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Adventitious regeneration from mature seed-derived tissues of Prunus cerasifera and Prunus insititia
Scientia Horticulturae 259 (2020) 108746
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Short communication
Adventitious regeneration from mature seed-derived tissues of Prunus cerasifera and Prunus insititia Elisabeth Carmona-Martin, Cesar Petri
T
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Departamento de Fruticultura Subtropical y Mediterránea, Instituto de Hortofruticultura Subtropical y Mediterránea, UMA-CSIC, Avenida Dr. Wienberg, s/n. 29750 Algarrobo-Costa, Málaga, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Rosaceae Auxin pulses Woody plants Organogenesis 2,4-dichlorophenoxyacetic acid Rootstocks
An essential preliminary step in the application of genetic engineering is the development of robust and effective procedures of adventitious regeneration. We have developed an efficient regeneration procedure from mature seeds hypocotyl sections and cotyledons of Prunus rootstocks: Four genotypes of Prunus cerasifera (‘Adara’, ‘Ademir’, ‘Myrobolan B’ and ‘Myrobalan 29C’) and one genotype of Prunus insititia (‘Pixi’). A pulse of 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) significantly stimulated adventitious organogenesis from both sort of explants. However, the 2,4-D treatment effect relied on the genotype and the type of explant. Best regeneration rates achieved from hypocotyl sections were 16.28%, 31.25%, 13.79%, 46.86% and 53.03% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. Best regeneration rates achieved from cotyledons were 60.00%, 51.61%, 42.86%, 75.00%, 90.48% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. Rooting and acclimatization steps were successfully achieved. The whole process, from initial culture to established plants in the greenhouse, took approximately 15 weeks.
1. Introduction Stone fruits (Prunus) are economically important worldwide. This genus comprises apricots, almonds, cherries, plums, prunes, peaches, etc. In the commercial cultivation of these species, the desired cultivar, vegetatively propagated, is grafted onto a rootstock. Seedling rootstocks have been traditionally used. Nevertheless, more and more commercial clonal Prunus rootstocks are being released and used in the orchards as a result from private and public rootstock breeding programs (Beckman and Lang, 2003). Particularly remarkable among recent releases has been the incorporation of resistance to soil borne diseases, nematodes, waterlogging, iron chlorosis and vigor control (Beckman and Lang, 2003; Moreno, 2004; Moreno et al., 1995a, 1995b; Reig et al., 2018). These genotypes are quite interesting as a starting material to apply genetic engineering techniques in order to stack new agronomical traits that cannot be otherwise introduced by hybridization and selection. This could be the case of resistance traits non-available into the species or related wild species germplasms. A crucial preliminary step in the application of genetic engineering is the development of robust and effective procedures of adventitious
regeneration. Consequently, the goal of the present work was to establish methodologies to obtain adventitious regeneration from tissues of the Prunus rootstocks described in Table 1. For this purpose, we adapted procedures previously used successfully with other close-related species, such as apricot (P. armeniaca), European plum (P. domestica), Japanese plum (P. salicina) and cherry (P. avium) (Canli and Tian, 2009, 2008; Petri et al., 2008b; Urtubia et al., 2008; Wang et al., 2013a, 2011). 2. Materials and methods 2.1. Plant material and explant preparation Open pollinated mature seeds of the rootstocks ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ (P. cerasifera) and ‘Pixi’ (P. insititia) were collected at the “Estación Experimental Aula Dei” (EEAD-CSIC) in Zaragoza, Spain (Table 1). For seeds disinfection, the endocarp was removed with a nutcracker, and seeds were immersed in a beaker containing a solution of 20% commercial bleach (commercial bleach = 6.15% sodium hypochlorite) and 0.02% Tween-20 for 20 min and rinsed three times with sterile
Abbreviations: 2,4-D, 2,4- dichlorophenoxyacetic acid; BAP, benzylaminopurine; IBA, indole butyric acid; NAA, α-naphthaleneacetic; PG, phloroglucinol; TDZ, thidiazuron; RM, rooting medium; SGM, shoot growing medium; SRM, shoot regeneration medium ⁎ Corresponding author. E-mail address: [email protected] (C. Petri). https://doi.org/10.1016/j.scienta.2019.108746 Received 1 July 2019; Received in revised form 1 August 2019; Accepted 3 August 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 259 (2020) 108746
Dwarfing Vigorous
Wide range of compatibility Vigorous Vigorous
Tolerance to Pseudomonas syringae Resistance to Meloidogyne spp.
Non defined Tolerance to Verticilium, Phytophthora and Pseudomonas syringae Resistance to Meloidogyne spp. Tolerance to Armillaria, Phytophthora, Agrobacterium and Verticilium.
distilled water in a laminar flow bench. Disinfected seeds were soaked in sterile water overnight at 4 °C. Hypocotyl sections were prepared as previously described (Petri et al., 2012). The radicle and the epicotyl were discarded, and the hypocotyl was sliced into 1–4 cross sections (0.5–1 mm) (Fig. 1A). Cotyledon explants were prepared as described in Wang et al. (2013a) and placed onto the shoot regeneration medium (SRM) with the adaxial face down (Fig. 1A).
