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Improvement of plant recovery from avocado zygotic embryos by desiccation under high relative humidity conditions
Scientia Horticulturae 222 (2017) 169–174
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Improvement of plant recovery from avocado zygotic embryos by desiccation under high relative humidity conditions Belén Márquez-Martín, Fernando Pliego-Alfaro, Carolina Sánchez-Romero
MARK
⁎
Dpto. Biología Vegetal, Universidad de Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Avocado Desiccation Embryo rescue Maturation Germination Persea americana RITA®
Due to the high fruit abscission characterizing avocado, protocols for immature embryo rescue are an important tool in breeding programs based on hybridization of elite genotypes. The objective of this investigation was to evaluate the effect of in vitro desiccation under high relative humidity conditions on immature avocado embryo germination. The duration of the desiccation treatment significantly affected embryo germination, with optimum results achieved after a 14-day treatment (66.7% in desiccated embryos versus 15% in control, nontreated embryos). Other traits, such as recovery of complete plants and quality of the obtained plants, were also improved following desiccation. Trying to reproduce the final stages of avocado zygotic embryogenesis, the effects of desiccation after an in vitro maturation treatment was also evaluated. Results obtained revealed a significant effect of maturation stage, desiccation and the interaction between both factors; e.g., while desiccation significantly improved germination after in vivo maturation, a slight decline was observed in in vitro matured embryos, probably due to differences in embryo water status. However, recovery of complete plants and length of the structures formed were only significantly affected by maturation stage. Nevertheless, in embryos directly coming from trees, in vitro drying significantly improved rescue in all cases, independently of embryo developmental stage. Therefore, desiccation under high relative humidity conditions can be considered a valuable tool to be used in protocols for avocado embryo rescue. However, its use in conjunction with in vitro maturation treatments is not recommended.
1. Introduction Avocado is an evergreen tree cultivated for its nutritious fruits. World production has doubled in the last fifteen years to 4,717,102 t produced in 516,484 ha (FAOSTAT, 2013). Avocado cultivation extends throughout regions with tropical, subtropical and temperate climates including Mexico, Dominican Republic, Colombia, Peru and Indonesia (FAOSTAT, 2013). Avocado breeding programs based on hybridization of selected genotypes have been reported in multiple countries such as USA, Australia, South Africa, Mexico and Israel (Lahav and Lavi, 2009). However, the avocado is characterized by excessive fruit abscission (Garner and Lovatt, 2016), with peak abscission rates ranging from 50 to 280 immature fruits per day (Garner and Lovatt, 2008). Premature abortion of the developing embryos results in low fruit set (< 0.1%) (Whiley and Schaffer, 1994), which provokes a dramatic reduction of the viable hybrid progeny, significantly reducing the efficiency of avocado breeding programs (Sánchez-Romero et al., 2007). Previous studies carried out in avocado revealed that, under
standard conditions, acceptable germination rates were only obtained at advanced developmental stages (Perán-Quesada et al., 2005). Therefore, development of protocols for immature embryo rescue is becoming an important tool in avocado breeding programs. In vitro culture of immature embryos has been used as a rescue technique as it facilitates conversion of these embryos into plants (Raghavan, 2003). In most cases, development of in vitro rescue protocols has been focused on the optimisation of the germination process (Sánchez-Romero et al., 2007; Skene and Barlass, 1983). However, embryo germination is significantly influenced by events occurring during maturation and desiccation phases (Bewley and Black, 1994). The desiccation stage is characterized by an important loss of water which causes seed metabolism to decrease in preparation for a quiescent period and subsequent germination (Kermode and FinchSavage, 2002). This step occurs between two metabolically different phases. The anabolic phase is characterized by the synthesis and accumulation of reserve products (maturation), while in the catabolic these storage substances are metabolized in order to support development of the new plant (germination).
Abbreviations: DAP, days after pollination; LSD, least significant difference; RH, relative humidity ⁎ Corresponding author. E-mail address: [email protected] (C. Sánchez-Romero). http://dx.doi.org/10.1016/j.scienta.2017.05.018 Received 23 January 2017; Received in revised form 3 May 2017; Accepted 4 May 2017 Available online 15 May 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
Scientia Horticulturae 222 (2017) 169–174
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During development, orthodox seeds acquire desiccation tolerance and, therefore, during drying, moisture content can decline to very low levels. For most of these seeds, desiccation is considered a potent developmental switch signalling the cessation of seed development and their entry into the germinative programme upon cellular rehydration (Bewley et al., 2013). Although recalcitrant seeds, such as avocado, lack or do not express the various processes and mechanisms typifying the acquisition of tolerance (Berjak and Pammenter, 2013) and therefore are sensitive to desiccation, an important loss of water at the end of the maturation period has also been described (Sánchez-Romero et al., 2002). Previous investigations carried out in immature avocado embryos revealed that including an in vitro maturation phase prior to induction of germination greatly improves the efficiency of plant recovery as well as the quality of the resulting plants (Márquez-Martín et al., 2009). The objective of the present investigation was to study the effect of desiccation on the efficiency of avocado embryo rescue. Following the model of in vivo avocado embryo development (Perán-Quesada et al., 2005; Sánchez-Romero et al., 2002), we studied the effect of sequential application of maturation and desiccation in vitro treatments on subsequent embryo germination. 2. Material and methods 2.1. Plant material and culture conditions Avocado (Persea americana Mill.) fruits, cv. ‘Hass’, were harvested at random from open pollinated trees growing in a monovarietal orchard at IHSM La Mayora (Algarrobo Costa, Spain). After harvesting, fruits were surface sterilized by immersion for 10 min in a 0.5% (v/v) sodium hypochlorite solution containing 20 drops l−1 of Tween 20, followed by three rinses in sterile distilled water. Sterilized fruits were cut lengthwise under aseptic conditions and immature zygotic embryos carefully excised. In all media preparations, the pH was adjusted to 5.74 before autoclaving for 7 min. Subsequently, 25 ml were dispensed into 25 mm × 150 mm test tubes (Bellco Glass Inc., NJ, USA) or 50 ml into 85 mm × 80 mm cylindrical glass jars. Finally, media were sterilized by autoclaving at 121 °C and 0.1 MPa for 15–20 min. RITA® vessels were sterilized by autoclaving for 20 min at the same conditions the day before desiccation treatments were initiated. All cultures were incubated in a growth chamber at 25 ± 1 °C. Maturation and desiccation treatments were carried out in darkness, while germination was induced under a 16 h light photoperiod, provided by Grolux lamps (Sylvania, Germany) (40 μmol m−2 s−1).
