS medium containing 0, 2, 3, 4, or 5 mM 2,4-D and 0 or 0.5 mM BA. A two factorial (PGR callus response) in a completely randomized block design with three blocks (five calli within each block i.e. 15 explants per treatment) was used. Callus growth and somatic embryogenesis was recorded for the percentage of calli forming somatic embryos, weight of calli and callus score. Calli were scored as: (1) callus turned dark brown and no growth, (2) no differentiation of callus but remained yellow, (3) callus-formed plantlets with 1–4 leaves and 1–8 roots, (4) callusformed somatic embryos at proembryo, globular and heart shape stages and (5) callusformed somatic embryos at proembryo, globular, heart, torpedo and mature embryo stages. 2.5. Histology For histological observations, embryogenic calli at different developmental stages were fixed in FAA (formalin:glacial acetic acid:ethanol, 5:5:90, v/v) for 24 h, dehydrated through an ethanol–xylol series and embedded in paraffin wax. Tissues were sectioned at 3–4 mm and stained with 0.5% (w/v) fast green and 0.25% (w/v) safranin and examined under a microscope. 2.6. Statistical analysis Cultures were observed weekly. The percentage of explants forming callus, mean callus score (somatic embryo induction) and mean callus weight were analyzed using ANOVA and Duncan’s multiple range test.
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Table 1 Effect of 2,4-D and BA on explant growth in lotus 12 weeks of culture Bud
Auxin and cytokinin (mM)
Explant-formed callus (%) (S.E.)a
Cotyledon Explant and callus weight (mg) (S.E.)a
Explant-formed callus (%) (S.E.)a
Young leaf Explant and callus weight (mg) (S.E.)a
Explant-formed callus (%) (S.E.)a
Explant and callus weight (mg) (S.E.)a
2,4-D 0, BA 0 2,4-D 0, BA 0.5 2,4-D 0, BA 1
0.0 12.0 0.0 5.1 0.0 10.1
100 30 b 90 80 bc 50 50 bc
25.0 4.1 5.0 0.0 30.0 5.1
190 20 ab 130 90 cd 120 70 d
5.0 4.9 20.0 3.8 10.0 7.5
130 100 bc 450 80 a 190 70 b
2,4-D 4, BA 0 2,4-D 4, BA 0.5 2,4-D 4, BA 1
15.0 10.4 25.0 0.0 25.0 7.3
40 20 c 120 70 b 210 90 a
25.0 4.1 0.0 0.0 10.0 4.1
190 30 a–c 200 40 ab 220 70 a
25.0 3.2 25.0 0.0 20.0 3.8
80 50 c 100 30 bc 130 110 bc
2,4-D 6, BA 0 2,4-D 6, BA 0.5 2,4-D 6, BA 1
20.0 4.1 25.0 5.1 15.0 0.0
100 30 b 70 30 bc 130 100 b
5.0 4.6 15.0 8.2 0.0 0.0
190 20 ab 200 130 a–d 190 30 ab
55.0 2.8 20.0 3.2 20.0 8.6
90 30 bc 330 80 a 100 40 bc
2,4-D 8, BA 0 2,4-D 8, BA 0.5 2,4-D 8, BA 1
10.0 0.0 50.0 0.0 40.0 9.8
70 50 bc 70 80 bc 90 50 bc
0.0 7.5 0.0 11.2 25.09.2
190 30 a–c 220 40 a 140 40 b–d
60.0 0.0 20.0 5.9 20.0 0.0
90 20 bc 390 140 a 120 50 bc
2,4-D 10, BA 0 2,4-D 10, BA 0.5 2,4-D 10, BA 1
15.0 4.1 20.0 5.1 40.0 7.5
80 90 bc 90 120 bc 80 60 bc
5.05.0 15.09.6 15.05.1
140 30 b–d 200 50 ab 170 10 a–d
70.0 12.7 45.0 8.6 10.0 2.8
260 80 bc 120 60 bc 110 90 bc
F-test Regression CV (%)
ns L** 71.93
**
L ns 27.85
ns L ns 98.28
*
L ns 15.11
ns L** 57.42
ns: non-significant. a Values within a column followed by the same letter are not significantly different by Duncan’s multiple range test P < 0. * Significant at P < 0.05. ** Significant at P < 0.01.
