Plant Cell Tiss Organ Cult (2007) 89:75–81 DOI 10.1007/s11240-007-9210-y
RESEARCH NOTE
Somatic embryogenesis of Myrciaria aureana (Brazilian grape tree) Sergio Yoshimitsu Motoike Æ Edson Santana Saraiva Æ Marilia Contin Ventrella Æ Crislene Viana Silva Æ Luiz Carlos Chamhum Saloma˜o
Received: 31 July 2006 / Accepted: 21 January 2007 / Published online: 6 March 2007 Springer Science+Business Media B.V. 2007
Abstract The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation. For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl-L-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction and regeneration of somatic embryo in Myrciaria aureana. The combination of light
S. Y. Motoike (&) E. S. Saraiva C. V. Silva L. C. C. Saloma˜o Departamento de Fitotecnia, Universidade Federal de Vic¸osa (UFV), Vic¸osa, MG 36570-000, Brazil e-mail:
[email protected] M. C. Ventrella Departamento de Biologia Vegetal, Universidade Federal de Vic¸osa (UFV), Vic¸osa, MG 36570-000, Brazil
and PEG increased the number of mature embryos; however, charcoal was detrimental to the process. Keywords Charcoal In vitro culture Jaboticaba Light Myrtaceae Polyethylene glycol Abbreviations Ch Activated charcoal BM Basal medium 2,4-D 2,4-Dichlorophenoxyacetic acid IASP Indole-3-acetyl-L-aspartic acid IBA Indole-3-butyric acid JADS Correia et al. (1995) medium NN Nitsch and Nitsch (1969) medium PAR Photosynthetically active radiation PEG Polyethylene glycol PEM Pro-embryogenic mass PVP Polyvinyl-pyrrolidone RM1 Regeneration medium 1 RM2 Regeneration medium 2 Jaboticaba, known as Brazilian grape tree, is an Atlantic Forest species popular in Brazil. It is a multistemmed short tree exhibiting a cauliflower habit in which the flower cushions and berries are borne directly on the main trunk and old branches (Barros et al. 1996; Magallha˜es et al. 1996). The ripened berries resemble the size and shape of a
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common grape, and vary in colour from green to deep purple or blackish depending on the species. The berries are sweet and rich in anthocyanin with high antioxidant activity (Einbond et al. 2004). These are consumed fresh or transformed into fruit wine, liquor, and jelly. Their extract is also used to dye wine and vinegar. Most species of Jaboticaba are distributed within the genus Myrciaria of the family Myrtaceae. According to Mattos (1983), there are nine species of Brazilian grape tree, among these species, two Myrciaria cauliflora (Mart.) Berg and M. jaboticaba (Vell.) Berg, are the most widely cultivated. However, many species are not cultivated and threatened by extinction, including M. espı´rito-santenses Mattos, M. grandifolia Mattos, and M. aureana Mattos (Mattos 1983; Donadio 2000). Recently, cryopreservation was successfully applied to the conservation of Myrciaria spp. in our laboratory, but it was achieved only when somatic embryogenic cells were used. Among the genus Myrciaria, somatic embryogenesis was only reported for M. cauliflora (Litz 1984). For this species which is naturally polyembryonic, the in vitro somatic embryogenesis was achieved using ovules as explant. However, most threatened Myrciaria spp. are monoembryonic (Mendonc¸a 2000) and it is more difficult to obtain somatic embryogenesis. This paper reports the establishment of a longterm somatic embryogenic culture of White Jaboticaba M. aureana, a monoembryonic species of the genus Myrciaria, describing induction, regeneration, and germination of somatic embryos. This research was conducted in the Plant Cell and Tissue Culture Laboratory at the Federal University of Vicosa (UFV) in Brazil, using genetic material from the jaboticaba germplasm collection maintained by the Department of Plant Science at UFV. For the induction of somatic embryogenesis in M. aureana, cotyledons of zygotic embryos were used as explants. The zygotic embryos were obtained from mature seeds derived from ripe M. aureana fruits. The seeds were manually extracted from these fruits, rubbed against coarse hydrated lime powder for the elimination of their