Fig. 1. Immature avocado embryos during desiccation under 97.5% RH into a RITA® system.
and the water content calculated on a fresh weight basis (Pâques and Boxus, 1987). To evaluate the effect of desiccation on embryo conversion, embryo germination was induced by partial removal of the cotyledons and culture on M1 medium (Skene and Barlass, 1983; Perán-Quesada et al., 2005). M1 medium consisted of half strength MS formulation supplemented with 2.22 μM benzyladenine and gelled with 1.7 g l−1 Gelrite (G-1910, Sigma Chemical Co., St. Louis, MO, USA). Germination phase was carried out during 15 weeks with recultures onto fresh medium at 5-week intervals. 2.3. Effect of desiccation after an in vitro maturation treatment Trying to reproduce the last phases of zygotic embryogenesis in avocado (Perán-Quesada et al., 2005; Sánchez-Romero et al., 2002), the effects of desiccation after an in vitro maturation treatment were tested. In order to elucidate the effect of in vitro maturation on subsequent response to desiccation, two controls were included in this experiment: non-matured (control) and in vivo matured embryos. Very immature embryos, collected 65 DAP with 7 mm average length, were used in this experiment. Control embryos were taken directly from the tree at the beginning of the experiment. For in vitro maturation, embryos were cultured on B5m medium (consisting of major salts of the B5 formulation (Gamborg et al., 1968), MS minor salts and vitamins (Murashige and Skoog, 1962) and 88 mM sucrose) supplemented with the Jensen’s amino acids (Jensen, 1977), extra 88 mM sucrose and 6 g l−1 agar (A-1296, Sigma Chemical Co., St. Louis, MO, USA) (Márquez-Martín et al., 2009). Maturation was carried out during 12 weeks with reculture onto fresh medium after 6 weeks. For in vivo maturation, avocado embryos remained growing in the trees during the same time period. Thus, 12 weeks after experiment initiation (139 DAP), zygotic embryos averaging 25 mm long were collected and subjected to the same subsequent treatments than those in vitro matured. Half of control, in vivo and in vitro matured embryos were induced to germinate in M1 medium. The rest of embryos were subjected to a
2.2. Effect of desiccation treatment duration According to George (1996), atmospheres with specific relative humidities (RH) can be achieved in confined spaces containing saturated solutions of different salts. In the present investigation, a 97.5% RH was attained by adding 150 ml of a saturated K2SO4 solution in the lower compartment of a RITA® temporary immersion system (Cirad, Saint-Mathieu-de-Tréviers, France) and cancelling outside air exchange and solution movement to the upper compartment. Once isolated, zygotic embryos were quickly placed into the bottom half of a sterile 90 × 25 mm Petri dish divided into three sections, located in the upper part of the vessel (Fig. 1). For testing the effect of desiccation treatment duration, zygotic embryos collected 106 days after pollination (DAP) averaging 19 mm in length, were desiccated as indicated above for 7, 14 and 21 days. Control treatment consisted of embryos directly coming from trees, without having undergone any desiccation treatment. Variations in fresh and dry weight were determined throughout the desiccation process in a minimum of 6 embryos per treatment. Dry weight was measured after heating zygotic embryos at 75 °C for 48 h 170
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maturation (P = 0.0000) and desiccation (P = 2.501 10−6) (Fig. 4). Although maturation significantly improved germination regardless of whether it was carried out in vivo or in vitro, a significant interaction with desiccation was evident (P = 3.775 10−5). Thus, while desiccation significantly improved germination after in vivo maturation, under in vitro conditions a slight decline was observed. However, recovery of complete plants was only affected by maturation (P = 1.150 10−9), without any significant differences between in vivo and in vitro processes (Table 1). Only in control embryos was recovery of complete plants significantly affected by desiccation. Quality of plants obtained was only influenced by maturation (P = 0.000 for shoots and P = 0.044 for roots), with significantly better results achieved from in vivo matured embryos (Table 2). Germination rate clearly depended on the embryo developmental stage (P = 7.642 10−13), with desiccation under 97.5% RH for 14 days significantly (P = 2.972 10−6) improving germination independently of embryo size. However, recovery of complete plantlets was only significantly influenced by the embryo developmental stage (P = 1.732 10−8) and the interaction embryo stage × desiccation (P = 0.0420) (Table 1). Thus, desiccation improved recovery of plants with shoots and roots in very immature embryos (7 mm long), but not in embryos at later stages (25 mm long). Although plantlets developed from desiccated embryos were in general more vigorous, the length of the structures formed did not statistically differ from those of nondesiccated embryos (Table 2). Embryo developmental stage was the only factor influencing size of structures developed during germination (P = 0.001 for shoots and P = 0.022 for roots).
desiccation treatment for 14 days before inducing germination. 2.4. Data taken and statistical analysis In the first experiment, 15–20 embryos were used per treatment while in the second experiment 50 embryos were utilized. Embryos were considered germinated when shoot and/or root elongation was 2 mm. Data on percentage of germinated embryos, type of germination (shoot, root or shoot and root) and length of the shoots and roots formed were recorded at the end of each reculture. Percentage data were subjected to frequency analysis with an RxC test of independence or with a three-way log-linear analysis to evaluate interactions between variables. The rest of data were analyzed by ANOVA followed by mean comparison by the least significant difference (LSD) test. The significance level used was 0.05 in all cases (Sokal and Rohlf, 2003). 3. Results 3.1. Effect of desiccation treatment duration Despite only small variations in embryo water content observed during desiccation under an atmosphere with 97.5% RH, germination of avocado embryos was significantly affected by desiccation treatment (P = 0.0118) (Fig. 2). While only 15% of embryos germinated when directly cultured on M1 medium, in desiccated embryos germination rates ranged between 26.7 and 66.7%, depending on the treatment duration. The best results were obtained when embryos were desiccated for 14 days. Recovery of complete plants was also significantly improved by desiccation (P = 0.0139), with optimum results achieved after a 14-day treatment (33.3% of embryos developed plants with shoot and root versus 0% in non-desiccated embryos). Although no statistically significant differences were found in the length of shoots and roots formed (data not shown), control embryos gave rise to short and thin stems with small and hyperhidric leaves. However, plants obtained from partially desiccated embryos were more vigorous, exhibiting welldeveloped shoots (Fig. 3).