2.2. Adventitious bud regeneration, shoot elongation, rooting and acclimatization For all these steps previously described procedures, media and culture conditions were followed (Petri et al., 2012), with some modifications as described below. Briefly, media used were MS-based (Murashige and Skoog, 1962) with the following growth regulators: shoot regeneration medium (SRM) contained 7.5 μM thidiazuron (TDZ) and 0.25 μM indole butyric acid (IBA); shoot growing medium (SGM) contained 1.5 μM benzylaminopurine (BAP), instead of 3.0 μM as described in the original recipe, and 0.25 μM IBA; rooting medium (RM) contained 0.1 μM kinetin and 5.0 μM α-naphthaleneacetic acid (NAA). Additionally, in this occasion 100 mg l−1 phloroglucinol (PG) was added to RM. In order to study the effect of 2,4- dichlorophenoxyacetic acid (2,4-D) pulses, we tested a treatment described for Prunus domestica (Petri et al., 2008b). Explants were cultured on SRM supplemented with 9.05 μM 2,4-D during 3 days and then transferred to SRM, or explants were placed directly onto SRM. After 5–7 weeks, bud clusters were excised from the explant and placed onto SGM. When shoots reached 2–3 cm long they were separated from the cluster and transferred to Magenta type vessels with RM. Tissue culture chamber conditions, along the whole process (from initial culture to rooting), were set up to a 16/8 h light/dark cycle, light intensity of 45–50 μE m−2s−1, and a temperature of 25+1 °C. Regeneration was evaluated weekly, starting 2 weeks after the beginning of the experiment until adventitious bud regeneration stopped appearing for 2 consecutive weeks (approximately 6–7 weeks from the beginning of the experiment). An average of 9 hypocotyl slices and 6 cotyledon explants were used for each genotype and treatment. Regeneration experiments were repeated, at least, three times. Rooting data was collected 4 weeks after the shoots were placed in RM supplemented with 100 mg l−1 PG. Rooting, and subsequent acclimatization, were repeated twice. A pool of 87 rooted shoots belonging to the 5 genotypes was chosen for acclimatization. Rooted shoots were washed in water to eliminate agar residues and transferred to pots containing 60% coconut fiber, 30% commercial substrate (Turbas y Coco Mar Menor S.L., Spain) and 10% litonite. The potted plantlets were introduced into plastic bags which were sealed and maintained in a growth chamber with a 16/8 h light/dark cycle, light intensity of 45–50 μE m−2s−1, 70% relative humidity and a temperature of 27+1 °C. The plastic bags were opened gradually and, after 2 weeks, they were fully opened. Following 1 week in opened plastic bags during which time plants were irrigated as needed and finally, plants were transferred to a greenhouse.
EEAD-CSIC (Spain) East Malling (UK) University of California (USA) Prunus cerasifera Prunus cerasifera Prunus cerasifera Ademir Myrobalan B Myrobalan 29C
Non defined Resistance to root asphyxia and iron-induced chlorosis Resistance to iron chlorosis and root asphyxia Tolerance to drought Tolerance to root asphyxia East Malling (UK) EEAD-CSIC (Spain) Prunus insititia Prunus cerasifera Pixi Adara
Abiotic stress resilience Origin Species Rootstock
Table 1 Brief description of the Prunus rootstocks used as the source of explants in this study.
Biotic stress resilience
Other characteristics
E. Carmona-Martin and C. Petri
2.3. Experimental design and statistical analyses Regeneration and rooting experiments were performed in a completely randomized design. To test for differences among more than two independent groups, data were transformed as needed to fit to a Normal distribution and ANOVA was performed. To compare treatments within a given genotype Student’s t-test (No. of roots per rooted shoot, fresh weight of explants) or Pearson’s Chi-test (regeneration percentages) were applied.
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Fig. 1. (A) Source of explants. Cotyledon explant as described in Wang et al. (2013a). The hypocotyl was sliced into 1 to 4 cross sections (0.5–1 mm). The radicle and the epicotyl were discarded. Bar represents 1 mm. (B) Adventitious regeneration from a ‘Pixi’ hypocotyl section. Picture was taken at 5 weeks from the beginning of the experiment. Bar represents 1 mm. (C) Adventitious regeneration from a ‘Adara’ cotyledon. Picture was taken at 4 weeks from the beginning of the experiment. Bar represents 1 mm. (D) ‘Myrobalan 29C’ shoots after 4 weeks cultured in SGM. Bar represents 1 cm. (E) ‘Adara’ rooted shoot prompt for the acclimatization stage. Bar represents 1 cm. (F) Acclimatized shoots cultured in a greenhouse. (Color figure on line).
3. Results
the proximal surface of the explant (Fig. 1C).
All genotypes showed an identical regeneration, elongation and rooting pattern (Fig. 1B-E). First buds started appearing 2–3 weeks after the beginning of the experiment as direct adventitious regeneration (without the formation of an intermediated callus). In hypocotyl sections regeneration appeared on the explant border as bud clusters, and frequently a continuous crown along the border was observed (Fig. 1B). Regeneration in the cotyledons also appeared as bud clusters, mostly on
3.1. Effect of 2,4-D treatment Both types of explants (hypocotyls slices and cotyledons) were directly cultured on SRM or they were firstly cultured on SRM supplemented with 9.05 μM 2,4-D for 3 days and then transferred to SRM. The 2,4-D pulse significantly affected the regeneration rates from hypocotyl sections (P < 0.05) and cotyledons (P < 0.01). The 2,4-D treatment 3
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Fig. 2. Effect of 2,4-D treatment. Bars show Standard Errors. Asterisks indicate statistical significance between treatments within a given genotype (*P < 0.05; **P < 0.01). A: Regeneration rates from hypocotyl sections. A total of 453 explants were used in this study. B: Regeneration rates from cotyledons. A total of 184 explants were used in this study. C: Explants from ‘Pixi’ seeds cultured onto SRM for 5 weeks. D: Fresh weight of hypocotyl explants after 7 weeks cultured on SRM. A total of 184 explants were used in this study. (Color figure on line).
randomly chosen) was annotated. Data analyses showed that fresh weight of the 2,4-D treated explants was significantly higher (P < 0.05) compared to the fresh weight of the non-treated ones, except for ‘Myrobalan B’ (Fig. 2D).