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mucilage, and washed in a current of tap water until clean. After cleaning, the seeds were disinfested as follow: 15 min in a solution of captan 2.5% (w/v), 1 min in alcohol 70%, and an additional 60 min in a solution of sodium hypochlorite 2.5% (v/v) and Tween-20 0.01% (v/v). After disinfestation, the seeds were rinsed three times in sterile distilled water. The zygotic embryos were extracted by elimination of the seed coat with scalpel and forceps. The cotyledons were obtained by splitting the zygotic embryo longitudinally into half. The whole cotyledon was used as explants. Three doses (5.0, 10.0, and 20.0 lM) of 2,4-dichlorophenoxyacetic acid (2,4-D) were tested for the induction of somatic embryogenesis in M. aureana. The experiment was designed in a completely randomized fashion with five replications per treatment. Each replication was represented by one 100 · 15 mm2 disposable Petri dish containing 10 cotyledons. The basal medium (BM) used in this experiment consisted of Nitsch and Nitsch (Nitsch and Nitsch 1969) salts, 30 g L–1 sucrose, 100 mg L–1 myo-inositol, 800 mg L–1 polyvinyl-pyrrolidone (PVP), and Staba vitamins (Staba 1969). The pH of the medium was adjusted to 5.7 ± 0.1 and gelled with 7.5 g L–1 Bacto agar. The medium was autoclaved at 121C and 1.5 atm for 20 min, and 30 ml of the medium distributed in the Petri dishes. The Petri dishes containing cotyledons were incubated in the dark at 25 ± 1C for 50 days. After this period, the experiment was subjected to the first visual evaluation. After the first evaluation, the explants were halved with a scalpel and separated according to the induction treatment received initially. Then the halved explants were subcultured on two different media: the regeneration medium 1 (RM1) and regeneration medium 2 (RM2). RM1 and RM2 contained the nutrients and organic compounds of the BM medium, but different growth regulator combinations. RM1 included 30 lM 2,4-D and RM2 30 lM 2,4-D and 17 lM indole-3-acetyl-L-aspartic acid (IASP, dissolved in dimethyl sulfoxide). The pH of the media was adjusted to 5.7 ± 0.1 and autoclaved and distributed in Petri dishes as described above. The Petri dishes containing halved explants were incubated
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in the dark at 25 ± 1C. After 17 and 33 days, the experiment was subjected to the second and third visual evaluations. At the third evaluation, samples of embryogenic and nonembryogenic explants were randomly collected for anatomical studies. These samples were fixed in FAA50 (Johansen 1940) and conserved in alcohol 70% (v/v). For the preparation of permanent slides, the samples were dehydrated in an ethanol series and embedded in methacrylate (Historesin Leica) and sectioned transversally and longitudinally in 6.0 lm slices with a rotatory microtome. The sections were then stained with toluidine blue O (O’Brien et al. 1964) for 10 min, washed in water and dried at room temperature, and stained with Lugol reagent (Johansen 1940) for starch detection, and washed in water and dried again. The slides were mounted in synthetic resin (Permount) and
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analysed with a light microscope, and photos were obtained with a photomicroscope with a U-Photo system. For long-term maintenance of pro-embryogenic masses (PEMs), 10 small colonies of embryogenic tissue (2.0 mm in diameter) were selectively scooped from the cracked region of the explants (Fig. 1b) and subcultured onto RM2 in 100 · 15 mm2 Petri dishes. These cultures were incubated in the dark at 25C and subcultured every 30 days. The development and maturation of M. aureana somatic embryos occurred on BM medium without growth regulators. The combination of three factors were tested in a 2 · 2 · 2 factorial: 0 and 50 lmol m–2 s–1 photosynthetically active radiation (PAR) at the level of the medium surface provided by 40 W Cool-White fluorescent tubes (Sylvania), 0 and 50 g L–1 polyethylene glycol
Fig. 1 M. aureana Mattos somatic embryogenesis: (a) direct embryogenesis on the adaxial surface of cotyledon (arrows pointing to somatic embryos); (b) crack on cotyledon surface and development of embryogenic tissue (arrow); (c) somatic embryo regeneration and development from longterm pro-embryogenic mass; (d) matured somatic embryos; (e) comparing somatic embryos (white arrows) vs. zygotic embryo (black arrow); (f) somatic embryo germination