4. Discussion Desiccation under high RH conditions significantly affected avocado embryo germination, although the results obtained clearly depended on treatment duration. The role of desiccation on the germination process has previously been described both in zygotic (Kermode et al., 1986) and somatic embryos (Hazubska-Przybył et al., 2015; Roberts et al., 1989). For most orthodox seeds, maturation drying clearly switches the seed from a developmental to a germinative mode upon subsequent rehydration (Bewley et al., 2013). Although it has been proposed that recalcitrant seeds, such as avocado, can achieve this switch without dehydration (Bewley et al., 2013), the results obtained revealed that desiccation also appears to play a regulatory role on germination in this type of
3.2. Effect of desiccation after an in vitro maturation treatment Results revealed a significant influence on embryo recovery of both
Fig. 2. Effect of duration of desiccation under high RH conditions on water content and subsequent germination of avocado embryos harvested 106 DAP. Water content data correspond to mean ± standard error. Different letters in germination data indicate significant differences by frequency analysis at P = 0.05.
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Fig. 3. Plantlet recovery from avocado embryos harvested 106 DAP. Seedlings obtained from (a) control, non-desiccated embryos and (b) embryos desiccated under 97.5% RH for 14 days.
Fig. 4. Effect of in vitro or in vivo maturation and partial desiccation under high RH conditions on subsequent germination of avocado embryos harvested 65 DAP. Different letters indicate significant differences by frequency analysis at P = 0.05. Table 1 Effect of in vitro or in vivo maturation and partial desiccation under high RH conditions on subsequent germination of avocado embryos harvested 65 DAP. Different letters within each column indicate significant differences obtained by frequency analysis (α = 0.05). Treatment
Control −D Control +D In vitro mat −D In vitro mat +D In vivo mat −D In vivo mat +D
Table 2 Effect of in vitro or in vivo maturation and partial desiccation under high RH conditions on subsequent germination type of avocado embryos harvested 65 DAP. Different letters within each column indicate significant differences by ANOVA (α = 0.05).
Germination (%)
Treatment
Shoot
Root
6.00 c 26.00 b 30.00 b 22.50 b 18.75 b 51.02 a
0.00 2.00 5.00 2.50 2.08 4.08
Shoot and root a a a a a a
Length (cm) Shoot
Control −D Control +D In vitro mat −D In vitro mat +D In vivo mat −D In vivo mat +D
0.00 c 6.00 b 40.00 a 30.00 a 33.33 a 30.61 a
11.67 11.00 30.46 30.18 46.88 47.97
Root bc c b b a a
– 18.25 41.39 45.14 42.94 44.41
a a a a a
−D: Non desiccated; +D: desiccated.
−D: Non desiccated; +D: desiccated.
embryos. According to Angelovici et al. (2010), seed desiccation implies more than a physical process of drying and it must be considered an active stage in which transcriptional, post-transcriptional and metabolic
processes take place. In Arabidopsis, transcript profiling analysis showed that the expression of approximately 30% of its genes significantly change during seed desiccation. Metabolism also changes from a general decline of free metabolites during the maturation phase to 172
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observed in different parameters, such as final germination and recovery of complete plants. Final germination was affected by desiccation and embryo stage, but a significant interaction between both factors was not evident. However, the effect of desiccation on plant recovery was variable depending on embryo stage. Desiccation significantly improved recovery of complete plants in embryos at early stages (7 and 19 mm long), but no effect was observed in embryos at more advanced stages (25 mm long). Vigour of newly developed plants was only determined by the embryo size, with longer shoots and roots obtained from more developed embryos. In conclusion, desiccation under RH conditions can be considered a valuable tool to be incorporated in protocols focused on rescue of immature avocado embryos. Although in vitro maturation also represent an improvement of this process, application of both techniques in conjunction is not recommended. Nevertheless, further optimization would be possible by testing different RH conditions and treatment durations so that desiccation better fits water characteristics of embryos obtained after an in vitro maturation phase.
accumulation of specific compounds, such as sugars, secondary metabolites, TCA-cycle intermediates, ɣ-aminobutyric acid as well as some amino acids and free fatty acids. Various events observed during desiccation (Angelovici et al., 2010) could explain how processes occurring during this phase may assist subsequent germination: (a) some of the proteins operating during seed germination derive from precursors (mRNAs or proteins) synthesized during seed desiccation, (b) a coordinated regulation of genes throughout desiccation and early germination has been described, and (c) preparation for seed germination appears to begin during the desiccation phase, as an important proportion of gene expression and metabolic signatures observed during drying resemble those characterizing seed germination. Partial desiccations with slight water losses, as that recorded in avocado embryos, have significantly increased embryo germination in others species such as Laurus nobilis (Takos et al., 2002) or Ricinus communis (Kermode and Bewley, 1989). Such effects with a low variation of water content have been explained by an apparent capacity of partial desiccation to replace total desiccation treatments or because embryos do not require more drastic treatments, that may lead to a severe disruption of cellular structure or metabolism (Kermode and Bewley, 1989). According to these authors, during partial drying a redistribution or relocation of water may occur within the seed and, as consequence, drying of the outer layers. During desiccation of wheat embryos at high RH, Mitchell (1980) reported water losses of 80% at the outer pericarp layer, but an increase in water content within the inner parts. Studies on water location using scanning nuclear magnetic resonance confirmed a water redistribution in the wheat grain during partial drying (Armstrong et al., 1982). Although water content has been used as a physical parameter to characterize embryo development, it has proven to be inadequate to indicate the seed water status as it does not provide information about water availability or energy status. Water potential of seed and, more