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Adventitious Shoot Regeneration and Agrobacterium Tumefaciens-Mediated Transient Transformation of Almond 3 Peach Hybrid Rootsto

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Adventitious Shoot Regeneration and Agrobacterium Tumefaciens-Mediated Transient Transformation of Almond 3 Peach Hybrid Rootsto HORTSCIENCE 54(5):936–940. 2019. https://doi.org/10.21273/HORTSCI13930-19 sirable. Because the ‘Hansen 536’ rootstock is propagated clonally, genetic engineering is an effective approach to incorporate target Adventitious Shoot Regeneration and genes of interest for its further improvement. To achieve this, an efficient in vitro shoot Agrobacterium tumefaciens-mediated regeneration system is needed. Successful in vitro shoot regeneration Transient Transformation of from seed-derived or leaf explants has been reported for several Prunus species, such as 3 P. persica (Druart, 1999; Gentile et al., 2002; Almond Peach Hybrid Rootstock Hammerschlag et al., 1985; Mante et al., 1989; Pooler and Scorza, 1995; Scorza ‘Hansen 536’ et al., 1990), P. canescens (Antonelli and Druart, 1989), P. padus (Hammatt, 1993), P. Xiaojuan Zong domestica (Bassi and Cossio, 1991, 1994; Department of Horticulture, Michigan State University, Plant Biotechnology Yancheva, 1993), P. dulcis (Ainsley et al., Resource and Outreach Center, East Lansing, MI 48824; and Shandong 2000; Miguel and Oliveira, 1999; Miguel Institute of Pomology, Shandong Academy of Agricultural Sciences, Taian, et al., 1996), P. armeniaca (Perez-Tornero P.R. China et al., 2000), P. serotina (Espinosa et al., 2006; Hammatt and Grant, 1998), P. avium Brandon J. Denler (Bhagwat and Lane, 2004; Canli and Tian, Department of Horticulture, Michigan State University, Plant Biotechnology 2008; Feeney et al., 2007; Hammatt and Grant, 1998; Matt and Jehle, 2005; Tang Resource and Outreach Center, East Lansing, MI 48824 et al., 2002; Zong et al., 2019), P. cerasus Gharbia H. Danial (Mante et al., 1989; Sarropoulou et al., 2012; Song and Sink, 2006; Tang et al., 2002), and Department of Horticulture, Michigan State University, Plant Biotechnology several hybrid rootstocks (Hasan et al., 2010; Resource and Outreach Center, East Lansing, MI 48824; and Scientific Ochatt et al., 1988; Perez-Jimenez et al., Research Center, Faculty of Science, University of Duhok, Duhok, Kurdistan 2012, 2014; Pooler and Scorza, 1995; San Region, Iraq et al., 2015; Sarropoulou et al., 2012; Zhou et al., 2010). For most cultivars of Prunus Yongjian Chang species, leaf explants are preferable to seed- North American Plants, Inc., McMinnville, OR 97128 derived explants for maintaining an identical genetic background of the cultivars be- Guo-qing Song1 cause of their heterozygosity and self- Department of Horticulture, Michigan State University, Plant Biotechnology incompatibility. To date, plant regeneration Resource and Outreach Center, East Lansing, MI 48824 from leaf explants of the ‘Hansen 536’ rootstock has not been reported. Additional index words. leaf explants, micropropagation, organogenesis Peach and peach rootstocks are among the 3 most recalcitrant plants for genetic transfor- Abstract. ‘Hansen 536’ (Prunus dulcis Prunus persica) is an important commercial mation, although some efforts using particle rootstock for peach and almond. However, susceptibility to wet soil and bacterial canker bombardment or Agrobacterium-mediated has limited its use primarily to areas with less annual rainfall. Genetic engineering transformation in peach have been made techniques offer an attractive approach to improve effectively the current problems with (Escalettes et al., 1997; Padilla et al., 2006; this cultivar. To develop an efficient shoot regeneration system from leaf explants, 10 Perez-Clemente et al., 2005; Scorza et al., culture media containing Murashige and Skoog (MS) or woody plant medium (WPM) 1995; Ye et al., 1994). Only one study supplemented with different plant growth regulators were evaluated, and adventitious reported the success of obtaining stable shoot regeneration occurred at frequencies ranging from 0% to 36.1%. Optimal transgenic plants from an Agrobacterium- regeneration with a frequency of 32.3% to 36.1% occurred with WPM medium m m mediated transformation using embryo sec- containing 8.88 M 6-benzylamino-purine (BAP) and 0.98 to 3.94 M indole-3-butyric tions of mature seeds (Perez-Clemente et al., acid (IBA). The regenerated shoots had a high rooting ability, and 80% of the in vitro 2005). Lack of an efficient in vitro shoot shoots tested rooted and survived after being transplanted to substrate directly. b regeneration system remains the main obsta- Transient transformation showed an efficient delivery of the -glucuronidase (GUS) cle for stable transformation of peach and reporter gene (gusA) using all three Agrobacterium tumefaciens strains tested with a peach rootstocks. concentration of OD600 0.5 to 1.0 for 4 days of cocultivation. The protocols described We describe a protocol for in vitro