AOBPreview originally published online on May 21, 2003
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Annals of Botany 92: 129-136, 2003
© 2003 Annals of Botany Company
Proliferation, Maturation and Germination of Castanea sativa Mill. Somatic Embryos Originated from Leaf Explants
1 Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Apartado 122, 15080 Santiago de Compostela, Spain
* For correspondence: Fax: +34 981592504, e-mail: amvieitez{at}iiag.cesga.es
Received: 6 November 2002; Returned for revision: 7 February 2003; Accepted: 17 March 2003 Published electronically: 21 May 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experiments were performed to determine the influence of proliferation medium on the maintenance of embryogenic competence and on repetitive embryogenesis in Castanea sativa Mill. somatic embryos derived from leaf explants. Somatic embryo proliferation was carried out by both direct secondary embryogenesis and by the culture of nodular callus tissue originated from cotyledons of somatic embryos. Both systems led to the production of cotyledonary somatic embryos on Murashige and Skoog proliferation medium supplemented with 0·1 mg l–1 benzyladenine and 0·1 mg l–1 naphthaleneacetic acid. Carbon source and concentration had a marked influence on maturation and subsequent germination ability of chestnut somatic embryos. Plantlet conversion was achieved in embryos matured on media with 6 % sucrose, and on 3 or 6 % maltose, whereas mean shoot length, root length and leaf number of produced plants were not significantly affected by these maturation media. Overall, the best results were obtained with 3 % maltose-matured somatic embryos, giving rise to 6 % plant recovery in addition to 33 % of embryos exhibiting only shoot development. The application of a 2-month cold treatment at 4 °C to somatic embryos matured on medium with 3 % maltose was necessary for achieving plant conversion, while partial desiccation did not appear to influence this response. A total of 39 % of embryos eventually produced plants either through conversion to plantlets or indirectly through rooting of shoots. Shoots formed by somatic embryos could be excised, multiplied and rooted following the micropropagation procedures previously developed for chestnut.
Key words: Castanea sativa, chestnut, cold treatment, germination, plant conversion, secondary embryogenesis, somatic embryogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There is growing interest in developing somatic embryogenesis systems for chestnut (Holliday and Merkle, 2000; Ballester et al., 2001) as a useful and efficient method for clonal mass propagation of selected material, genetic transformation and germplasm cryopreservation. The regeneration of transgenic chestnut plants through somatic embryogenesis, with introduced fungus-resistant genes against blight and/or ink diseases would be of great importance for chestnut improvement (Maynard et al., 1998; Holliday and Merkle, 2000; San-José et al., 2001). However, attempts to produce such transgenics have failed owing to the poor regeneration rates of the somatic embryogenesis protocols used (Merkle and Carraway, 1994; Maynard et al., 1998). Another difficulty is related to the fact that in the genus Castanea, somatic embryogenesis has only been induced from immature zygotic embryos and, therefore, availability of starting material is limited (Merkle et al., 1991; Vieitez, 1995; Carraway and Merkle, 1997; Xing et al., 1999). However, Corredoira (2002) demonstrated that induction of somatic embryos is also possible from in vitro clonal material, i.e. in tissues other than zygotic embryos. In that case embryogenic lines were established from leaf explants of shoots multiplied in tissue cultures. The study focused on somatic embryo induction, and no detailed results were presented on proliferation, germination and plant recovery from these somatic embryos.
A major limitation of the embryogenic systems used in chestnut is the maintenance of embryogenic competence and the low conversion rate of somatic embryos into plants. Attempts to recover plants from C. dentata somatic embryos failed to achieve values higher than 9 % (Xing et al., 1999), whereas 30 % plant regeneration was reported in the Castanea sativa x C. crenata hybrid after exposure to a chilling treatment (Vieitez, 1995).