adequately, of the different seed structures would be a better indicator of water status (Villela, 1998). The effect of desiccation was significantly affected by previous treatments applied to seeds. Thus, desiccation for 14 days under a 97.5% RH atmosphere significantly improved germination when applied to embryos directly derived from trees, but opposite results were obtained after an in vitro maturation treatment. Although the in vitro maturation protocol applied in the present investigation reproduced the embryogenic developmental pattern previously established for avocado (Márquez-Martín et al., 2009), the effect of desiccation clearly differed from that obtained in in vivo matured embryos. Differences observed depending on maturation conditions, in vitro or in vivo, may be probably related to differences in embryo water status. Partial desiccation under high RH conditions significantly improved germination of avocado embryos, independently of embryo size. This effect is especially valuable in embryos at the earlier developmental stage (7 mm long), in which histodifferentiation was not accomplished and the maturation phase was far from being initiated. Previous investigations on avocado embryogenesis revealed that morphogenesis was not completed until embryos reached 16–18 mm in length, approximately 100 DAP (Perán-Quesada et al., 2005). Reserve products, which are utilized by the germinating embryo until the development of autotrophy (Merkle et al., 1995), have been found to be very scarce in 7 mm long embryos. Although these embryos contained small starch granules, their starch content was low and a significant accumulation of this storage carbohydrate was not observed until the initiation of the maturation phase, 125 DAP (Perán-Quesada et al., 2005; Sánchez-Romero et al., 2002). In relation to storage proteins, despite a group of proteins of 40–48 kD, classified as albumins, was evident since the earlier developmental stages, the larger part was deposited at more advanced stages of embryo development (SánchezRomero et al., 2002). In fact, protein bodies were not visible until the maturation phase had started (Perán-Quesada et al., 2005). Positive effects of desiccation on germinability of immature embryos was
Acknowledgements This work was supported by the Comisión Interministerial de Ciencia y Tecnología of Spain, grants AGL 2005-06347-C03-03 and AGL 2014-53518-C2-IR, and Grupo de Investigación PAIDI AGR-226. References Angelovici, R., Galili, G., Fernie, A.R., Fait, A., 2010. Seed desiccation: a bridge between maturation and germination. Trends Plant Sci. 15, 211–218. Armstrong, C., Black, M., Chapman, J.M., Norman, H.A., Angold, K., 1982. The induction of sensitivity to gibberelin in aleurone tissue of developing wheat grains. I. The effect of dehydration. Planta 154, 573–577. Berjak, P., Pammenter, N.W., 2013. Implications of the lack of desiccation tolerance in recalcitrant seeds. Front. Plant Sci. 4, 478. http://dx.doi.org/10.3389/fpls.2013. 00478. Bewley, J.D., Black, M., 1994. Seeds. Physiology of Development and Germination. Plenum Press, New York. Bewley, J.D., Bradford, K.J., Hilhorst, H.W.M., Nonogaki, H., 2013. Seeds. Physiology of Development Germination and Dormancy. Springer, New York. FAOSTAT, 2013. Online database available at link http://faostat3.fao.org (Accessed December, 14, 2016). Gamborg, O.L., Muller, R.A., Ojima, K., 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158. Garner, L.C., Lovatt, C.J., 2008. The relationship between flower and fruit abscission and alternate bearing of ‘Hass’ avocado. J. Am. Soc. Hortic. Sci. 133, 3–10. Garner, L.C., Lovatt, C.J., 2016. Physiological factors affecting flower and fruit abscission of ‘Hass’ avocado. Sci. Hortic. 199, 32–40. George, E.F., 1996. Plant Propagation by Tissue Culture, Part 2: In Practice. Exegetics Ltd., Westbury pp. 711–712. Hazubska-Przybył, T., Wawrzyniak, M., Obarska, A., Bojarczuk, K., 2015. Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce. Acta Physiol. Plant. 37, 1735. Jensen, C.J., 1977. Monoploid production by chromosome elimination. In: Reinert, J., Bajaj, Y.P.S. (Eds.), Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture. Springer-Verlag, Berlin, pp. 299–340. Kermode, A.R., Bewley, J.D., 1989. Developing seeds of Ricinus communis L., when detached and maintained in an atmosphere of high relative humidity, switch to a germinative mode without the requirement for complete desiccation. Plant Physiol. 90, 702–707. Kermode, A.R., Finch-Savage, B.E., 2002. Desiccation sensitivity in orthodox and recalcitrant seeds in relation to development. In: Black, M., Pritchard, H.W. (Eds.), Desiccation and Survival in Plants: Drying Without Dying. CABI Publishing, Wallinford, pp. 149–184. Kermode, A.R., Bewley, J.D., Dasgupta, J., Misra, S., 1986. The transition from seed development to germination: a key role for desiccation? HortSci 21, 1113–1118. Lahav, E., Lavi, U., 2009. Avocado genetics and breeding. In: Jain, S.M., Priyadarshan, P.M. (Eds.), Breeding Plantation Tree Crops: Tropical Species. Springer, New York, pp. 247–285. Márquez-Martín, B., Guzmán-García, E., Barceló-Muñoz, A., Pliego-Alfaro, F., SánchezRomero, C., 2009. Effects of an in vitro maturation treatment on plant recovery from avocado zygotic embryos. Sci. Hortic. 122, 532–539. Merkle, S.A., Parrott, W.A., Flinn, B.S., 1995. Morphogenic aspects of somatic embryogenesis. In: Thorpe, T.A. (Ed.), In Vitro Embryogenesis in Plants. Kluwer Academic Publishers, Dordrecht, pp. 155–203. Mitchell, B.A., 1980. The Control of Germination Ability in Developing Wheat Grains. PhD Thesis. University of London. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with
173
Scientia Horticulturae 222 (2017) 169–174
B. Márquez-Martín et al.
Alfaro, F., 2007. In vitro rescue of immature avocado (Persea americana Mill.) embryos. Sci. Hortic. 111, 365–370. Skene, K.G.M., Barlass, M., 1983. In vitro culture of abscissed immature avocado embryos. Ann. Bot. 52, 667–672. Sokal, R.R., Rohlf, F.J., 2003. Biometry. W.H. Freeman and Company, New York. Takos, I., Konstantinidou, E., Merou, T., 2002. The effect of desiccation on the seed germination of Laurus nobilis L. In: Thanos, C.A., Beardmore, T.L., Connor, K.F., Tolentino, E.L. (Eds.), Tree Seeds 2002, Annual Meeting of IUFRO. Chania Crete. pp. 178–182. Villela, F.A., 1998. Water relations in seed biology. Sci. Agric. 55, 98–101. Whiley, A.W., Schaffer, B., 1994. Avocado. In: Schaffer, B., Anderson, P.C. (Eds.), Handbook of Environmental Physiology of Fruit Crops: Vol II Sub-Tropical and Tropical Crops. CRC Press Inc., Boca Raton, pp. 3–35.