**
L ns 24.29
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Explants
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3. Results 3.1. Callus induction After 12 weeks of culture, bud explants produced better callus growth than cotyledon and young leaf explants cultured on media with either combination of auxin and cytokinin (2,4-D and kinetin or 2,4-D and BA). However, combinations of 2,4-D and BA produced more callus than combinations of 2,4-D and kinetin for all explants (data not shown). Explants displaying better growth were cultured on media with combinations of 4, 6, 8 and 10 mM 2,4-D and 0.5 and 1 mM BA. Buds cultured on a medium containing 4 mM 2,4D and 1 mM BA produced the most growth (0.21 g; Table 1). All explants grew callus within 4 weeks of culture and continued to grow until the 12th week. Growth was reduced after 12 weeks and calli finally turned brown or died within 16 weeks. Bud explants enlarged and formed white or pale yellow and friable callus with a shiny surface (Fig. 1A). Callus from cotyledons was clear and glandular, particularly at the cut surfaces. Young leaf explants exhibited white or light brown, soft callus, which formed at the cut surfaces. 3.2. Induction of plantlets and somatic embryogenesis There was no statistical difference between any of the culture media (Table 2). Plantlets (Fig. 1B) were obtained from calli which were initially grown on media containing 4 mM 2,4-D and 1 mM BA or 10 mM 2,4-D and 1 mM BA and then transferred to medium with 0.5 mM BA or 2 mM 2,4-D and 0.5 mM BA (Table 2). Somatic embryos were produced on media containing 2–4 mM 2,4-D alone or with 0.5 mM BA (Table 2). However, the calli initiated on medium with 6 mM 2,4-D and 1 mM BA and transferred to medium with 4 mM 2,4-D and 0.5 mM BA gave the highest yield of somatic embryos (1.94) and a high proportion (22%) of calli-forming embryoids. Calliformed proembryos representing the early stages of somatic embryogenesis (Figs. 2A and 3A) within 4 weeks of culture and developed globular (Figs. 2B and 3B) and heart shape stages (Fig. 2C) within 12 weeks. Torpedo stage (Fig. 2D) and mature embryos (Figs. 2C and 3C) were obtained within 16 weeks. After 16 weeks, calli in all media started to die and there was no further development of somatic embryos.
Fig. 1. (A) Calli derived from bud cultured on MS medium containing 4 mM 2,4-D and 1 mM BA (bar = 1 mm) and (B) plantlet obtained from MS medium with 2 mM 2,4-D and 0.5 mM BA (bar =1 cm).
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Table 2 Effect of 2,4-D and BA on somatic embryo induction in lotus 12 weeks of culture Calli from medium with 4 mM 2,4-D and 1 mM BA Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
0.0 0.0 1.8 0.4 0.0 0.0 1.4 0.4
Callus and embryoid weight (mg) (S.E.)
Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
Callus and embryoid weight (mg) (S.E.)
Calli from medium with 8 mM 2,4-D and 1 mM BA Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
Callus and embryoid weight (mg) (S.E.)
Calli from medium with 10 mM 2,4-D and 1 mM BA Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
340 10 440 170
0.0 0.0 1.1 0.2 0.0 0.0 1.2 0.3
380 70 400 90
2,4-D 2, BA 0 11.1 0.5 1.6 0.2 2,4-D 2, BA 0.5 33.3 0.8 1.7 0.5
410 100 0.0 0.0 1.3 0.3 470 170 11.1 0.5 1.6 0.5
550 270 290 100
0.0 0.0 1.2 0.2 0.0 0.0 1.3 0.3
370 10 0.0 0.0 1.2 0.2 370 100 22.2 0.5 1.6 0.4
220 100 290 50
2,4-D 3, BA 0 0.0 0.0 1.4 0.2 2,4-D 3, BA 0.5 11.1 0.5 1.6 0.3
310 50 490 70
0.0 0.0 1.3 0.3 0.0 0.0 1.6 0.2
350 150 0.0 0.0 1.5 0.3 540 330 11.1 0.5 1.7 0.6
400 70 0.0 0.0 1.3 0.3 470 120 11.1 0.5 1.3 0.3
310 80 460 10
2,4-D 4, BA 0 0.0 0.0 1.3 0.3 2,4-D 4, BA 0.5 11.1 0.5 1.6 0.4
290 30 22.2 0.5 1.8 0.3 420 210 22.2 0.5 1.9 0.7
300 70 11.1 0.5 1.6 0.6 420 110 0.0 0.0 1.2 0.2
340 20 390 10
0.0 0.0 1.6 0.2 11.1 0.5 1.1 0.2
310 50 310 50
260 80 390 100
400 100 280 130
300 90 260 30
0.0 0.0 1.1 0.2 0.0 0.0 1.1 0.2
260 220 370 30
0.0 0.0 1.2 0.2 0.0 0.0 1.5 0.2
2,4-D 5, BA 0 2,4-D 5, BA 0.5 F-test Regression CV (%)
ns L ns 16.21
ns: non-significant.