In a number of species, low conversion rates have been shown to be due to poor somatic embryo quality and a lack of maturation and desiccation tolerance (Etienne et al., 1993). A common procedure for the maturation of somatic embryos in conifers is the culture of embryogenic tissue on medium with decreased osmotic water potential compared with the maintenance medium (Klimaszewska et al., 2000). The maturation of somatic embryos of conifers, in the presence of osmotic agents, gives rise to hydric stress and the induction of storage product synthesis (Yeung, 1995). It is conceivable that in Castanea somatic embryogenesis is a maturation stage allowing the synthesis and accumulation of storage compounds, and the acquisition of desiccation tolerance has to be completed prior to germination. The use of an appropriate concentration and type of carbohydrate in the culture medium has increased the maturation and germination ability of somatic embryos of a variety of species including the related species Quercus robur (Sánchez et al., 2003), but this aspect has not been extensively investigated in chestnut.
The aim of this study was to develop protocols for the maintenance of embryogenic competence, maturation and germination of C. sativa somatic embryos derived from leaf explants, by investigating the effects of plant growth regulators, and carbohydrate type and concentration. The suitability of applying pre-germination treatments, such as cold treatment and partial desiccation, was also evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plant material and culture conditions
Embryogenic cultures were initiated from leaf explants excised from in vitro-cloned shoots of Castanea sativa Mill. cultured on induction MS (Murashige and Skoog, 1962) medium supplemented with 1 mg l–1 benzyladenine (BA) and 1 mg l–1 1-naphthaleneacetic acid (NAA) (Corredoira, 2002). Somatic embryos used for proliferation, germination and conversion studies were taken from a repetitively embryogenic line. These embryogenic cultures were maintained for more than 2 years with sequential subculture at 6-week intervals. Unless otherwise indicated, all cultures were incubated under a 16-h photoperiod (provided by cool-white fluorescent lamps at a photon flux density of 30 µmol m–2 s–1) and 25 °C light/20 °C dark temperatures.
Maintenance of embryogenic competence
Preliminary experiments revealed the low efficiency of MS medium without plant growth regulators (PGR) or medium supplemented with zeatin, indole-3-butyric acid (IBA), 2,4-dichorephenoxyacetic acid (2,4-D) or BA on secondary embryo productivity. Given that the somatic embryogenesis induction medium contained BA and NAA (Corredoira, 2002), different combinations of these two growth regulators were tested to optimize secondary embryo production. Secondary (repetitive) embryogenesis was induced by culturing somatic embryos on proliferation medium consisting of MS mineral salts (half-strength macronutrients) and vitamins, 30 g l–1 sucrose and 8 g l–1 agar (Sigma A-1296, St Louis, MO, USA) (basal medium), supplemented with 3 mM glutamine, and different concentrations of BA (0·1 and 1 mg l–1) and NAA (0·1 and 1 mg l–1). The pH of the medium was adjusted to 5·7 before autoclaving at 121 °C for 20 min. Each treatment was applied to 30 cotyledonary somatic embryo explants, distributed six per dish among five replicate 94 x 16 mm Petri dishes, with 25 ml medium per dish. The experiment was repeated twice. After 6 weeks of culture, the number of explants forming callus, secondary embryos and both callus and secondary embryos, as well as the number of secondary embryos per embryogenic explant and the multiplication coefficient (m.c.) were recorded. The multiplication coefficient was defined as the product of the proportion of explants producing secondary embryos and the mean number of embryos per embryogenic explant.
Proliferation of somatic embryos from callus tissue was evaluated using 50–60 mg callus explants cultured on the same proliferation media used for inducing secondary embryogenesis. The experiment consisted of five replicate Petri dishes with six explants each per treatment, and was repeated twice. After 6 weeks of culture the number of explants with somatic embryos, the number of somatic embryos per embryogenic explant and the m.c. were recorded.
Maturation and germination of somatic embryos
The effect of various maturation media was evaluated in terms of subsequent somatic embryo germination and plantlet conversion ability. White opaque cotyledonary somatic embryos, 4–6 mm in length, were isolated from embryogenic cultures and cultured on different maturation media, consisting of PGR-free basal medium supplemented either with sucrose (3 or 6 %), maltose (3 or 6 %), 3 % sucrose + 6 % sorbitol or 3 % sucrose + 0·5 % activated charcoal (Sigma C-3790). Each treatment was applied to 24 somatic embryos distributed six per dish among four replicate Petri dishes, and the experiment was repeated twice. After 4 weeks of culture on maturation medium, somatic embryos were transferred to basal medium with 3 % sucrose and stored at 4 °C for 2 months. They were then transferred into test tubes containing 16 ml germination medium (basal medium with 0·1 mg l–1 BA) for 8 weeks.