Table 2 Rooting results. Genotype
No. of shoots placed in RM
Rooting rate (% + S.E)
Number of roots/ rooted shoot (Mean + S.E.)
Ademir Adara Myrobalan B Myrobalan 29C Pixi
21 20 18 18 19
100.0 + 0.0 100.0 + 0.0 92.8 + 6.9 100.0 + 0.0 89.4 + 7.1
4.6 3.8 5.1 4.7 3.7
+ + + + +
3.2. Shoot elongation, rooting and acclimatization
0.4 0.4 0.5 0.4 0.5
One week after buds/clusters appeared they were excised from the explant and transferred to SGM. This medium resulted adequate for shoot elongation for all genotypes tested. In approximately 3–4 weeks, shoots of about 2–3 cm long were available (Fig. 1D) and ready to be separated and placed in RM. Adventitious roots started being visible after 2 weeks of culture in RM supplemented with 100 mg l−1 PG. After two more additional weeks, shoots showed a well-developed root system (Fig. 1E). At this point rooting data was collected. Statistical differences among genotypes were not detected for any of both parameters measured; rooting rate and number of roots per rooted shoot (Table 2). Overall, rooting rate was nearly 100% with 4–5 roots per shoot (Table 2; Fig. 1E). A pool of shoots belonging to the five genotypes was chosen to proceed with the acclimatization step. 86 shoots, out of 87, survived and were transferred to the greenhouse (Fig. 1F).
Data was collected 4 weeks after shoots were placed in RM. Statistical differences among genotypes were not detected for any of the two parameters measured. Rooting experiment was repeated twice.
increased regeneration percentages from hypocotyl slices compared to the regeneration observed without the 2,4-D pulse in all genotypes tested. Nevertheless, the augmentation only resulted statistically significant for ‘Adara’ (P < 0.05), ‘Myrobalan 29C’ (P < 0.01) and ‘Pixi’ (P < 0.01) (Fig. 2A). Best regeneration rates achieved from mature seeds hypocotyl sections were 16.28 + 5.63%, 31.25 + 6.69%, 13.79 + 6.42%, 46.86 + 8.82% and 53.03 + 6.14% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. In cotyledons, regeneration was significantly higher in the 2,4-D treated explants compared to the non-treated ones for all genotypes tested (Fig. 2B). For ‘Ademir’ and ‘Myrobalan B’ cotyledons, the 2,4-D treatment was critical and adventitious regeneration was only observed when the auxin pulse was applied (Fig. 2B). Best regeneration rates achieved from mature seeds cotyledons were 60.00 + 8.98%, 51.61 + 9.8%, 42.86 + 18.7%, 75.00 + 15.31%, 90.48 + 6.4% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. Along the regeneration experiments, explants that were cultured for 3 days with 9.05 μM 2,4-D seemed larger and healthier than those without the treatment (Fig. 2C). After regeneration data was collected (7 weeks from the beginning of the experiment), fresh weight of some hypocotyl explants (approximately 20 per genotype-treatment,
4. Discussion To our knowledge, this is the first report on direct organogenesis from P. cerasifera and P. insititia. As the source of explants, we have chosen rootstock genotypes which accumulate many interesting agronomical traits, such as biotic and abiotic stress resistance (Table 1). We report in vitro adventitious shoot regeneration from two different mature seed-derived tissues. Data for the number of shoots were not presented since adventitious shoots appeared as clusters and it was difficult to accurately count them. Regeneration patterns and efficiencies, from both sort of explants, are comparable to those reported for another Prunus species (Canli and Tian, 2009, 2008; Mante et al., 1989; Petri et al., 2008b; Tian et al., 2007; Urtubia et al., 2008; Wang et al., 2013a, 4
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et al., 2008b; Tian et al., 2009; Wang et al., 2013b), Japanese plum (Urtubia et al., 2008) and peach (Perez-Clemente et al., 2005). A remarkable aspect is that these protocols have been shown to be transferable among genotypes. The regeneration pattern described in those procedures, as direct regeneration of bud clusters, is similar to the pattern observed in this study (Fig. 1B, C). It is not possible to improve a known rootstock/variety by transforming seed-derived tissues. Nevertheless, seed material could be very useful to accomplish several objectives in fundamental research areas such as gene silencing, gene expression studies, resistance assessments to insects, diseases and herbicides, and promoter gene assessments. Also, transformation of nonclonal material could be used to incorporate a new trait (such as disease resistance genes) in the gene pool of the species, making the trait available for breeders. We have used open-pollinated seeds in the current research. However, by using seeds from controlled crosses of a breeding program, it is assumable that the new engineered trait could be incorporated to an advanced/elite selection, which could become a new clonal rootstock or be used for further breeding. For some important soil pests, such as the flat-headed borers (Capnodis spp.), that affects the cultivation of stone fruits in the Mediterranean basin (Gindin et al., 2014), sources of genetic resilience has not been found yet into the Prunus germplasm (Dicenta et al., 2002; Mendel et al., 2006).