ns L ns 26.74
ns L** 4.41
0.0 0.0 1.2 0.3 0.0 0.0 1.1 0.2 ns L ns 12.25
ns L ns 28.53
ns L ns 5.65
0.0 0.0 1.4 0.1 0.0 0.0 1.1 0.2 ns L ns 10.41
ns L ns 29.09
ns L ns 3.15
0.0 0.0 1.1 0.2 0.0 0.0 1.3 0.5
Callus and embryoid weight (mg) (S.E.)
0.0 0.0 1.0 0.0 0.0 0.0 1.6 0.1
2,4-D 0, BA 0 2,4-D 0, BA 0.5
590 240 680 70
Calli from medium with 6 mM 2,4-D and 1 mM BA
ns L ns 12.41
ns L ns 25.21
330 50 430 70
ns L ns 3.53
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Auxin and cytokinin (mM)
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Fig. 2. Stage of somatic embryos obtained from calli after 4–12 weeks culturing on MS medium with 2,4-D and BA: (A) proembryo (arrow), (B) globular shaped (arrow), (C) heart shaped (arrow), (D) torpedo shaped and (E) mature embryo. Each bar = 1 mm.
4. Discussion The initiation of somatic embryogenic cultures is influenced by many factors such as explant types and plant growth regulators especially auxins and cytokinins (Jain et al., 1995). In the present studies, bud explants produced better callus growth and somatic embryo formation than cotyledon and young leaf explants. This is similar to the results of in vitro shoot multiplication in which bud explants provided best shoot multiplication
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Fig. 3. Sections of somatic embryo: (A) proembryo (bar = 100 mm), (B) globular shaped (bar = 100 mm) and (C) mature embryo (bar = 250 mm).
(Arunyanart, 1998). Thus, buds of mature lotus plants, appear to be a better explant source than explants from seeds (juvenile). This is contrary to what is most frequently reported where juvenile explants are most responsive (Merkle et al., 1987; Jain et al., 1995; George, 1996). In most dicotyledons, the addition of a low concentration of cytokinin to media containing auxin tend to increase the growth rate of embryogenic callus (George, 1996). According to our results, bud explant on medium with 2,4-D and BA gave better growth of callus and somatic embryogenesis than media with 2,4-D and kinetin. Therefore, among the different cytokinins tested in combination with 2,4-D, BA was found superior; this result confirms earlier observations in other species (Ishii et al., 1998; Nhut et al., 2000; Ashok Kumar et al., 2003).
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Somatic embryogenesis can be accomplished in two stages: in the first, an embryogenic callus is obtained, and in the second, the callus or sometimes recognizable somatic embryos, are transferred to a different medium, where the embryos develop. In most cases, the concentrations of auxins and cytokinins used at the second stage have been less than the first stage (Ammirato, 1983; George, 1996). This is supported by our data where the highest somatic embryo production was obtained when calli were first cultured on a medium with 6 mM 2,4-D and 1 mM BA and then subsequently transferred to a medium with 4 mM 2,4-D and 0.5 mM BA. Also, the calli derived from medium with 10 mM 2,4-D and 1 mM BA and transferred to medium with only 4 mM 2,4-D also showed a similar response. This supports the general trend that a decrease of 2,4-D (auxin) and BA (cytokinin) in the second medium promotes somatic embryo development (Merkle et al., 1987; Kim and Soh, 1996; Fei et al., 2002). The present study successfully describes somatic embryogenesis from bud explant of lotus over a range of 2,4-D and BA concentrations. The optimum conditions for somatic embryo induction were achieved when calli produced from MS medium supplemented with 6 mM 2,4-D and 1 mM BA were sub-cultured to a medium containing 4 mM 2,4-D and 0.5 mM BA. Although, the frequency of somatic embryogenesis is still low, this protocol might help in the genetic improvement of this plant especially when combined with other developments in the tissue culture of lotus (Arunyanart and Soontronyatara, 2002). Further experiments, with other exogeneous plant growth regulators (such as NAA or TDZ) and different conditions (such as light effects), may be helpful in enhancing the frequencies.