Somatic embryo lengths were measured after culture on maturation media and cold storage. After 8 weeks of culture in germination medium, performance was evaluated in terms of percentage of embryos developing only roots or shoots or both roots and shoots (plantlet conversion) longer than 5 mm. The length of roots and shoots and the number of leaves per regenerated plantlet were also determined.
Effect of pre-germination treatments
The following pre-germination treatments were tested on somatic embryos matured for 4 weeks on 3 % maltose medium, as this was the optimal maturation medium from the above maturation experiments. (1) Desiccation: mature embryos were partially desiccated (14 % water loss after desiccation) by placing them in a sealed quadrant Petri dish with sterile water in two quadrants; after 2 weeks (25 °C, dark) they were transferred to germination medium. (2) Cold treatment: mature embryos were transferred to basal medium and placed at 4 °C in the dark for 2 months before culture on germination medium. (3) Desiccation plus cold treatment: mature embryos were partially desiccated as described above and then exposed to treatment at 4 °C in the dark for 2 months. (4) Control: mature embryos were directly transferred to germination medium.
Each treatment was applied to a total of 36 embryos in three replicates of 12 embryos each, and the experiment was repeated twice. After 8 weeks on germination medium, performance of somatic embryos was evaluated according to the same parameters as described above.
Statistical analysis
Treatment effects were evaluated using ANOVA, followed by the least significant difference (LSD) test at the P = 0·05 level to compare means. Percentages were subjected to arcsine transformation prior to analysis. Non-transformed data are presented in tables and figures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Maintenance of embryogenic competence
Three responses were observed when cotyledonary somatic embryos were cultured on proliferation medium supplemented with different concentrations of BA and NAA (Table 1): (1) secondary embryogenesis from the hypocotyl–root axis and nodular compact callogenesis on the cotyledons of the primary embryos; (2) secondary embryogenesis alone (Fig. 1A and B); and (3) compact nodular callogenesis alone (Fig. 1C). All parameters were significantly affected by the treatment, with the exception of the frequency of explants producing only secondary embryos. The best treatment in terms of somatic embryo productivity (m.c.) was the one including 0·1 mg l–1 BA + 0·1 mg l–1 NAA.
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When nodular callus explants (50–60 mg) were subcultured on media of the same composition as those used for induction of secondary embryogenesis, callus growth occurred in all treatments during the first 3 weeks of culture. After 4 weeks, the calli gave rise to a number of somatic embryos at various developmental stages, including translucent or opaque cotyledonary embryos (Fig. 1D). The frequency of callus explants producing somatic embryos ranged from 31 to 50 %, with the mean number of embryos per explant ranging from 4·2 to 11·3, both parameters being significantly influenced by the treatment (Table 2). As for the secondary embryogenesis experiment (Table 1), the best results, in terms of m.c., were obtained on 0·1 mg l–1 BA + 0·1 mg l–1 NAA medium.
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These data indicate that the capacity of chestnut somatic embryos for secondary embryogenesis is relatively low, although the multiplication efficiency can be improved by subculturing of callus tissue formed on the cotyledons of primary embryos. These calli retained competence for somatic embryo formation for only two or three subcultures, but new nodular callus explants could be excised from cotyledons of subcultured somatic embryos and continuously used to maintain embryogenic competence.
Maturation and germination of somatic embryos
After 4 weeks of culture on different maturation media (Table 3), somatic embryos increased in size with significant differences (P < 0·001) according to the treatment. The greatest length was exhibited by somatic embryos matured on media supplemented with 3 % sucrose or 3 % maltose. A further increase occurred after 2 months of cold treatment (P < 0·001) applied to all mature embryos, with significant differences between treatments, similar to those observed after 4 weeks on the maturation media (Table 3). Embryos treated with 6 % sorbitol + 3 % sucrose showed little or no variation in size. In general, no secondary embryo formation was observed during maturation and cold storage periods, although a few secondary embryos occasionally were differentiated in maltose-supplemented media.