tobacco tissue cultures. Physiol. Plant. 15, 473–497. Pâques, M., Boxus, Ph., 1987. A model to learn ‘vitrification’, the rootstock apple M.26 present results. Acta Hortic. 212, 193–210. Perán-Quesada, R., Sánchez-Romero, C., Pliego-Alfaro, F., Barceló-Muñoz, A., 2005. Histological aspects of avocado embryo development and effect of developmental stages on germination. Seed Sci. Res. 15, 125–132. Raghavan, V., 2003. One hundred years of zygotic embryo culture investigations. In Vitro Cell. Dev. Biol. Plant 39, 437–442. Roberts, D.R., Flinn, B.S., Sutton, B.C.S., 1989. Desiccation promotes and synchronizes the germination of white spruce somatic embryos. Plant Physiol. 89 (Suppl), 8. Sánchez-Romero, C., Perán-Quesada, R., Barceló-Muñoz, A., Pliego-Alfaro, F., 2002. Variations in storage protein and carbohydrate levels during development of avocado zygotic embryos. Plant Physiol. Biochem. 40, 1043–1049. Sánchez-Romero, C., Perán-Quesada, R., Márquez-Martín, B., Barceló-Muñoz, A., Pliego-
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Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Improvement of plant recovery from avocado zygotic embryos by desiccation under high relative humidity conditions Belén Márquez-Martín, Fernando Pliego-Alfaro, Carolina Sánchez-Romero
MARK
⁎
Dpto. Biología Vegetal, Universidad de Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Avocado Desiccation Embryo rescue Maturation Germination Persea americana RITA®
Due to the high fruit abscission characterizing avocado, protocols for immature embryo rescue are an important tool in breeding programs based on hybridization of elite genotypes. The objective of this investigation was to evaluate the effect of in vitro desiccation under high relative humidity conditions on immature avocado embryo germination. The duration of the desiccation treatment significantly affected embryo germination, with optimum results achieved after a 14-day treatment (66.7% in desiccated embryos versus 15% in control, nontreated embryos). Other traits, such as recovery of complete plants and quality of the obtained plants, were also improved following desiccation. Trying to reproduce the final stages of avocado zygotic embryogenesis, the effects of desiccation after an in vitro maturation treatment was also evaluated. Results obtained revealed a significant effect of maturation stage, desiccation and the interaction between both factors; e.g., while desiccation significantly improved germination after in vivo maturation, a slight decline was observed in in vitro matured embryos, probably due to differences in embryo water status. However, recovery of complete plants and length of the structures formed were only significantly affected by maturation stage. Nevertheless, in embryos directly coming from trees, in vitro drying significantly improved rescue in all cases, independently of embryo developmental stage. Therefore, desiccation under high relative humidity conditions can be considered a valuable tool to be used in protocols for avocado embryo rescue. However, its use in conjunction with in vitro maturation treatments is not recommended.
1. Introduction Avocado is an evergreen tree cultivated for its nutritious fruits. World production has doubled in the last fifteen years to 4,717,102 t produced in 516,484 ha (FAOSTAT, 2013). Avocado cultivation extends throughout regions with tropical, subtropical and temperate climates including Mexico, Dominican Republic, Colombia, Peru and Indonesia (FAOSTAT, 2013). Avocado breeding programs based on hybridization of selected genotypes have been reported in multiple countries such as USA, Australia, South Africa, Mexico and Israel (Lahav and Lavi, 2009). However, the avocado is characterized by excessive fruit abscission (Garner and Lovatt, 2016), with peak abscission rates ranging from 50 to 280 immature fruits per day (Garner and Lovatt, 2008). Premature abortion of the developing embryos results in low fruit set (< 0.1%) (Whiley and Schaffer, 1994), which provokes a dramatic reduction of the viable hybrid progeny, significantly reducing the efficiency of avocado breeding programs (Sánchez-Romero et al., 2007). Previous studies carried out in avocado revealed that, under
standard conditions, acceptable germination rates were only obtained at advanced developmental stages (Perán-Quesada et al., 2005). Therefore, development of protocols for immature embryo rescue is becoming an important tool in avocado breeding programs. In vitro culture of immature embryos has been used as a rescue technique as it facilitates conversion of these embryos into plants (Raghavan, 2003). In most cases, development of in vitro rescue protocols has been focused on the optimisation of the germination process (Sánchez-Romero et al., 2007; Skene and Barlass, 1983). However, embryo germination is significantly influenced by events occurring during maturation and desiccation phases (Bewley and Black, 1994). The desiccation stage is characterized by an important loss of water which causes seed metabolism to decrease in preparation for a quiescent period and subsequent germination (Kermode and FinchSavage, 2002). This step occurs between two metabolically different phases. The anabolic phase is characterized by the synthesis and accumulation of reserve products (maturation), while in the catabolic these storage substances are metabolized in order to support development of the new plant (germination).
Abbreviations: DAP, days after pollination; LSD, least significant difference; RH, relative humidity ⁎ Corresponding author. E-mail address: [email protected] (C. Sánchez-Romero). http://dx.doi.org/10.1016/j.scienta.2017.05.018 Received 23 January 2017; Received in revised form 3 May 2017; Accepted 4 May 2017 Available online 15 May 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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During development, orthodox seeds acquire desiccation tolerance and, therefore, during drying, moisture content can decline to very low levels. For most of these seeds, desiccation is considered a potent developmental switch signalling the cessation of seed development and their entry into the germinative programme upon cellular rehydration (Bewley et al., 2013). Although recalcitrant seeds, such as avocado, lack or do not express the various processes and mechanisms typifying the acquisition of tolerance (Berjak and Pammenter, 2013) and therefore are sensitive to desiccation, an important loss of water at the end of the maturation period has also been described (Sánchez-Romero et al., 2002). Previous investigations carried out in immature avocado embryos revealed that including an in vitro maturation phase prior to induction of germination greatly improves the efficiency of plant recovery as well as the quality of the resulting plants (Márquez-Martín et al., 2009). The objective of the present investigation was to study the effect of desiccation on the efficiency of avocado embryo rescue. Following the model of in vivo avocado embryo development (Perán-Quesada et al., 2005; Sánchez-Romero et al., 2002), we studied the effect of sequential application of maturation and desiccation in vitro treatments on subsequent embryo germination. 2. Material and methods 2.1. Plant material and culture conditions Avocado (Persea americana Mill.) fruits, cv. ‘Hass’, were harvested at random from open pollinated trees growing in a monovarietal orchard at IHSM La Mayora (Algarrobo Costa, Spain). After harvesting, fruits were surface sterilized by immersion for 10 min in a 0.5% (v/v) sodium hypochlorite solution containing 20 drops l−1 of Tween 20, followed by three rinses in sterile distilled water. Sterilized fruits were cut lengthwise under aseptic conditions and immature zygotic embryos carefully excised. In all media preparations, the pH was adjusted to 5.74 before autoclaving for 7 min. Subsequently, 25 ml were dispensed into 25 mm × 150 mm test tubes (Bellco Glass Inc., NJ, USA) or 50 ml into 85 mm × 80 mm cylindrical glass jars. Finally, media were sterilized by autoclaving at 121 °C and 0.1 MPa for 15–20 min. RITA® vessels were sterilized by autoclaving for 20 min at the same conditions the day before desiccation treatments were initiated. All cultures were incubated in a growth chamber at 25 ± 1 °C. Maturation and desiccation treatments were carried out in darkness, while germination was induced under a 16 h light photoperiod, provided by Grolux lamps (Sylvania, Germany) (40 μmol m−2 s−1).