Acknowledgements We thank Dr. I. Bennett and Mr. G.P. Baxter-Jones for critical reading of this manuscript.
References Ammirato, P.V., 1983. Embryogenesis. In: Evans, D.A., William, R.S., Ammirato, P.V., Yamada, Y. (Eds.), Handbook of Plant Cell Culture, vol. 1. Macmillan Publishing Company, New York, pp. 82–123. Arunyanart, S., 1998. In vitro culture of lotus (Nelumbo nucifera Gaernt.). In: Proceedings of the Symposium and 1998 Spring Meeting of Japanese Society for Horticultural Science. J. Jpn. Soc. Hort. Sci. 67 (Suppl.), 257. Arunyanart, S., Soontronyatara, S., 2002. Mutation induction by and X-ray irradiation in tissue cultured lotus. Plant Cell Tiss. Org. Cult. 70, 119–122. Ashok Kumar, H.G., Muethy, H.N., Paek, K.Y., 2003. Embryogenesis and plant regeneration from anther cultures of Cucumis sativas L.. Sci. Hort. 98, 213–222. Dandekar, A.M., 1995. Genetic transformation of angiosperm. In: Jain, S.M., Gupta, P.K. (Eds.), Somatic Embryogenesis in Woody Plants, vol. 1. Kluwer Acadamic Publishers, The Netherlands, pp. 193–226. Dusadeemaetha, P., 2003. Import values of lotus and waterlily in Thailand. In: Proceedings of the Waterlily as an Economic Crop of Thailand Conference. Kasetsart University, Bangkok, Thailand, (in Thai), pp. 56–59. Fei, S.Z., Riordan, T., Read, P., 2002. Stepwise decrease of 2,4-D and addition of BA in sub-culture medium stimulated shoot regeneration and somatic embryogenesis in buffalo grass. Plant Cell Tiss. Org. Cult. 70, 275– 279. George, E.F., 1996. Plant Propagation by Tissue Culture. Part 2. In Practice. Exegetics Ltd., Great Britain.
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Ishii, Y., Takamura, T., Goi, M., Tanaka, M., 1998. Callus induction and somatic embryogenesis of Phalaenopsis. Plant Cell Rep. 17, 446–450. Jain, S.M., Gupta, P.K., Newman, R.J., 1995. Somatic Embryogenesis in Woody Plants, vol. 2. Kluwer Acadamic Publishers, The Netherlands. Kim, J.W., Soh, W.Y., 1996. Plant regeneration through somatic embryogenesis from suspension cultures of Allium fistulosum L.. Plant Sci. 114, 215–220. Merkle, S.A., Wetzstein, H.Y., Sommer, H.E., 1987. Somatic embryogenesis in tissue cultures of pecan. HortScience 22, 128–130. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant 15, 473–497. Nhut, D.T., Le, B.V., Van, T.T., 2000. Somatic embryogenesis and direct shoot regeneration of rice (Oryza sativa L.) using thin cell layer cultures of apical meristematic tissue. J. Plant Physiol. 157, 559–565. Neumann, K.H., 2000. Some studies on somatic embryogenesis, a tool in plant biotechnology. In: Proceedings of the 87th Indian Science Congress, Pune, India. Suvatabandhu, K., 1958. On Nympheaceae of Thailand. Nat. Hist. Bull. 17, 11–15.