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Following 2 months in cold storage, somatic embryos were transferred to germination medium, where germination rates were significantly affected by the previous maturation treatment (Fig. 2). The frequency of somatic embryos with only root development was highest in treatments supplemented with 6 % maltose and sucrose (3–6 %), whereas embryos on media containing 6 % sorbitol exhibited the lowest germination ability. This treatment also yielded germinated embryos with the shortest roots, whereas the longest roots were observed in embryos matured on charcoal medium (Table 4).
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The percentage of somatic embryos with only shoot development (Fig. 1E) was also significantly affected by the maturation treatment, with best results occurring on media with 3 % maltose (27 % shoot development), and 6 % maltose (Fig. 2). The shoot length of these partially converted somatic embryos was not significantly affected by the maturation media, with values ranging from 8·3 to 13·4 mm (Table 4).
Plantlet conversion (Fig. 1F) was only obtained in somatic embryos matured on media supplemented with 6 % sucrose or 3 % and 6 % maltose, with slightly higher values in maltose media (Fig. 2). Among treatments leading to plant recovery, mean shoot length, root length and leaf number of produced plants were not significantly affected by the treatment, although higher values were observed after maturation on 6 % maltose (Table 4). Overall, the best results were achieved with 3 % maltose-treated embryos, which converted to plants at 6 % in addition to 33 % of somatic embryos that developed only shoots. These shoots could be multiplied and rooted as microcuttings (Fig. 1G).
Effect of pre-germination treatments on plantlet conversion
After experiments in which the maturation medium was optimized by including 3 % maltose, experiments on pre-germination treatments were carried out (Fig. 3). Whereas the frequency of somatic embryos that developed only roots was not affected by the pre-germination treatments, the percentage of embryos developing only shoots was significantly (P < 0·05) higher compared with the control. Plantlet conversion was only observed in embryos subjected to cold treatments (14–17 %). Mean root length, shoot length and leaf number of partially converted somatic embryos and plantlets (data not shown) were not significantly affected by the pre-germination condition.
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Considering both the percentage of somatic embryos developing plants and the percentage of embryos developing only shoots, the best results were achieved by application of cold treatment, with or without a partial desiccation treatment, giving a total of 41·7 and 38·9 % of mature embryos eventually producing plants, respectively. Somatic embryos that developed shoots only were multiplied and rooted following the micropropagation procedure previously described for chestnut (Vieitez et al., 1986; Sánchez et al., 1997).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two processes of somatic embryogenesis were observed in chestnut: (1) direct repetitive (secondary) embryogenesis; and (2) subculture of embryogenic nodular callus originated from cotyledons of somatic embryos. These two processes led to the production of cotyledonary somatic embryos and the maintenance of embryogenic competence for more than 3 years. Secondary embryos arose directly from the hypocotyl–root zone of somatic embryos, where some epidermal and/or subepidermal cells may have already been embryogenically determined (Williams and Maheswaran, 1986). The formation of somatic embryos from the nodular callus suggested that cotyledon cells divided and proliferated before some of the callus cells had reached embryogenic competence; a callogenesis stage occurred prior to initiation of the embryogenic process. A similar indirect pathway for embryo proliferation was reported for the related species, Quercus robur, where secondary embryos were formed from mitotically active cells within callus which developed from the cortical tissues of primary somatic embryos (Zegzouti et al., 2001).