Fig. 1. Immature avocado embryos during desiccation under 97.5% RH into a RITA® system.
and the water content calculated on a fresh weight basis (Pâques and Boxus, 1987). To evaluate the effect of desiccation on embryo conversion, embryo germination was induced by partial removal of the cotyledons and culture on M1 medium (Skene and Barlass, 1983; Perán-Quesada et al., 2005). M1 medium consisted of half strength MS formulation supplemented with 2.22 μM benzyladenine and gelled with 1.7 g l−1 Gelrite (G-1910, Sigma Chemical Co., St. Louis, MO, USA). Germination phase was carried out during 15 weeks with recultures onto fresh medium at 5-week intervals. 2.3. Effect of desiccation after an in vitro maturation treatment Trying to reproduce the last phases of zygotic embryogenesis in avocado (Perán-Quesada et al., 2005; Sánchez-Romero et al., 2002), the effects of desiccation after an in vitro maturation treatment were tested. In order to elucidate the effect of in vitro maturation on subsequent response to desiccation, two controls were included in this experiment: non-matured (control) and in vivo matured embryos. Very immature embryos, collected 65 DAP with 7 mm average length, were used in this experiment. Control embryos were taken directly from the tree at the beginning of the experiment. For in vitro maturation, embryos were cultured on B5m medium (consisting of major salts of the B5 formulation (Gamborg et al., 1968), MS minor salts and vitamins (Murashige and Skoog, 1962) and 88 mM sucrose) supplemented with the Jensen’s amino acids (Jensen, 1977), extra 88 mM sucrose and 6 g l−1 agar (A-1296, Sigma Chemical Co., St. Louis, MO, USA) (Márquez-Martín et al., 2009). Maturation was carried out during 12 weeks with reculture onto fresh medium after 6 weeks. For in vivo maturation, avocado embryos remained growing in the trees during the same time period. Thus, 12 weeks after experiment initiation (139 DAP), zygotic embryos averaging 25 mm long were collected and subjected to the same subsequent treatments than those in vitro matured. Half of control, in vivo and in vitro matured embryos were induced to germinate in M1 medium. The rest of embryos were subjected to a
2.2. Effect of desiccation treatment duration According to George (1996), atmospheres with specific relative humidities (RH) can be achieved in confined spaces containing saturated solutions of different salts. In the present investigation, a 97.5% RH was attained by adding 150 ml of a saturated K2SO4 solution in the lower compartment of a RITA® temporary immersion system (Cirad, Saint-Mathieu-de-Tréviers, France) and cancelling outside air exchange and solution movement to the upper compartment. Once isolated, zygotic embryos were quickly placed into the bottom half of a sterile 90 × 25 mm Petri dish divided into three sections, located in the upper part of the vessel (Fig. 1). For testing the effect of desiccation treatment duration, zygotic embryos collected 106 days after pollination (DAP) averaging 19 mm in length, were desiccated as indicated above for 7, 14 and 21 days. Control treatment consisted of embryos directly coming from trees, without having undergone any desiccation treatment. Variations in fresh and dry weight were determined throughout the desiccation process in a minimum of 6 embryos per treatment. Dry weight was measured after heating zygotic embryos at 75 °C for 48 h 170
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maturation (P = 0.0000) and desiccation (P = 2.501 10−6) (Fig. 4). Although maturation significantly improved germination regardless of whether it was carried out in vivo or in vitro, a significant interaction with desiccation was evident (P = 3.775 10−5). Thus, while desiccation significantly improved germination after in vivo maturation, under in vitro conditions a slight decline was observed. However, recovery of complete plants was only affected by maturation (P = 1.150 10−9), without any significant differences between in vivo and in vitro processes (Table 1). Only in control embryos was recovery of complete plants significantly affected by desiccation. Quality of plants obtained was only influenced by maturation (P = 0.000 for shoots and P = 0.044 for roots), with significantly better results achieved from in vivo matured embryos (Table 2). Germination rate clearly depended on the embryo developmental stage (P = 7.642 10−13), with desiccation under 97.5% RH for 14 days significantly (P = 2.972 10−6) improving germination independently of embryo size. However, recovery of complete plantlets was only significantly influenced by the embryo developmental stage (P = 1.732 10−8) and the interaction embryo stage × desiccation (P = 0.0420) (Table 1). Thus, desiccation improved recovery of plants with shoots and roots in very immature embryos (7 mm long), but not in embryos at later stages (25 mm long). Although plantlets developed from desiccated embryos were in general more vigorous, the length of the structures formed did not statistically differ from those of nondesiccated embryos (Table 2). Embryo developmental stage was the only factor influencing size of structures developed during germination (P = 0.001 for shoots and P = 0.022 for roots).
desiccation treatment for 14 days before inducing germination. 2.4. Data taken and statistical analysis In the first experiment, 15–20 embryos were used per treatment while in the second experiment 50 embryos were utilized. Embryos were considered germinated when shoot and/or root elongation was 2 mm. Data on percentage of germinated embryos, type of germination (shoot, root or shoot and root) and length of the shoots and roots formed were recorded at the end of each reculture. Percentage data were subjected to frequency analysis with an RxC test of independence or with a three-way log-linear analysis to evaluate interactions between variables. The rest of data were analyzed by ANOVA followed by mean comparison by the least significant difference (LSD) test. The significance level used was 0.05 in all cases (Sokal and Rohlf, 2003). 3. Results 3.1. Effect of desiccation treatment duration Despite only small variations in embryo water content observed during desiccation under an atmosphere with 97.5% RH, germination of avocado embryos was significantly affected by desiccation treatment (P = 0.0118) (Fig. 2). While only 15% of embryos germinated when directly cultured on M1 medium, in desiccated embryos germination rates ranged between 26.7 and 66.7%, depending on the treatment duration. The best results were obtained when embryos were desiccated for 14 days. Recovery of complete plants was also significantly improved by desiccation (P = 0.0139), with optimum results achieved after a 14-day treatment (33.3% of embryos developed plants with shoot and root versus 0% in non-desiccated embryos). Although no statistically significant differences were found in the length of shoots and roots formed (data not shown), control embryos gave rise to short and thin stems with small and hyperhidric leaves. However, plants obtained from partially desiccated embryos were more vigorous, exhibiting welldeveloped shoots (Fig. 3).