2011). Additionally, the process length was also satisfactory. The method takes about 15 weeks from initial culture to the establishment of the plants in the greenhouse, which is a reasonable time for woody plants. It is also notable to mention the high rooting rates and acclimatization survival achieved. We used a RM designed for P. domestica (Gonzalez Padilla et al., 2003) with the addition of 100 mg l−1 PG. PG is a rooting cofactor that has been previously reported to stimulate rhizogenesis in Prunus and other fruit tree species (Hammerschlag et al., 1987; James and Thurbon, 2013; Jones and Hatfield, 1976; Petri and Scorza, 2010). In peach and European plum, the addition of 100 mg l−1 to the RM significantly increased rooting efficiency for all genotypes tested (Hammerschlag et al., 1987; Petri and Scorza, 2010). Our experience, acquired over the years, in Prunus micropropagation, regeneration and genetic transformation allowed us to accomplish the acclimatization step without further difficulties. We applied well established procedures used routinely for P. domestica and P. armeniaca (Petri et al., 2014, 2012). Our results indicate that 2,4-D stimulates cell division and/or adventitious organogenesis from mature seed-derived tissues in P. cerasifera and P. insititia. Previous studies in Prunus spp. also stated pulses with 2,4-D as beneficial for organogenesis from leaf or hypocotyl explants (Pascual and Marín, 2005; Petri et al., 2008b, 2005; Petri and Scorza, 2010). In some cases, authors described an increment in callus formation when explants were exposed to auxin pulses (Petri et al., 2005; Wang et al., 2011), which agrees with the fresh weight increment observed in our experiments (Fig. 2C, D). Regeneration from mature cotyledons in different Prunus species has been reported (Canli and Tian, 2009, 2008; Hokanson and Pooler, 2000; Mante et al., 1989; Wang et al., 2013a). Auxin pulses were not assayed. However, most of them described a dark incubation period at the initial stage of the culture as a critical factor for obtaining higher regeneration efficiencies (Canli and Tian, 2009, 2008; Wang et al., 2013a). It has been reported that dark incubation influences endogenous levels of auxins such as indole-3-acetic acid (Lopez-Carbonell et al., 1992). The increase of auxins levels during the dark period at the early stage of the regeneration process may be comparable to the exogenous addition of 2,4-D during 3 days assayed in the current study. From our results it is notable that the effect of the 2,4-D treatment was genotype and explant dependent. The 2,4-D treatment significantly improved regeneration from hypocotyl slices in 3 out of 5 genotypes tested (Fig. 2A). Similarly, regeneration from apricot (cv.’Canino’) mature hypocotyl slices was not affected or negatively affected by 2,4-D pulses or dark incubation initial period, respectively (Wang et al., 2011). On the other side, the 2,4-D pulse significantly increased regeneration percentages from cotyledons in all the 5 genotypes tested (Fig. 2A). It is widely known that optimal regeneration conditions are highly genotype dependent. A study on peach (Prunus persica) cultivars and P. persica x P. dulcis hybrids showed as differences in regeneration among genotypes were related to the endogenous differences in the hormonal content (Pérez-Jiménez et al., 2014). Therefore, organogenesis depends on the interaction between exogenously added plant growth regulators over the concentration of endogenous hormones, together with the sensitivity of the tissues (explant type) to particular hormone groups (Reviewed by Gaspar et al., 1996; Jiménez, 2005). In further studies, different 2,4-D concentrations and periods of application might be tested in order to optimize its usage for each explantgenotype tandem. To date in Prunus, there are scarce transformation procedures available from clonal tissues and they are limited to few genotypes; one apricot cultivar (‘Helena’), one sour cherry cultivar (‘Montmorency’), one almond clone (a seedling from’ Boa Casta’), one European plum cultivar (‘Startovaya’) and two cherry rootstocks (‘Gisela 6’ and ‘Gisela 7’) (Costa et al., 2006; Petri et al., 2008a; Sidorova et al., 2017; Song and Sink, 2006; Song et al., 2013). Many of the transformation procedures in Prunus spp. involve the use of seed-derived explants: Apricot (Câmara Machado et al., 1992; Petri et al., 2015), European plum (Petri
5. Conclusions The goal of this study was accomplished. We have developed an efficient regeneration procedure from mature seeds hypocotyl sections and cotyledons of Prunus rootstocks (four genotypes of P. cerasifera and one genotype of P. insititia). The ability to consistently regenerate plants from Prunus rootstock seed-derived tissues has a number of practical applications. The method allows rapid shoot multiplication as numerous shoots are produced from each seed. Other potential application includes the improvement of Prunus rootstocks through the introduction of engineered genes. The high quality lineage of the plant material used in this study (Table 1) makes this last application of particular interest to stack more agronomical traits in elite genotypes. The hypocotyl regeneration detailed in this manuscript is similar to that described in the highly efficient Agrobacterium-mediated transformation procedure of P. domestica (Petri et al., 2008b). The procedure has been successfully applied for the production of new clones with agronomical interesting traits, such as pests or diseases resistance (Petri et al., 2018). To our knowledge, in Prunus there is not a current transformation procedure that uses mature cotyledons as the source of explants. Further research will be conducted to study the possibility of combining these regeneration procedures with gene transfer techniques. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments Authors deeply thanks Elena García Martín for providing ‘Adara’, ‘Ademir’ ‘Myrobalan B’ and ‘Myrobalan 29C’ fruits. References Beckman, T.G., Lang, G.A., 2003. Rootstock breeding for stone fruits. Acta Hortic. 622, 531–551. https://doi.org/10.17660/ActaHortic.2003.622.58. Câmara Machado, M.L da, Câmara Machado, A. da, Hanzer, V., Weiss, H., Regner, F., Steinkellner, H., Mattanovich, D., Plail, R., Knapp, E., Kalthoff, B., 1992. Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of plum pox virus. Plant Cell Rep. 11, 25–29. Canli, F.A., Tian, L., 2009. Regeneration of adventitious shoots from mature stored cotyledons of Japanese plum (Prunus salicina Lind1). Sci. Hortic. (Amst.) 120, 64–69. https://doi.org/10.1016/j.scienta.2008.09.017. Canli, F.A., Tian, L., 2008. In vitro shoot regeneration from stored mature cotyledons of
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Short communication
Adventitious regeneration from mature seed-derived tissues of Prunus cerasifera and Prunus insititia Elisabeth Carmona-Martin, Cesar Petri
T
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Departamento de Fruticultura Subtropical y Mediterránea, Instituto de Hortofruticultura Subtropical y Mediterránea, UMA-CSIC, Avenida Dr. Wienberg, s/n. 29750 Algarrobo-Costa, Málaga, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Rosaceae Auxin pulses Woody plants Organogenesis 2,4-dichlorophenoxyacetic acid Rootstocks
An essential preliminary step in the application of genetic engineering is the development of robust and effective procedures of adventitious regeneration. We have developed an efficient regeneration procedure from mature seeds hypocotyl sections and cotyledons of Prunus rootstocks: Four genotypes of Prunus cerasifera (‘Adara’, ‘Ademir’, ‘Myrobolan B’ and ‘Myrobalan 29C’) and one genotype of Prunus insititia (‘Pixi’). A pulse of 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) significantly stimulated adventitious organogenesis from both sort of explants. However, the 2,4-D treatment effect relied on the genotype and the type of explant. Best regeneration rates achieved from hypocotyl sections were 16.28%, 31.25%, 13.79%, 46.86% and 53.03% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. Best regeneration rates achieved from cotyledons were 60.00%, 51.61%, 42.86%, 75.00%, 90.48% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. Rooting and acclimatization steps were successfully achieved. The whole process, from initial culture to established plants in the greenhouse, took approximately 15 weeks.