The effect of different combinations of BA and NAA were tested to optimize secondary embryo production. These growth regulators were used for the initiation of embryogenic cultures from chestnut leaf explants (Corredoira, 2002), and they were also effective for maintaining embryogenic competence, with multiplication coefficients ranging from 3·9 to 4·6 (Tables 1 and 2). Similarly, a combination of NAA and BA was also employed for secondary embryo production in oak embryogenic lines derived from leaf and internode explants (Cuenca et al., 1999). Although the suitability of PGR-free medium to sustain secondary embryogenesis has also been reported in different oak embryogenic systems (Fernández-Guijarro et al., 1995; Cuenca et al., 1999), this was not observed in chestnut. The maintenance of embryogenic cultures derived from immature zygotic embryos of Castanea sativa x C. crenata (Vieitez, 1995) and C. dentata (Carraway and Merkle, 1997; Xing et al., 1999) was carried out by culturing proembryogenic masses in medium containing 2,4-D. Subsequently, the cultures were transferred to a PGR-free embryo-development medium (Carraway and Merkle, 1997) or to a medium supplemented with low concentrations of auxins and cytokinins to yield embryos beyond the cotyledonary stage (Vieitez, 1995; Xing et al., 1999).
It has been suggested that direct secondary embryogenesis and indirect proliferation by proembryogenic masses can be considered as two extremes of a continuum (Williams and Maheswaran, 1986), and the occurrence of one or the other will depend on the explant type or growth regulator supplied (Akhtar et al., 2000). The occurrence of both processes in the present study may be caused by different competence of hypocotyls and cotyledon cells of somatic embryos.
Cellular expansion and accumulation of storage products occur during embryo maturation, generally leading to an increase in embryo size (Merkle et al., 1995); therefore, variation in embryo length could be a good marker for monitoring the maturation process. However, in this study, maturation treatments that led to production of longer embryos were not always related to the best frequencies of plant recovery, e.g. somatic embryos matured on 3 % sucrose medium were similar in length to those treated with 3 % maltose, but the former did not develop whole plantlets.
Carbon source and concentration had a marked influence on maturation, and subsequent germination and conversion ability of chestnut somatic embryos, with maltose (3 %) promoting the greatest number of somatic embryos developing either shoots or plantlets. On the contrary, maltose was very poor in supporting development of somatic embryos from proembryogenic masses of American chestnut (Carraway and Merkle, 1997), whereas 6 % sucrose was used for somatic embryo maturation in this species (Xing et al., 1999). The reason for the different maltose effect might be related to the species as well as to the different application time of the maltose: cotyledonary embryos in this study on C. sativa vs. proembryogenic masses in C. dentata.
The positive effect of maltose on somatic embryo maturation has been mentioned in various woody species including Prunus incisa x P. serrula (Druart, 1990), Theobroma cacao (Alemanno et al., 1997) and Pinus taeda (Li et al., 1998), but its mechanism of action is not well known. Strickland et al. (1987) reported that maltose improved the development of alfalfa somatic embryos, reaching the conclusion that the maltose effect was primarily of nutritional and not of osmotic nature. 
ørgaard (1997) assumed that the beneficial effect of maltose on maturation of Abies nordmanniana somatic embryos came from its slow hydrolysis. Moreover, Blanc et al. (1999) suggested that maltose could induce a nutritional stress linked to its low absorption ability and slow metabolism. These authors also suggested that maltose could be a signal molecule with a specific effect on cell physiology.
The application of sorbitol plus sucrose to chestnut was based on results reported for oak, where this treatment promoted maturation and plantlet conversion (Sánchez et al., 2003). However, the sorbitol treatment had a negative effect on the growth and subsequent germination of chestnut embryos, suggesting a problem of concentration or possible toxicity for this species; it was also observed that the addition of activated charcoal had no positive effect on germination and plantlet development. Charcoal enhanced yield and growth of embryos from post-embryogenic masses of C. dentata, although it did not appear to influence germination response (Merkle and Carraway, 1994; Carraway and Merkle, 1997).
It is important to note that on germination medium with BA, root growth was inhibited in germinating embryos producing only roots and also in those showing conversion. In contrast, root growth was not prevented in germinating oak somatic embryos when a similar germination medium supplemented with 0·1 mg l–1 BA was used (Sánchez et al., 2003). Preliminary germination experiments in chestnut indicated that the incorporation of 0·1 mg l–1 BA was more effective than PGR-free medium in terms of shoot development. The application of a rooting treatment might counteract the poor root growth observed in some regenerated plantlets.