4. Discussion Desiccation under high RH conditions significantly affected avocado embryo germination, although the results obtained clearly depended on treatment duration. The role of desiccation on the germination process has previously been described both in zygotic (Kermode et al., 1986) and somatic embryos (Hazubska-Przybył et al., 2015; Roberts et al., 1989). For most orthodox seeds, maturation drying clearly switches the seed from a developmental to a germinative mode upon subsequent rehydration (Bewley et al., 2013). Although it has been proposed that recalcitrant seeds, such as avocado, can achieve this switch without dehydration (Bewley et al., 2013), the results obtained revealed that desiccation also appears to play a regulatory role on germination in this type of
3.2. Effect of desiccation after an in vitro maturation treatment Results revealed a significant influence on embryo recovery of both
Fig. 2. Effect of duration of desiccation under high RH conditions on water content and subsequent germination of avocado embryos harvested 106 DAP. Water content data correspond to mean ± standard error. Different letters in germination data indicate significant differences by frequency analysis at P = 0.05.
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Fig. 3. Plantlet recovery from avocado embryos harvested 106 DAP. Seedlings obtained from (a) control, non-desiccated embryos and (b) embryos desiccated under 97.5% RH for 14 days.
Fig. 4. Effect of in vitro or in vivo maturation and partial desiccation under high RH conditions on subsequent germination of avocado embryos harvested 65 DAP. Different letters indicate significant differences by frequency analysis at P = 0.05. Table 1 Effect of in vitro or in vivo maturation and partial desiccation under high RH conditions on subsequent germination of avocado embryos harvested 65 DAP. Different letters within each column indicate significant differences obtained by frequency analysis (α = 0.05). Treatment
Control −D Control +D In vitro mat −D In vitro mat +D In vivo mat −D In vivo mat +D
Table 2 Effect of in vitro or in vivo maturation and partial desiccation under high RH conditions on subsequent germination type of avocado embryos harvested 65 DAP. Different letters within each column indicate significant differences by ANOVA (α = 0.05).
Germination (%)
Treatment
Shoot
Root
6.00 c 26.00 b 30.00 b 22.50 b 18.75 b 51.02 a
0.00 2.00 5.00 2.50 2.08 4.08
Shoot and root a a a a a a
Length (cm) Shoot
Control −D Control +D In vitro mat −D In vitro mat +D In vivo mat −D In vivo mat +D
0.00 c 6.00 b 40.00 a 30.00 a 33.33 a 30.61 a
11.67 11.00 30.46 30.18 46.88 47.97
Root bc c b b a a
– 18.25 41.39 45.14 42.94 44.41
a a a a a
−D: Non desiccated; +D: desiccated.
−D: Non desiccated; +D: desiccated.
embryos. According to Angelovici et al. (2010), seed desiccation implies more than a physical process of drying and it must be considered an active stage in which transcriptional, post-transcriptional and metabolic
processes take place. In Arabidopsis, transcript profiling analysis showed that the expression of approximately 30% of its genes significantly change during seed desiccation. Metabolism also changes from a general decline of free metabolites during the maturation phase to 172
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observed in different parameters, such as final germination and recovery of complete plants. Final germination was affected by desiccation and embryo stage, but a significant interaction between both factors was not evident. However, the effect of desiccation on plant recovery was variable depending on embryo stage. Desiccation significantly improved recovery of complete plants in embryos at early stages (7 and 19 mm long), but no effect was observed in embryos at more advanced stages (25 mm long). Vigour of newly developed plants was only determined by the embryo size, with longer shoots and roots obtained from more developed embryos. In conclusion, desiccation under RH conditions can be considered a valuable tool to be incorporated in protocols focused on rescue of immature avocado embryos. Although in vitro maturation also represent an improvement of this process, application of both techniques in conjunction is not recommended. Nevertheless, further optimization would be possible by testing different RH conditions and treatment durations so that desiccation better fits water characteristics of embryos obtained after an in vitro maturation phase.
accumulation of specific compounds, such as sugars, secondary metabolites, TCA-cycle intermediates, ɣ-aminobutyric acid as well as some amino acids and free fatty acids. Various events observed during desiccation (Angelovici et al., 2010) could explain how processes occurring during this phase may assist subsequent germination: (a) some of the proteins operating during seed germination derive from precursors (mRNAs or proteins) synthesized during seed desiccation, (b) a coordinated regulation of genes throughout desiccation and early germination has been described, and (c) preparation for seed germination appears to begin during the desiccation phase, as an important proportion of gene expression and metabolic signatures observed during drying resemble those characterizing seed germination. Partial desiccations with slight water losses, as that recorded in avocado embryos, have significantly increased embryo germination in others species such as Laurus nobilis (Takos et al., 2002) or Ricinus communis (Kermode and Bewley, 1989). Such effects with a low variation of water content have been explained by an apparent capacity of partial desiccation to replace total desiccation treatments or because embryos do not require more drastic treatments, that may lead to a severe disruption of cellular structure or metabolism (Kermode and Bewley, 1989). According to these authors, during partial drying a redistribution or relocation of water may occur within the seed and, as consequence, drying of the outer layers. During desiccation of wheat embryos at high RH, Mitchell (1980) reported water losses of 80% at the outer pericarp layer, but an increase in water content within the inner parts. Studies on water location using scanning nuclear magnetic resonance confirmed a water redistribution in the wheat grain during partial drying (Armstrong et al., 1982). Although water content has been used as a physical parameter to characterize embryo development, it has proven to be inadequate to indicate the seed water status as it does not provide information about water availability or energy status. Water potential of seed and, more adequately, of the different seed structures would be a better indicator of water status (Villela, 1998). The effect of desiccation was significantly affected by previous treatments applied to seeds. Thus, desiccation for 14 days under a 97.5% RH atmosphere significantly improved germination when applied to embryos directly derived from trees, but opposite results were obtained after an in vitro maturation treatment. Although the in vitro maturation protocol applied in the present investigation reproduced the embryogenic