1. Introduction Stone fruits (Prunus) are economically important worldwide. This genus comprises apricots, almonds, cherries, plums, prunes, peaches, etc. In the commercial cultivation of these species, the desired cultivar, vegetatively propagated, is grafted onto a rootstock. Seedling rootstocks have been traditionally used. Nevertheless, more and more commercial clonal Prunus rootstocks are being released and used in the orchards as a result from private and public rootstock breeding programs (Beckman and Lang, 2003). Particularly remarkable among recent releases has been the incorporation of resistance to soil borne diseases, nematodes, waterlogging, iron chlorosis and vigor control (Beckman and Lang, 2003; Moreno, 2004; Moreno et al., 1995a, 1995b; Reig et al., 2018). These genotypes are quite interesting as a starting material to apply genetic engineering techniques in order to stack new agronomical traits that cannot be otherwise introduced by hybridization and selection. This could be the case of resistance traits non-available into the species or related wild species germplasms. A crucial preliminary step in the application of genetic engineering is the development of robust and effective procedures of adventitious
regeneration. Consequently, the goal of the present work was to establish methodologies to obtain adventitious regeneration from tissues of the Prunus rootstocks described in Table 1. For this purpose, we adapted procedures previously used successfully with other close-related species, such as apricot (P. armeniaca), European plum (P. domestica), Japanese plum (P. salicina) and cherry (P. avium) (Canli and Tian, 2009, 2008; Petri et al., 2008b; Urtubia et al., 2008; Wang et al., 2013a, 2011). 2. Materials and methods 2.1. Plant material and explant preparation Open pollinated mature seeds of the rootstocks ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ (P. cerasifera) and ‘Pixi’ (P. insititia) were collected at the “Estación Experimental Aula Dei” (EEAD-CSIC) in Zaragoza, Spain (Table 1). For seeds disinfection, the endocarp was removed with a nutcracker, and seeds were immersed in a beaker containing a solution of 20% commercial bleach (commercial bleach = 6.15% sodium hypochlorite) and 0.02% Tween-20 for 20 min and rinsed three times with sterile
Abbreviations: 2,4-D, 2,4- dichlorophenoxyacetic acid; BAP, benzylaminopurine; IBA, indole butyric acid; NAA, α-naphthaleneacetic; PG, phloroglucinol; TDZ, thidiazuron; RM, rooting medium; SGM, shoot growing medium; SRM, shoot regeneration medium ⁎ Corresponding author. E-mail address: [email protected] (C. Petri). https://doi.org/10.1016/j.scienta.2019.108746 Received 1 July 2019; Received in revised form 1 August 2019; Accepted 3 August 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 259 (2020) 108746
Dwarfing Vigorous
Wide range of compatibility Vigorous Vigorous
Tolerance to Pseudomonas syringae Resistance to Meloidogyne spp.
Non defined Tolerance to Verticilium, Phytophthora and Pseudomonas syringae Resistance to Meloidogyne spp. Tolerance to Armillaria, Phytophthora, Agrobacterium and Verticilium.
distilled water in a laminar flow bench. Disinfected seeds were soaked in sterile water overnight at 4 °C. Hypocotyl sections were prepared as previously described (Petri et al., 2012). The radicle and the epicotyl were discarded, and the hypocotyl was sliced into 1–4 cross sections (0.5–1 mm) (Fig. 1A). Cotyledon explants were prepared as described in Wang et al. (2013a) and placed onto the shoot regeneration medium (SRM) with the adaxial face down (Fig. 1A).