Generally, the objective of partial desiccation and cold treatments is to break the dormancy imposed by ABA and/or osmotic stress, to promote germination and to synchronize shoot and root development (Merkle et al., 1995). Castanea seeds require a cold period to break zygotic embryo dormancy (Hartmann et al., 1990), so it was expected that the application of a cold treatment might enable recovery of plantlets from somatic embryos (Carraway and Merkle, 1997). This phenomenon has been attributed to an increase of endogenous levels of gibberellic acid (Deng and Cornu, 1992). Chilling at 4 °C enhanced root development from somatic embryos of American chestnut, but epicotyl elongation and formation of true leaves was infrequent (Carraway and Merkle, 1997). On the contrary, no desiccation or chilling treatments were used by Xing et al. (1999), who reported 3·3 % of embryos directly converting into plantlets, and an additional 6·3 % of embryos forming only shoots. When a chilling treatment was applied to European chestnut, growth rates of both shoots and whole plantlets were higher, with the total potential plantlet regeneration rate increasing to 41 %.
The present study has clearly demonstrated that the application of a cold-treatment to European chestnut somatic embryos matured on maltose-supplemented medium is necessary for achieving plant conversion, while partial desiccation does not appear to influence this response when both treatments are applied together.
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ACKNOWLEDGEMENTS |
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We thank N. Vidal and M. T. Martínez for technical assistance. This research was partially supported by DGI (MCYT) and Xunta de Galicia (Spain) through the projects AGL2000-1073 and PGIDT00BIO40001PR, respectively.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Akhtar N, Kumari N, Pandey S, Ara H, Singh M, Jaiswal U, Jaiswal VS, Jain SM. 2000. Somatic embryogenesis in tropical fruit trees. In: Jain SM, Gupta PK, Newton RJ, eds. Somatic embryogenesis in woody plants. Vol. 6. Dordrecht: Kluwer Academic Publishers, 93–140.
Alemanno L, Berthouly M, Michaux-Ferrière N. 1997. A comparison between Theobroma cacao L. zygotic embryogenesis and somatic embryogenesis from floral explants. In vitro Cellular and Developmental Biology – Plant 33: 163–172.
Ballester A, Bourrain L, Corredoira E, Gonçalves JC, Lê C-L, Miranda ME, San-José MC, Sauer U, Vieitez AM, Wilhelm E. 2001. Improving chestnut micropropagation through axillary shoot development and somatic embryogenesis. Forest Snow Landscape Research 76: 460–467.
Blanc G, Michaux-Ferrière N, Teisson C, Lardet L, Carron MP. 1999. Effects of carbohydrate addition on the induction of somatic embryogenesis in Hevea brasiliensis. Plant Cell Tissue and Organ Culture 59: 103–112.
Carraway DT, Merkle SA. 1997. Plantlet regeneration from somatic embryos of American chestnut. Canadian Journal of Forest Research 27: 1805–1812.[CrossRef]
Corredoira E. 2002. Desarrollo de sistemas embriogénicos en olmo y castaño. Doctoral Thesis. Universidad de Santiago de Compostela, Spain.
Cuenca B, San-José MC, Martínez MT, Ballester A, Vieitez AM. 1999. Somatic embryogenesis from stem and leaf explants of Quercus robur L. Plant Cell Reports 18: 538–543.[CrossRef]
Deng MD, Cornu D. 1992. Maturation and germination of walnut somatic embryos. Plant Cell Tissue and Organ Culture 28: 195–202.[CrossRef]
Druart, P. 1990. Improvement of somatic embryogenesis of cherry dwarf rootstocks INMIL/GM9 by use of different carbon source. Acta Horticulturae 280: 125–129.
Etienne H, Montoro P, Michaux-Ferrière N, Carron MP. 1993. Effects of desiccation, medium osmolarity and abscisic acid on the maturation of Hevea brasiliensis somatic embryos. Journal of Experimental Botany 44: 1613–1619.