developmental pattern previously established for avocado (Márquez-Martín et al., 2009), the effect of desiccation clearly differed from that obtained in in vivo matured embryos. Differences observed depending on maturation conditions, in vitro or in vivo, may be probably related to differences in embryo water status. Partial desiccation under high RH conditions significantly improved germination of avocado embryos, independently of embryo size. This effect is especially valuable in embryos at the earlier developmental stage (7 mm long), in which histodifferentiation was not accomplished and the maturation phase was far from being initiated. Previous investigations on avocado embryogenesis revealed that morphogenesis was not completed until embryos reached 16–18 mm in length, approximately 100 DAP (Perán-Quesada et al., 2005). Reserve products, which are utilized by the germinating embryo until the development of autotrophy (Merkle et al., 1995), have been found to be very scarce in 7 mm long embryos. Although these embryos contained small starch granules, their starch content was low and a significant accumulation of this storage carbohydrate was not observed until the initiation of the maturation phase, 125 DAP (Perán-Quesada et al., 2005; Sánchez-Romero et al., 2002). In relation to storage proteins, despite a group of proteins of 40–48 kD, classified as albumins, was evident since the earlier developmental stages, the larger part was deposited at more advanced stages of embryo development (SánchezRomero et al., 2002). In fact, protein bodies were not visible until the maturation phase had started (Perán-Quesada et al., 2005). Positive effects of desiccation on germinability of immature embryos was
Acknowledgements This work was supported by the Comisión Interministerial de Ciencia y Tecnología of Spain, grants AGL 2005-06347-C03-03 and AGL 2014-53518-C2-IR, and Grupo de Investigación PAIDI AGR-226. References Angelovici, R., Galili, G., Fernie, A.R., Fait, A., 2010. Seed desiccation: a bridge between maturation and germination. Trends Plant Sci. 15, 211–218. Armstrong, C., Black, M., Chapman, J.M., Norman, H.A., Angold, K., 1982. The induction of sensitivity to gibberelin in aleurone tissue of developing wheat grains. I. The effect of dehydration. Planta 154, 573–577. Berjak, P., Pammenter, N.W., 2013. Implications of the lack of desiccation tolerance in recalcitrant seeds. Front. Plant Sci. 4, 478. http://dx.doi.org/10.3389/fpls.2013. 00478. Bewley, J.D., Black, M., 1994. Seeds. Physiology of Development and Germination. Plenum Press, New York. Bewley, J.D., Bradford, K.J., Hilhorst, H.W.M., Nonogaki, H., 2013. Seeds. Physiology of Development Germination and Dormancy. Springer, New York. FAOSTAT, 2013. Online database available at link http://faostat3.fao.org (Accessed December, 14, 2016). Gamborg, O.L., Muller, R.A., Ojima, K., 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158. Garner, L.C., Lovatt, C.J., 2008. The relationship between flower and fruit abscission and alternate bearing of ‘Hass’ avocado. J. Am. Soc. Hortic. Sci. 133, 3–10. Garner, L.C., Lovatt, C.J., 2016. Physiological factors affecting flower and fruit abscission of ‘Hass’ avocado. Sci. Hortic. 199, 32–40. George, E.F., 1996. Plant Propagation by Tissue Culture, Part 2: In Practice. Exegetics Ltd., Westbury pp. 711–712. Hazubska-Przybył, T., Wawrzyniak, M., Obarska, A., Bojarczuk, K., 2015. Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce. Acta Physiol. Plant. 37, 1735. Jensen, C.J., 1977. Monoploid production by chromosome elimination. In: Reinert, J., Bajaj, Y.P.S. (Eds.), Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture. Springer-Verlag, Berlin, pp. 299–340. Kermode, A.R., Bewley, J.D., 1989. Developing seeds of Ricinus communis L., when detached and maintained in an atmosphere of high relative humidity, switch to a germinative mode without the requirement for complete desiccation. Plant Physiol. 90, 702–707. Kermode, A.R., Finch-Savage, B.E., 2002. Desiccation sensitivity in orthodox and recalcitrant seeds in relation to development. In: Black, M., Pritchard, H.W. (Eds.), Desiccation and Survival in Plants: Drying Without Dying. CABI Publishing, Wallinford, pp. 149–184. Kermode, A.R., Bewley, J.D., Dasgupta, J., Misra, S., 1986. The transition from seed development to germination: a key role for desiccation? HortSci 21, 1113–1118. Lahav, E., Lavi, U., 2009. Avocado genetics and breeding. In: Jain, S.M., Priyadarshan, P.M. (Eds.), Breeding Plantation Tree Crops: Tropical Species. Springer, New York, pp. 247–285. Márquez-Martín, B., Guzmán-García, E., Barceló-Muñoz, A., Pliego-Alfaro, F., SánchezRomero, C., 2009. Effects of an in vitro maturation treatment on plant recovery from avocado zygotic embryos. Sci. Hortic. 122, 532–539. Merkle, S.A., Parrott, W.A., Flinn, B.S., 1995. Morphogenic aspects of somatic embryogenesis. In: Thorpe, T.A. (Ed.), In Vitro Embryogenesis in Plants. Kluwer Academic Publishers, Dordrecht, pp. 155–203. Mitchell, B.A., 1980. The Control of Germination Ability in Developing Wheat Grains. PhD Thesis. University of London. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with
173
Scientia Horticulturae 222 (2017) 169–174
B. Márquez-Martín et al.
Alfaro, F., 2007. In vitro rescue of immature avocado (Persea americana Mill.) embryos. Sci. Hortic. 111, 365–370. Skene, K.G.M., Barlass, M., 1983. In vitro culture of abscissed immature avocado embryos. Ann. Bot. 52, 667–672. Sokal, R.R., Rohlf, F.J., 2003. Biometry. W.H. Freeman and Company, New York. Takos, I., Konstantinidou, E., Merou, T., 2002. The effect of desiccation on the seed germination of Laurus nobilis L. In: Thanos, C.A., Beardmore, T.L., Connor, K.F., Tolentino, E.L. (Eds.), Tree Seeds 2002, Annual Meeting of IUFRO. Chania Crete. pp. 178–182. Villela, F.A., 1998. Water relations in seed biology. Sci. Agric. 55, 98–101. Whiley, A.W., Schaffer, B., 1994. Avocado. In: Schaffer, B., Anderson, P.C. (Eds.), Handbook of Environmental Physiology of Fruit Crops: Vol II Sub-Tropical and Tropical Crops. CRC Press Inc., Boca Raton, pp. 3–35.
tobacco tissue cultures. Physiol. Plant. 15, 473–497. Pâques, M., Boxus, Ph., 1987. A model to learn ‘vitrification’, the rootstock apple M.26 present results. Acta Hortic. 212, 193–210. Perán-Quesada, R., Sánchez-Romero, C., Pliego-Alfaro, F., Barceló-Muñoz, A., 2005. Histological aspects of avocado embryo development and effect of developmental stages on germination. Seed Sci. Res. 15, 125–132. Raghavan, V., 2003. One hundred years of zygotic embryo culture investigations. In Vitro Cell. Dev. Biol. Plant 39, 437–442. Roberts, D.R., Flinn, B.S., Sutton, B.C.S., 1989. Desiccation promotes and synchronizes the germination of white spruce somatic embryos. Plant Physiol. 89 (Suppl), 8. Sánchez-Romero, C., Perán-Quesada, R., Barceló-Muñoz, A., Pliego-Alfaro, F., 2002. Variations in storage protein and carbohydrate levels during development of avocado zygotic embryos. Plant Physiol. Biochem. 40, 1043–1049. Sánchez-Romero, C., Perán-Quesada, R., Márquez-Martín, B., Barceló-Muñoz, A., Pliego-
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