2.2. Adventitious bud regeneration, shoot elongation, rooting and acclimatization For all these steps previously described procedures, media and culture conditions were followed (Petri et al., 2012), with some modifications as described below. Briefly, media used were MS-based (Murashige and Skoog, 1962) with the following growth regulators: shoot regeneration medium (SRM) contained 7.5 μM thidiazuron (TDZ) and 0.25 μM indole butyric acid (IBA); shoot growing medium (SGM) contained 1.5 μM benzylaminopurine (BAP), instead of 3.0 μM as described in the original recipe, and 0.25 μM IBA; rooting medium (RM) contained 0.1 μM kinetin and 5.0 μM α-naphthaleneacetic acid (NAA). Additionally, in this occasion 100 mg l−1 phloroglucinol (PG) was added to RM. In order to study the effect of 2,4- dichlorophenoxyacetic acid (2,4-D) pulses, we tested a treatment described for Prunus domestica (Petri et al., 2008b). Explants were cultured on SRM supplemented with 9.05 μM 2,4-D during 3 days and then transferred to SRM, or explants were placed directly onto SRM. After 5–7 weeks, bud clusters were excised from the explant and placed onto SGM. When shoots reached 2–3 cm long they were separated from the cluster and transferred to Magenta type vessels with RM. Tissue culture chamber conditions, along the whole process (from initial culture to rooting), were set up to a 16/8 h light/dark cycle, light intensity of 45–50 μE m−2s−1, and a temperature of 25+1 °C. Regeneration was evaluated weekly, starting 2 weeks after the beginning of the experiment until adventitious bud regeneration stopped appearing for 2 consecutive weeks (approximately 6–7 weeks from the beginning of the experiment). An average of 9 hypocotyl slices and 6 cotyledon explants were used for each genotype and treatment. Regeneration experiments were repeated, at least, three times. Rooting data was collected 4 weeks after the shoots were placed in RM supplemented with 100 mg l−1 PG. Rooting, and subsequent acclimatization, were repeated twice. A pool of 87 rooted shoots belonging to the 5 genotypes was chosen for acclimatization. Rooted shoots were washed in water to eliminate agar residues and transferred to pots containing 60% coconut fiber, 30% commercial substrate (Turbas y Coco Mar Menor S.L., Spain) and 10% litonite. The potted plantlets were introduced into plastic bags which were sealed and maintained in a growth chamber with a 16/8 h light/dark cycle, light intensity of 45–50 μE m−2s−1, 70% relative humidity and a temperature of 27+1 °C. The plastic bags were opened gradually and, after 2 weeks, they were fully opened. Following 1 week in opened plastic bags during which time plants were irrigated as needed and finally, plants were transferred to a greenhouse.
EEAD-CSIC (Spain) East Malling (UK) University of California (USA) Prunus cerasifera Prunus cerasifera Prunus cerasifera Ademir Myrobalan B Myrobalan 29C
Non defined Resistance to root asphyxia and iron-induced chlorosis Resistance to iron chlorosis and root asphyxia Tolerance to drought Tolerance to root asphyxia East Malling (UK) EEAD-CSIC (Spain) Prunus insititia Prunus cerasifera Pixi Adara
Abiotic stress resilience Origin Species Rootstock
Table 1 Brief description of the Prunus rootstocks used as the source of explants in this study.
Biotic stress resilience
Other characteristics
E. Carmona-Martin and C. Petri
2.3. Experimental design and statistical analyses Regeneration and rooting experiments were performed in a completely randomized design. To test for differences among more than two independent groups, data were transformed as needed to fit to a Normal distribution and ANOVA was performed. To compare treatments within a given genotype Student’s t-test (No. of roots per rooted shoot, fresh weight of explants) or Pearson’s Chi-test (regeneration percentages) were applied.
2
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Fig. 1. (A) Source of explants. Cotyledon explant as described in Wang et al. (2013a). The hypocotyl was sliced into 1 to 4 cross sections (0.5–1 mm). The radicle and the epicotyl were discarded. Bar represents 1 mm. (B) Adventitious regeneration from a ‘Pixi’ hypocotyl section. Picture was taken at 5 weeks from the beginning of the experiment. Bar represents 1 mm. (C) Adventitious regeneration from a ‘Adara’ cotyledon. Picture was taken at 4 weeks from the beginning of the experiment. Bar represents 1 mm. (D) ‘Myrobalan 29C’ shoots after 4 weeks cultured in SGM. Bar represents 1 cm. (E) ‘Adara’ rooted shoot prompt for the acclimatization stage. Bar represents 1 cm. (F) Acclimatized shoots cultured in a greenhouse. (Color figure on line).
3. Results
the proximal surface of the explant (Fig. 1C).
All genotypes showed an identical regeneration, elongation and rooting pattern (Fig. 1B-E). First buds started appearing 2–3 weeks after the beginning of the experiment as direct adventitious regeneration (without the formation of an intermediated callus). In hypocotyl sections regeneration appeared on the explant border as bud clusters, and frequently a continuous crown along the border was observed (Fig. 1B). Regeneration in the cotyledons also appeared as bud clusters, mostly on
3.1. Effect of 2,4-D treatment Both types of explants (hypocotyls slices and cotyledons) were directly cultured on SRM or they were firstly cultured on SRM supplemented with 9.05 μM 2,4-D for 3 days and then transferred to SRM. The 2,4-D pulse significantly affected the regeneration rates from hypocotyl sections (P < 0.05) and cotyledons (P < 0.01). The 2,4-D treatment 3
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Fig. 2. Effect of 2,4-D treatment. Bars show Standard Errors. Asterisks indicate statistical significance between treatments within a given genotype (*P < 0.05; **P < 0.01). A: Regeneration rates from hypocotyl sections. A total of 453 explants were used in this study. B: Regeneration rates from cotyledons. A total of 184 explants were used in this study. C: Explants from ‘Pixi’ seeds cultured onto SRM for 5 weeks. D: Fresh weight of hypocotyl explants after 7 weeks cultured on SRM. A total of 184 explants were used in this study. (Color figure on line).
randomly chosen) was annotated. Data analyses showed that fresh weight of the 2,4-D treated explants was significantly higher (P < 0.05) compared to the fresh weight of the non-treated ones, except for ‘Myrobalan B’ (Fig. 2D).