Short communication
Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.) Sumay Arunyanart a,*, Montira Chaitrayagun b a
Department of Horticulture, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology, Ladkrabang, Bangkok 10520, Thailand b Programme Agriculture, Faculty of Agricultural Technology, Rajabhat Institute Phuket, Phuket 83000, Thailand Received 5 July 2004; received in revised form 6 January 2005; accepted 31 January 2005
Abstract Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 mM 2,4dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 mM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 mM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 mM 2,4-D and 1 mM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 mM 2,4-D and 1 mM BA and subsequently transferred to media containing 2–4 mM 2,4-D and 0 or 0.5 mM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 mM 2,4-D and 1 mM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 mM 2,4-D and 0.5 mM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks. # 2005 Elsevier B.V. All rights reserved. Keywords: Somatic embryogenesis; 2,4-Dichlorophenoxy acetic acid (2,4-D); Benzyladenine (BA); 6-Furfuryl aminopurine (kinetin); Lotus
* Corresponding author. Tel.: +66 2 3264318; fax: +66 2 3264318. E-mail address: [email protected] (S. Arunyanart). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.01.034
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S. Arunyanart, M. Chaitrayagun / Scientia Horticulturae 105 (2005) 411–420
1. Introduction Lotus (Nelumbo nucifera Gaertn.) is a south-east Asian aquatic edible plant known for the delicate beauty of its water flowers. Tubers, nuts, young leaves, embryos and stamen are all edible and produced for local market in Thailand and flowers and rhizomes are used for medicinal purposes (Suvatabandhu, 1958). The production of edible parts in Thailand is low and seed and rhizome products are imported from China (Dusadeemaetha, 2003). The flowers are considered sacred by Buddhists and are used for decorative purposes but they exhibit a narrow range of colours and shapes. They are produced for local and export markets. There are four commercial varieties of lotus available in Thailand which have had limited development through breeding programmes. These varieties are vegetatively propagated through the use of rhizomes. There is considerable potential for improvement of these plants through the use of tissue culture and preliminary work on micropropagation (Arunyanart, 1998) and mutagenesis (Arunyanart and Soontronyatara, 2002) has been useful. The use of somatic embryogenesis in developmental studies, crop improvement and genetic transformation is well recognized (Dandekar, 1995; Jain et al., 1995; Neumann, 2000). This process allows not only clonal plant propagation but also specific changes that provide desirable, elite individuals through genetic engineering. The development of a somatic embryogenesis protocol for lotus will provide useful tool for the further development of this plant. So far, there are no reports on an efficient culture system for somatic embryo induction in this species. This work was undertaken to study the effect of different explant types and plant growth regulators on the induction of somatic embryogenesis in lotus.
2. Materials and methods 2.1. Explant sources and surface sterilization Seeds of lotus were rinsed in 70% ethanol for 1 min, then surface sterilized in 5% (v/v) NaOCl (plus two drops of tween20) for 20 min and finally rinsed three times in sterile distilled water. Two-week-old seedlings were used as a source of cotyledons and young leaves. The aquatic habit of lotus makes obtaining aseptic cultures from field-growing plants very difficult, therefore, surface sterilization was carried out using a sequence of rinses in sterilizing agents. Buds from rhizomes of field-grown plants of lotus cv. Satabankacha were initially rinsed in 70% ethanol for 1 min. Explants were then transferred to 0.1% (w/ v) mercuric chloride (plus two drops of tween20) for 10 min followed by 30 min in 5% (w/ v) calcium hypochlorite (plus two drops of tween20) and then 10 min in 1% calcium hypochlorite (plus two drops of tween20). Finally, the explants were rinsed three times in sterile distilled water. 2.2. Culture medium Murashige and Skoog (1962) (MS) medium supplemented with 2 g l 1 phytagel was used as the basal medium for all experiments. Media were adjusted to pH 5.5 before
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autoclaving at 121 8C for 20 min. Cultures were maintained in the dark at 25 8C for 16 weeks and sub-cultured every 4 weeks. 2.3. Callus induction Callus was initiated by culturing buds from rhizome, cotyledon and young leaf explants on media containing combinations of various plant growth regulators (PGR) using two independent experiments. In the first experiment, the effect of MS medium containing a combination of 0, 4, 6, 8 and 10 mM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 mM 6-furfuryl aminopurine (kinetin) was examined. In the second experiment, MS medium supplemented with a combination of 0, 4, 6, 8 and 10 mM 2,4-D and 0, 0.5 and 1.0 mM benzyl adenine (BA) was examined. A two factorial (PGR explant), randomized complete block design with five blocks (four explants per block) was used in each experiment (i.e. 20 explants per treatment). The growth of callus was recorded as the percent explants forming callus and callus weight. 2.4. Induction of somatic embryogenesis To induce somatic embryos, friable calli derived from buds explants on media with 4, 6, 8 or 10 mM 2,4-D and 1 mM BA (callus induction experiment 2) were transferred to MS medium containing 0, 2, 3, 4, or 5 mM 2,4-D and 0 or 0.5 mM BA. A two factorial (PGR callus response) in a completely randomized block design with three blocks (five calli within each block i.e. 15 explants per treatment) was used. Callus growth and somatic embryogenesis was recorded for the percentage of calli forming somatic embryos, weight of calli and callus score. Calli were scored as: (1) callus turned dark brown and no growth, (2) no differentiation of callus but remained yellow, (3) callus-formed plantlets with 1–4 leaves and 1–8 roots, (4) callusformed somatic embryos at proembryo, globular and heart shape stages and (5) callusformed somatic embryos at proembryo, globular, heart, torpedo and mature embryo stages. 2.5. Histology For histological observations, embryogenic calli at different developmental stages were fixed in FAA (formalin:glacial acetic acid:ethanol, 5:5:90, v/v) for 24 h, dehydrated through an ethanol–xylol series and embedded in paraffin wax. Tissues were sectioned at 3–4 mm and stained with 0.5% (w/v) fast green and 0.25% (w/v) safranin and examined under a microscope. 2.6. Statistical analysis Cultures were observed weekly. The percentage of explants forming callus, mean callus score (somatic embryo induction) and mean callus weight were analyzed using ANOVA and Duncan’s multiple range test.