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(PEG, MW 3,350 Sigma-Aldrich), and 0 and 2.5 g L–1 activated charcoal (Synth, Brazil). The experiment was conducted in a completely randomized design with six replications. Each replication consisted of one Petri dish (100 · 15 mm2) with 10 PEMs of approximately 2.0 mm in diameter. The plates were incubated in a 25 ± 1C culture room. After 40 days of incubation, the experiment was evaluated, and the number of matured embryos obtained per PEM was determined. The germination of somatic embryos was obtained in a germination medium containing JADS salt (Correia et al. 1995), Staba vitamins (Staba 1969), 100 mg L–1 myo-inositol, 30 g L–1 sucrose, 2.5 g L–1 activated charcoal (Synth, Brazil), 800 mg L–1 PVP, 5.0 lM indole-3-butyric acid (IBA), and 8.0 g L–1 Bacto agar. The pH of the medium was adjusted to 5.7 ± 0.1 and autoclaved and distributed in Petri dishes as described above. Ten Petri dishes with 10 somatic embryos each were used in this assay. The Petri dishes were incubated in a culture room maintained at 25 ± 1C and 50 lmol m–2 s–1 PAR (40 W Cool-White fluorescent tubes, Sylvania) and 16 hday photoperiod for 60 days. The induction of somatic embryogenesis was achieved for Brazilian grape tree, M. aureana, using cotyledons of zygotic embryos as explants. Embryogenesis was observed after sequential passages of the explants on two different media: BM with 20 lM 2,4-D for 50 days and RM2 for another 50 days. None of the other treatments resulted in embryogenesis. In the first 50 days of incubation in BM with different levels of 2,4-D, the explants swelled doubling the original size. However, there was no sign of embryogenesis or callus formation during this period. The first sign of embryogenesis was observed 17 days after the transfer of the halved cotyledons from the BM medium with 2 lM 2,4-D into RM2. Initially, the somatic embryos developed as small protuberances formed at different regions on the adaxial face of the cotyledon associated or not with callus (Fig. 1a and b). The continuous growth of the explants led eventually to cracks on the surface of the cotyledons from where additional embryogenic tissue developed (Fig. 1b).
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The histological studies of the embryogenic explants showed that they are rich in starch. Abundant amyloplasts were found in two distinct regions of the explant: the peripheric region, which includes cells of the protoderm, and inner regions, constituted by storage parenchyma (Fig. 2b and c). These explants were also rich in phenolic compounds that had accumulated primarily in a subsuperficial zone between layers of cells of the peripheric region and storage parenchyma (Fig. 2b and c). Nonembryogenic explants were also rich in starch, but only in the storage parenchyma. Cells of the peripheric and subsuperficial zones in these explants accumulated phenolic compounds instead (Fig. 2a). The histological studies also showed that somatic embryos originated primarily from cells of the peripheric region (Fig. 2d). In most cases, these cells developed directly into somatic embryos without a callus interphase (Fig. 1a). Meristematic activity was observed in the cells between the subsuperficial zone rich in phenolic compounds and the storage parenchyma. The cells divided rapidly (Fig. 2b and c) which eventually lead to the cracks in the surface from which a mass of tissue with high embryogenic activity appeared (Fig. 1b). After 50 days of incubation, an average of 20% (2.0 ± 1.58 per Petri dish) of the explants coming from BM with 20 lM 2,4-D developed somatic embryos in RM2. The embryogenesis in M. aureana was asynchronous, showing different stages of embryo development simultaneously, including globular (Fig. 2d), heart (Fig. 2e), torpedo, and cotyledonary embryos (Fig. 2f). The developing embryos had a defined protoderm and a developing procambium in the heart, torpedo, and cotyledonary stage. They were also easily separated from the maternal explants and associated with a large amount of phenolic compounds (Fig. 2d), but no amyloplasts were found in their cells. Isolated PEMs have been maintained for up to 2 years by subculture on RM2 every 30 days. These PEMs grew up to four times in volume during 30 days of incubation. After several subcultures, they also became more synchronous. The maturation of somatic embryos taken from frequently subcultured PEMs was achieved after
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Fig. 2 Histological sections of M. aureana Mattos cotyledons in several stages through somatic embryogenesis. Transversal sections of nonembryogenic (a) and embryogenic (b–d) explants of M. aureana; longitudinal sections of M. aureana somatic embryos in heart (e), topedo and cotiledonary (f) stage. Large arrows show amiloplasts in black and small arrows show fenolic compounds in green. ET embryogenic tissue, GE globular embryo, PC procambium