Table 2 Rooting results. Genotype
No. of shoots placed in RM
Rooting rate (% + S.E)
Number of roots/ rooted shoot (Mean + S.E.)
Ademir Adara Myrobalan B Myrobalan 29C Pixi
21 20 18 18 19
100.0 + 0.0 100.0 + 0.0 92.8 + 6.9 100.0 + 0.0 89.4 + 7.1
4.6 3.8 5.1 4.7 3.7
+ + + + +
3.2. Shoot elongation, rooting and acclimatization
0.4 0.4 0.5 0.4 0.5
One week after buds/clusters appeared they were excised from the explant and transferred to SGM. This medium resulted adequate for shoot elongation for all genotypes tested. In approximately 3–4 weeks, shoots of about 2–3 cm long were available (Fig. 1D) and ready to be separated and placed in RM. Adventitious roots started being visible after 2 weeks of culture in RM supplemented with 100 mg l−1 PG. After two more additional weeks, shoots showed a well-developed root system (Fig. 1E). At this point rooting data was collected. Statistical differences among genotypes were not detected for any of both parameters measured; rooting rate and number of roots per rooted shoot (Table 2). Overall, rooting rate was nearly 100% with 4–5 roots per shoot (Table 2; Fig. 1E). A pool of shoots belonging to the five genotypes was chosen to proceed with the acclimatization step. 86 shoots, out of 87, survived and were transferred to the greenhouse (Fig. 1F).
Data was collected 4 weeks after shoots were placed in RM. Statistical differences among genotypes were not detected for any of the two parameters measured. Rooting experiment was repeated twice.
increased regeneration percentages from hypocotyl slices compared to the regeneration observed without the 2,4-D pulse in all genotypes tested. Nevertheless, the augmentation only resulted statistically significant for ‘Adara’ (P < 0.05), ‘Myrobalan 29C’ (P < 0.01) and ‘Pixi’ (P < 0.01) (Fig. 2A). Best regeneration rates achieved from mature seeds hypocotyl sections were 16.28 + 5.63%, 31.25 + 6.69%, 13.79 + 6.42%, 46.86 + 8.82% and 53.03 + 6.14% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. In cotyledons, regeneration was significantly higher in the 2,4-D treated explants compared to the non-treated ones for all genotypes tested (Fig. 2B). For ‘Ademir’ and ‘Myrobalan B’ cotyledons, the 2,4-D treatment was critical and adventitious regeneration was only observed when the auxin pulse was applied (Fig. 2B). Best regeneration rates achieved from mature seeds cotyledons were 60.00 + 8.98%, 51.61 + 9.8%, 42.86 + 18.7%, 75.00 + 15.31%, 90.48 + 6.4% for ‘Ademir’, ‘Adara’, ‘Myrobalan B’, ‘Myrobalan 29C’ and ‘Pixi’, respectively. Along the regeneration experiments, explants that were cultured for 3 days with 9.05 μM 2,4-D seemed larger and healthier than those without the treatment (Fig. 2C). After regeneration data was collected (7 weeks from the beginning of the experiment), fresh weight of some hypocotyl explants (approximately 20 per genotype-treatment,
4. Discussion To our knowledge, this is the first report on direct organogenesis from P. cerasifera and P. insititia. As the source of explants, we have chosen rootstock genotypes which accumulate many interesting agronomical traits, such as biotic and abiotic stress resistance (Table 1). We report in vitro adventitious shoot regeneration from two different mature seed-derived tissues. Data for the number of shoots were not presented since adventitious shoots appeared as clusters and it was difficult to accurately count them. Regeneration patterns and efficiencies, from both sort of explants, are comparable to those reported for another Prunus species (Canli and Tian, 2009, 2008; Mante et al., 1989; Petri et al., 2008b; Tian et al., 2007; Urtubia et al., 2008; Wang et al., 2013a, 4
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et al., 2008b; Tian et al., 2009; Wang et al., 2013b), Japanese plum (Urtubia et al., 2008) and peach (Perez-Clemente et al., 2005). A remarkable aspect is that these protocols have been shown to be transferable among genotypes. The regeneration pattern described in those procedures, as direct regeneration of bud clusters, is similar to the pattern observed in this study (Fig. 1B, C). It is not possible to improve a known rootstock/variety by transforming seed-derived tissues. Nevertheless, seed material could be very useful to accomplish several objectives in fundamental research areas such as gene silencing, gene expression studies, resistance assessments to insects, diseases and herbicides, and promoter gene assessments. Also, transformation of nonclonal material could be used to incorporate a new trait (such as disease resistance genes) in the gene pool of the species, making the trait available for breeders. We have used open-pollinated seeds in the current research. However, by using seeds from controlled crosses of a breeding program, it is assumable that the new engineered trait could be incorporated to an advanced/elite selection, which could become a new clonal rootstock or be used for further breeding. For some important soil pests, such as the flat-headed borers (Capnodis spp.), that affects the cultivation of stone fruits in the Mediterranean basin (Gindin et al., 2014), sources of genetic resilience has not been found yet into the Prunus germplasm (Dicenta et al., 2002; Mendel et al., 2006).