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Table 1 Effect of 2,4-D and BA on explant growth in lotus 12 weeks of culture Bud
Auxin and cytokinin (mM)
Explant-formed callus (%) (S.E.)a
Cotyledon Explant and callus weight (mg) (S.E.)a
Explant-formed callus (%) (S.E.)a
Young leaf Explant and callus weight (mg) (S.E.)a
Explant-formed callus (%) (S.E.)a
Explant and callus weight (mg) (S.E.)a
2,4-D 0, BA 0 2,4-D 0, BA 0.5 2,4-D 0, BA 1
0.0 12.0 0.0 5.1 0.0 10.1
100 30 b 90 80 bc 50 50 bc
25.0 4.1 5.0 0.0 30.0 5.1
190 20 ab 130 90 cd 120 70 d
5.0 4.9 20.0 3.8 10.0 7.5
130 100 bc 450 80 a 190 70 b
2,4-D 4, BA 0 2,4-D 4, BA 0.5 2,4-D 4, BA 1
15.0 10.4 25.0 0.0 25.0 7.3
40 20 c 120 70 b 210 90 a
25.0 4.1 0.0 0.0 10.0 4.1
190 30 a–c 200 40 ab 220 70 a
25.0 3.2 25.0 0.0 20.0 3.8
80 50 c 100 30 bc 130 110 bc
2,4-D 6, BA 0 2,4-D 6, BA 0.5 2,4-D 6, BA 1
20.0 4.1 25.0 5.1 15.0 0.0
100 30 b 70 30 bc 130 100 b
5.0 4.6 15.0 8.2 0.0 0.0
190 20 ab 200 130 a–d 190 30 ab
55.0 2.8 20.0 3.2 20.0 8.6
90 30 bc 330 80 a 100 40 bc
2,4-D 8, BA 0 2,4-D 8, BA 0.5 2,4-D 8, BA 1
10.0 0.0 50.0 0.0 40.0 9.8
70 50 bc 70 80 bc 90 50 bc
0.0 7.5 0.0 11.2 25.09.2
190 30 a–c 220 40 a 140 40 b–d
60.0 0.0 20.0 5.9 20.0 0.0
90 20 bc 390 140 a 120 50 bc
2,4-D 10, BA 0 2,4-D 10, BA 0.5 2,4-D 10, BA 1
15.0 4.1 20.0 5.1 40.0 7.5
80 90 bc 90 120 bc 80 60 bc
5.05.0 15.09.6 15.05.1
140 30 b–d 200 50 ab 170 10 a–d
70.0 12.7 45.0 8.6 10.0 2.8
260 80 bc 120 60 bc 110 90 bc
F-test Regression CV (%)
ns L** 71.93
**
L ns 27.85
ns L ns 98.28
*
L ns 15.11
ns L** 57.42
ns: non-significant. a Values within a column followed by the same letter are not significantly different by Duncan’s multiple range test P < 0. * Significant at P < 0.05. ** Significant at P < 0.01.