40 days of incubation on growth regulator free BM and exposure to different treatments (Fig. 3). Embryo maturation was preceded by fast growth of PEMs which tripled in volume. Subsequently, maturing embryos arose from PEMs (Fig. 1c and d), however, embryo development was not synchronized. Embryos at different stages of development were observed after 40 days of incubation. The number of matured embryos obtained differed according to the treatments applied, however, no significant effect of the various experimental factors were noted when they were applied singly. The best combination was 50 lmol m–2 s–1 PAR and 50 g L–1 PEG, which resulted in the highest number of embryos, i.e., 207.83 embryos per inoculated PEM. The combination of activated charcoal with the other factors resulted in a significant reduction in embryo numbers (Fig. 3). The somatic embryos that developed from the PEM were initially yellowish to translucid (Fig. 1c). Later in maturation, they became green
to purplish (Fig. 1d), which appeared more intense in the presence of light than in the dark. The somatic embryos obtained were three to four times smaller than zygotic embryos not surpassing 2.5 mm in size (Fig. 1e), however, they morphologically resembled the zygotic embryos presenting large cotyledons and a reduced embryo axis. Abnormal development was common among somatic embryos, many presenting fused or multiple cotyledons. The germination of somatic embryos (Fig. 1f) was observed after 40 days of incubation on the germination medium. At 60 days of incubation, the germination index of somatic embryos was still low reaching maximum 20% (2.0 ± 1.15 embryos per Petri dish). No embryos germinated after 60 days of incubation. Several embryos started germination but did not complete the conversion. Those embryos presented roots but did not develop apical shoots. Although 2,4-D alone has been successfully used for the induction of somatic embryogenesis in several plant species, including M. cauliflora
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Number embryos
A 250 200
Light Dark
150 100 50
Number embryos
B 250 200
EG
G *
Ch +P
PE
Ch ns
*
ns
Co nt ro l
0
Ch No ch
150 100 50
Number embryos
C 250 200
* P EG +L ig ht
ns
Li gh t
G PE ns
ns
Co nt ro l
0
PEG No PEG
150 100 50
Li gh t *
Ch ns
Ch +L ig ht *
ns
Co nt ro l
0
Fig. 3 The effect of light (a), activated charcoal (b), and polyethylene glycol (c) on the number of matured somatic embryos of M. aureana Mattos. Ch activated charcoal, PEG polyethylene glycol
(Litz 1984), in M. aureana somatic embryogenesis was observed only when explants were cultured at the highest dose of 2,4-D and were subcultured onto medium supplemented with 2,4-D + IASP. In this study, embryogenic explants were associated with the accumulation of starch in the cells of peripheral regions from where direct embry-
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ogenesis occurred. Accumulation of starch has been correlated with early embryogenic stages in several species. It has been suggested that starch accumulation can be used as an early embryogeneic marker (Ho and Vasil 1983). However, this is not universally the case. For instance, Canhoto et al. (1996) found that nonembryogenic cells of Feijoa sellowiana (Myrtaceae) accumulate much higher quantities of starch than embryogenic cells, and young embryos originated from epidermal cells that did not contain starch. In M. aureana, starch was also not found in developing embryos. Light and PEG stimulated the development and maturation of somatic embryos. When these factors were combined, a higher number of somatic embryos was obtained. PEG is a nonplasmolysing osmoticum with an effect on embryogenic tissue that mimics the naturally occurring water stress in seeds during late stages of maturation. This stress causes a major change in gene expression of developing embryos (Stasolla et al. 2003). The application of PEG also increases the deposition of storage proteins similar to those that accumulate in zygotic embryos (Misra et al. 1993), increasing embryo number and quality. Activated charcoal often favours the development of somatic embryos (Motoike et al. 2001). However, in M. aureana charcoal was found to be detrimental to the process. According to Pan and Staden (1998), the effects of charcoal on the growth and development in vitro may be attributed to the establishment of a darkened environment, adsorption of inhibitory substances, and adsorption of growth regulators and other organic compounds. Acknowledgment This research was supported by a grant from Fundac¸a˜o de Amparo a Pesquisa de Minas Gerais (FAPEMIG).
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