Fernández-Guijarro B, Celestino C, Toribio M. 1995. Influence of external factors on secondary embryogenesis and germination in somatic embryos from leaves of Quercus suber. Plant Cell Tissue and Organ Culture 41: 99–106.[CrossRef]
Hartmann HT, Kester DE, Davies Jr FT. 1990. Plant propagation. Principles and practices, 5th edn. New Jersey: Prentice-Hall International.
Holliday C, Merkle SA. 2000. Preservation of American chestnut germplasm by cryostorage of embryogenic cultures. Journal of the American Chestnut Foundation 14: 46–52.
Klimaszewska K, Bernier-Cardou M, Cyr DR, Sutton CS. 2000. Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L. In vitro Cellular and Developmental Biology – Plant 36: 279–286.[CrossRef]
Li XY, Feng H, Huang H, Murphy JB, Gbur Jr EE. 1998. Polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (Pinus taeda L.). In vitro Cellular and Developmental Biology – Plant 34: 22–26.
Maynard C, Xing Z, Bickel S, Powell W. 1998. Using genetic engineering to help save the American chestnut: a progress report. Journal of the American Chestnut Foundation 12: 40–56.
Merkle SA, Carraway DT. 1994. Somatic embryogenesis and gene transfer in American chestnut. In: Pardos JA, Ahuja MR, Rosello E, eds. Biotechnology of trees. Madrid: INIA, 199–208.
Merkle SA, Parrot WA, Flinn BS. 1995. Morphogenic aspects of somatic embryogenesis. In: Thorpe TA, ed. In vitro embryogenesis in plants. Dordrecht: Kluwer Academic Publishers, 155–203.
Merkle SA, Wiecko AT, Watson-Pauley BA. 1991. Somatic embryogenesis in American chestnut. Canadian Journal of Forest Research 21: 1698–1701.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497.[CrossRef]
ørgaard JV. 1997. Somatic embryo maturation and plant regeneration in Abies nordmanniana Lk. Plant Science 124: 211–221.[CrossRef]
Sánchez MC, Martínez MT, Valladares S, Ferro E, Vieitez AM. 2003. Maturation and germination of oak somatic embryos originated from leaf and stem explants. RAPD markers for genetic analysis of regenerants. Journal of Plant Physiology 6: 699–707.[CrossRef]
Sánchez MC, San-José MC, Ferro E, Ballester A, Vieitez AM. 1997. Improving micropropagation conditions for adult-phase shoots of chestnut. Journal of Horticultural Science 72: 433–443.
San-José MC, Ballester A, Vieitez AM. 2001. Effect of thidiazuron on multiple shoot induction and plant regeneration from cotyledonary nodes of chestnut. Journal of Horticultural Science and Bio technology 76: 588–595.
Strickland SG, Nichol JW, Call CMM, Stuart DA. 1987. Effect of carbohydrate source on alfalfa somatic embryogenesis. Plant Science 48: 113–121.[CrossRef]
Vieitez AM, Vieitez ML, Vieitez E. 1986. Chestnut (Castanea spp.). In: Bajaj YPS, ed. Biotechnology in agriculture and forestry, Vol. 1: Trees I. Berlin: Springer-Verlag, 393–414.
Vieitez FJ. 1995. Somatic embryogenesis in chestnut. In: Jain SM, Gupta PK, Newton RJ, eds. Somatic embryogenesis in woody plants. Vol. 2. Angiosperms. Dordrecht: Kluwer Academic Publishers, 375–407.
Williams EG, Maheswaran G. 1986. Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Annals of Botany 57: 443–462.
Xing Z, Powell WA, Maynard CA. 1999. Development and germination of American chestnut somatic embryos. Plant Cell Tissue and Organ Culture 57: 47–55.[CrossRef]
Yeung EC. 1995. Structural and developmental patterns in somatic embryogenesis. In: Thorpe TA, ed. In vitro embryogenesis in plants. Dordrecht: Kluwer Academic Publishers, 205–247.
Zegzouti R, Arnould MF, Favre JM. 2001. Histological investigation of the multiplication step in secondary somatic embryogenesis of Quercus robur L. Annals of Forest Science 58: 681–690.[CrossRef]
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