**
L ns 24.29
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Explants
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3. Results 3.1. Callus induction After 12 weeks of culture, bud explants produced better callus growth than cotyledon and young leaf explants cultured on media with either combination of auxin and cytokinin (2,4-D and kinetin or 2,4-D and BA). However, combinations of 2,4-D and BA produced more callus than combinations of 2,4-D and kinetin for all explants (data not shown). Explants displaying better growth were cultured on media with combinations of 4, 6, 8 and 10 mM 2,4-D and 0.5 and 1 mM BA. Buds cultured on a medium containing 4 mM 2,4D and 1 mM BA produced the most growth (0.21 g; Table 1). All explants grew callus within 4 weeks of culture and continued to grow until the 12th week. Growth was reduced after 12 weeks and calli finally turned brown or died within 16 weeks. Bud explants enlarged and formed white or pale yellow and friable callus with a shiny surface (Fig. 1A). Callus from cotyledons was clear and glandular, particularly at the cut surfaces. Young leaf explants exhibited white or light brown, soft callus, which formed at the cut surfaces. 3.2. Induction of plantlets and somatic embryogenesis There was no statistical difference between any of the culture media (Table 2). Plantlets (Fig. 1B) were obtained from calli which were initially grown on media containing 4 mM 2,4-D and 1 mM BA or 10 mM 2,4-D and 1 mM BA and then transferred to medium with 0.5 mM BA or 2 mM 2,4-D and 0.5 mM BA (Table 2). Somatic embryos were produced on media containing 2–4 mM 2,4-D alone or with 0.5 mM BA (Table 2). However, the calli initiated on medium with 6 mM 2,4-D and 1 mM BA and transferred to medium with 4 mM 2,4-D and 0.5 mM BA gave the highest yield of somatic embryos (1.94) and a high proportion (22%) of calli-forming embryoids. Calliformed proembryos representing the early stages of somatic embryogenesis (Figs. 2A and 3A) within 4 weeks of culture and developed globular (Figs. 2B and 3B) and heart shape stages (Fig. 2C) within 12 weeks. Torpedo stage (Fig. 2D) and mature embryos (Figs. 2C and 3C) were obtained within 16 weeks. After 16 weeks, calli in all media started to die and there was no further development of somatic embryos.
Fig. 1. (A) Calli derived from bud cultured on MS medium containing 4 mM 2,4-D and 1 mM BA (bar = 1 mm) and (B) plantlet obtained from MS medium with 2 mM 2,4-D and 0.5 mM BA (bar =1 cm).
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Table 2 Effect of 2,4-D and BA on somatic embryo induction in lotus 12 weeks of culture Calli from medium with 4 mM 2,4-D and 1 mM BA Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
0.0 0.0 1.8 0.4 0.0 0.0 1.4 0.4
Callus and embryoid weight (mg) (S.E.)
Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
Callus and embryoid weight (mg) (S.E.)
Calli from medium with 8 mM 2,4-D and 1 mM BA Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
Callus and embryoid weight (mg) (S.E.)
Calli from medium with 10 mM 2,4-D and 1 mM BA Callus formed embryoid (%) (S.E.)
Score of callus and embryoid growth (S.E.)
340 10 440 170
0.0 0.0 1.1 0.2 0.0 0.0 1.2 0.3
380 70 400 90
2,4-D 2, BA 0 11.1 0.5 1.6 0.2 2,4-D 2, BA 0.5 33.3 0.8 1.7 0.5
410 100 0.0 0.0 1.3 0.3 470 170 11.1 0.5 1.6 0.5
550 270 290 100
0.0 0.0 1.2 0.2 0.0 0.0 1.3 0.3
370 10 0.0 0.0 1.2 0.2 370 100 22.2 0.5 1.6 0.4
220 100 290 50
2,4-D 3, BA 0 0.0 0.0 1.4 0.2 2,4-D 3, BA 0.5 11.1 0.5 1.6 0.3
310 50 490 70
0.0 0.0 1.3 0.3 0.0 0.0 1.6 0.2
350 150 0.0 0.0 1.5 0.3 540 330 11.1 0.5 1.7 0.6
400 70 0.0 0.0 1.3 0.3 470 120 11.1 0.5 1.3 0.3
310 80 460 10
2,4-D 4, BA 0 0.0 0.0 1.3 0.3 2,4-D 4, BA 0.5 11.1 0.5 1.6 0.4
290 30 22.2 0.5 1.8 0.3 420 210 22.2 0.5 1.9 0.7
300 70 11.1 0.5 1.6 0.6 420 110 0.0 0.0 1.2 0.2
340 20 390 10
0.0 0.0 1.6 0.2 11.1 0.5 1.1 0.2
310 50 310 50
260 80 390 100
400 100 280 130
300 90 260 30
0.0 0.0 1.1 0.2 0.0 0.0 1.1 0.2
260 220 370 30
0.0 0.0 1.2 0.2 0.0 0.0 1.5 0.2
2,4-D 5, BA 0 2,4-D 5, BA 0.5 F-test Regression CV (%)
ns L ns 16.21
ns: non-significant.