2011). Additionally, the process length was also satisfactory. The method takes about 15 weeks from initial culture to the establishment of the plants in the greenhouse, which is a reasonable time for woody plants. It is also notable to mention the high rooting rates and acclimatization survival achieved. We used a RM designed for P. domestica (Gonzalez Padilla et al., 2003) with the addition of 100 mg l−1 PG. PG is a rooting cofactor that has been previously reported to stimulate rhizogenesis in Prunus and other fruit tree species (Hammerschlag et al., 1987; James and Thurbon, 2013; Jones and Hatfield, 1976; Petri and Scorza, 2010). In peach and European plum, the addition of 100 mg l−1 to the RM significantly increased rooting efficiency for all genotypes tested (Hammerschlag et al., 1987; Petri and Scorza, 2010). Our experience, acquired over the years, in Prunus micropropagation, regeneration and genetic transformation allowed us to accomplish the acclimatization step without further difficulties. We applied well established procedures used routinely for P. domestica and P. armeniaca (Petri et al., 2014, 2012). Our results indicate that 2,4-D stimulates cell division and/or adventitious organogenesis from mature seed-derived tissues in P. cerasifera and P. insititia. Previous studies in Prunus spp. also stated pulses with 2,4-D as beneficial for organogenesis from leaf or hypocotyl explants (Pascual and Marín, 2005; Petri et al., 2008b, 2005; Petri and Scorza, 2010). In some cases, authors described an increment in callus formation when explants were exposed to auxin pulses (Petri et al., 2005; Wang et al., 2011), which agrees with the fresh weight increment observed in our experiments (Fig. 2C, D). Regeneration from mature cotyledons in different Prunus species has been reported (Canli and Tian, 2009, 2008; Hokanson and Pooler, 2000; Mante et al., 1989; Wang et al., 2013a). Auxin pulses were not assayed. However, most of them described a dark incubation period at the initial stage of the culture as a critical factor for obtaining higher regeneration efficiencies (Canli and Tian, 2009, 2008; Wang et al., 2013a). It has been reported that dark incubation influences endogenous levels of auxins such as indole-3-acetic acid (Lopez-Carbonell et al., 1992). The increase of auxins levels during the dark period at the early stage of the regeneration process may be comparable to the exogenous addition of 2,4-D during 3 days assayed in the current study. From our results it is notable that the effect of the 2,4-D treatment was genotype and explant dependent. The 2,4-D treatment significantly improved regeneration from hypocotyl slices in 3 out of 5 genotypes tested (Fig. 2A). Similarly, regeneration from apricot (cv.’Canino’) mature hypocotyl slices was not affected or negatively affected by 2,4-D pulses or dark incubation initial period, respectively (Wang et al., 2011). On the other side, the 2,4-D pulse significantly increased regeneration percentages from cotyledons in all the 5 genotypes tested (Fig. 2A). It is widely known that optimal regeneration conditions are highly genotype dependent. A study on peach (Prunus persica) cultivars and P. persica x P. dulcis hybrids showed as differences in regeneration among genotypes were related to the endogenous differences in the hormonal content (Pérez-Jiménez et al., 2014). Therefore, organogenesis depends on the interaction between exogenously added plant growth regulators over the concentration of endogenous hormones, together with the sensitivity of the tissues (explant type) to particular hormone groups (Reviewed by Gaspar et al., 1996; Jiménez, 2005). In further studies, different 2,4-D concentrations and periods of application might be tested in order to optimize its usage for each explantgenotype tandem. To date in Prunus, there are scarce transformation procedures available from clonal tissues and they are limited to few genotypes; one apricot cultivar (‘Helena’), one sour cherry cultivar (‘Montmorency’), one almond clone (a seedling from’ Boa Casta’), one European plum cultivar (‘Startovaya’) and two cherry rootstocks (‘Gisela 6’ and ‘Gisela 7’) (Costa et al., 2006; Petri et al., 2008a; Sidorova et al., 2017; Song and Sink, 2006; Song et al., 2013). Many of the transformation procedures in Prunus spp. involve the use of seed-derived explants: Apricot (Câmara Machado et al., 1992; Petri et al., 2015), European plum (Petri
5. Conclusions The goal of this study was accomplished. We have developed an efficient regeneration procedure from mature seeds hypocotyl sections and cotyledons of Prunus rootstocks (four genotypes of P. cerasifera and one genotype of P. insititia). The ability to consistently regenerate plants from Prunus rootstock seed-derived tissues has a number of practical applications. The method allows rapid shoot multiplication as numerous shoots are produced from each seed. Other potential application includes the improvement of Prunus rootstocks through the introduction of engineered genes. The high quality lineage of the plant material used in this study (Table 1) makes this last application of particular interest to stack more agronomical traits in elite genotypes. The hypocotyl regeneration detailed in this manuscript is similar to that described in the highly efficient Agrobacterium-mediated transformation procedure of P. domestica (Petri et al., 2008b). The procedure has been successfully applied for the production of new clones with agronomical interesting traits, such as pests or diseases resistance (Petri et al., 2018). To our knowledge, in Prunus there is not a current transformation procedure that uses mature cotyledons as the source of explants. Further research will be conducted to study the possibility of combining these regeneration procedures with gene transfer techniques. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments Authors deeply thanks Elena García Martín for providing ‘Adara’, ‘Ademir’ ‘Myrobalan B’ and ‘Myrobalan 29C’ fruits. References Beckman, T.G., Lang, G.A., 2003. Rootstock breeding for stone fruits. Acta Hortic. 622, 531–551. https://doi.org/10.17660/ActaHortic.2003.622.58. Câmara Machado, M.L da, Câmara Machado, A. da, Hanzer, V., Weiss, H., Regner, F., Steinkellner, H., Mattanovich, D., Plail, R., Knapp, E., Kalthoff, B., 1992. Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of plum pox virus. Plant Cell Rep. 11, 25–29. Canli, F.A., Tian, L., 2009. Regeneration of adventitious shoots from mature stored cotyledons of Japanese plum (Prunus salicina Lind1). Sci. Hortic. (Amst.) 120, 64–69. https://doi.org/10.1016/j.scienta.2008.09.017. Canli, F.A., Tian, L., 2008. In vitro shoot regeneration from stored mature cotyledons of
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