ns L ns 26.74
ns L** 4.41
0.0 0.0 1.2 0.3 0.0 0.0 1.1 0.2 ns L ns 12.25
ns L ns 28.53
ns L ns 5.65
0.0 0.0 1.4 0.1 0.0 0.0 1.1 0.2 ns L ns 10.41
ns L ns 29.09
ns L ns 3.15
0.0 0.0 1.1 0.2 0.0 0.0 1.3 0.5
Callus and embryoid weight (mg) (S.E.)
0.0 0.0 1.0 0.0 0.0 0.0 1.6 0.1
2,4-D 0, BA 0 2,4-D 0, BA 0.5
590 240 680 70
Calli from medium with 6 mM 2,4-D and 1 mM BA
ns L ns 12.41
ns L ns 25.21
330 50 430 70
ns L ns 3.53
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Auxin and cytokinin (mM)
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Fig. 2. Stage of somatic embryos obtained from calli after 4–12 weeks culturing on MS medium with 2,4-D and BA: (A) proembryo (arrow), (B) globular shaped (arrow), (C) heart shaped (arrow), (D) torpedo shaped and (E) mature embryo. Each bar = 1 mm.
4. Discussion The initiation of somatic embryogenic cultures is influenced by many factors such as explant types and plant growth regulators especially auxins and cytokinins (Jain et al., 1995). In the present studies, bud explants produced better callus growth and somatic embryo formation than cotyledon and young leaf explants. This is similar to the results of in vitro shoot multiplication in which bud explants provided best shoot multiplication
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Fig. 3. Sections of somatic embryo: (A) proembryo (bar = 100 mm), (B) globular shaped (bar = 100 mm) and (C) mature embryo (bar = 250 mm).
(Arunyanart, 1998). Thus, buds of mature lotus plants, appear to be a better explant source than explants from seeds (juvenile). This is contrary to what is most frequently reported where juvenile explants are most responsive (Merkle et al., 1987; Jain et al., 1995; George, 1996). In most dicotyledons, the addition of a low concentration of cytokinin to media containing auxin tend to increase the growth rate of embryogenic callus (George, 1996). According to our results, bud explant on medium with 2,4-D and BA gave better growth of callus and somatic embryogenesis than media with 2,4-D and kinetin. Therefore, among the different cytokinins tested in combination with 2,4-D, BA was found superior; this result confirms earlier observations in other species (Ishii et al., 1998; Nhut et al., 2000; Ashok Kumar et al., 2003).
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Somatic embryogenesis can be accomplished in two stages: in the first, an embryogenic callus is obtained, and in the second, the callus or sometimes recognizable somatic embryos, are transferred to a different medium, where the embryos develop. In most cases, the concentrations of auxins and cytokinins used at the second stage have been less than the first stage (Ammirato, 1983; George, 1996). This is supported by our data where the highest somatic embryo production was obtained when calli were first cultured on a medium with 6 mM 2,4-D and 1 mM BA and then subsequently transferred to a medium with 4 mM 2,4-D and 0.5 mM BA. Also, the calli derived from medium with 10 mM 2,4-D and 1 mM BA and transferred to medium with only 4 mM 2,4-D also showed a similar response. This supports the general trend that a decrease of 2,4-D (auxin) and BA (cytokinin) in the second medium promotes somatic embryo development (Merkle et al., 1987; Kim and Soh, 1996; Fei et al., 2002). The present study successfully describes somatic embryogenesis from bud explant of lotus over a range of 2,4-D and BA concentrations. The optimum conditions for somatic embryo induction were achieved when calli produced from MS medium supplemented with 6 mM 2,4-D and 1 mM BA were sub-cultured to a medium containing 4 mM 2,4-D and 0.5 mM BA. Although, the frequency of somatic embryogenesis is still low, this protocol might help in the genetic improvement of this plant especially when combined with other developments in the tissue culture of lotus (Arunyanart and Soontronyatara, 2002). Further experiments, with other exogeneous plant growth regulators (such as NAA or TDZ) and different conditions (such as light effects), may be helpful in enhancing the frequencies.
Acknowledgements We thank Dr. I. Bennett and Mr. G.P. Baxter-Jones for critical reading of this manuscript.
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