shoot provide a foundation for further studies to improve shoot regeneration and stable regeneration from leaf explants of the ‘Han- transformation of the important peach and almond rootstock ‘Hansen 536’. sen 536’ rootstock. Several factors affecting in vitro shoot regeneration were optimized. Under optimal in vitro shoot regeneration The almond · peach hybrid rootstock 1986). It is an important commercial root- conditions, two key factors for A. tumefa- ‘Hansen 536’ was introduced from the stock for peach, almond, prune, and plum in ciens-mediated gene delivery—Agrobacte- Hesse-Hansen program (Kester and Asay, California. ‘Hansen 536’ rootstocks show rium strains and time of cocultivation— medium size and short dormancy, and have were evaluated. The overall results provide Received for publication 6 Feb. 2019. Accepted for a relatively high adventitious root system that guidelines for stable transformation of the publication 5 Mar. 2019. provides excellent anchorage in orchards ‘Hansen 536’ rootstock. This work was supported by Michigan State (Kester and Asay, 1986). However, because University under Grant of Project GREEEN (Gen- of its high susceptibility to root rot fungus erating Research and Extension to meet Economic infection and wet soils, this rootstock cultivar and Environmental Needs), Michigan State Uni- Materials and Methods versity, AgBioResearch; and the National Natural is not suitable for areas with high annual Science Foundation of China (no. 31601732). rainfall (Kester and Asay, 1986). Therefore, Plant materials. The ‘Hansen 536’ root- 1Corresponding author. E-mail: [email protected]. further improvement of this rootstock is de- stock cultures were provided by North 936 HORTSCIENCE VOL. 54(5) MAY 2019 American Plants, Inc (McMinnville, OR). tham, MA). In vitro shoot induction was every 3 d to keep the humidity inside the The stock cultures, four to five internode conducted in the dark at 25 °C for 2 weeks bag. After 4 weeks, the bags were opened segments each with one to three nodes, were and then was transferred to a 16-h photope- gradually for a couple of hours daily for 1 cultured on 50-mL shoot proliferation me- riod. Subcultures to the same fresh regener- week and subsequently removed com- dium (SPM) in a MagentaÒ GA7 box (Sigma- ation media were performed every 3 weeks. pletely. The total number of surviving plants Aldrich, St. Louis, MO). All basal media, The number of explants that produced at least was recorded at the end of the 4-week hormones, and antibiotics used in this study one shoot (length, >0.5 cm) was recorded at acclimation. were purchased from PhytoTechnology Lab- 4, 8, and 12 weeks. The number of shoots Optimizing A. tumefaciens-mediated trans- oratories (Shawnee Mission, KS). The SPM (length, >0.5 cm) per leaf were counted after formation factors via transient expression contained MS medium (Murashige and 8 or 12 weeks. experiments. The experimental design was Skoog, 1962), 4.44 mM BAP and 0.49 mM Three types of basal media WPM (Lloyd based on previous studies of cherry root- IBA. All cultures were maintained at 25 °C and McCown, 1980), MS, and ½ MS, each stocks and sour cherry (Song, 2015; Song with a 16-h photoperiod (40 mmol·s–1·m–2) containing 8.88 mM thidiazuron (TDZ) and and Sink, 2005, 2006). Briefly, A. tumefa- and cool-white fluorescent lighting unless 1.97mM IBA, were evaluated for in vitro shoot ciens strains EHA105, LBA4404, and otherwise noted, and they were subcultured induction from leaf explants (Table 2). The GV3101, each harboring the binary vector every 4 weeks using the same culture me- leaf explants were placed with the abaxial pBISN1 (Narasimhulu et al., 1996), were dium. The fully expanded leaves of 5- to side either up or down on the media to used. pBISN1 contains the neomycin phos- 6-week-old stock cultures were used for re- evaluate the effects of leaf orientation on in photransferase gene (nptII) directed by the generation studies. vitro shoot regeneration. nos promoter and a plant intron interrupted Optimizing shoot regeneration from leaf Rooting and acclimatization. The in vitro GUS directed by the chimeric super promoter explants. Young, fully expanded leaves regenerated shoots were transferred to 50 (Aocs)3AmasPmas (Ni et al., 1995). Single (length, 1.5–2.0 cm) that included the petiole mL SPM in each MagentaÒ GA7 box colonies of the three strains were each cul- from in vitro regenerated shoots were (Sigma-Aldrich) to increase the number of tured in 10 mL liquid YEB + 50 mg·L–1 wounded by several cuts transversely along shoots. After 6 weeks of culture in SPM, the kanamycin monosulfate at 28 °C in the dark the midrib while leaving the sections intact. elongated shoots (length, >3 cm) were ex- for 48 h. Bacterial cells were collected by a Ten regeneration media [woody plant regen- cised and transferred directly to a 4-inch 2-min centrifuge at 2500 gn and resuspended eration medium (WPRM) 1–WPRM10] were plastic pot (10.2 · 10.2 · 12.7 cm) contain- in liquid coculture medium (WPM + 8.88 mM designed to evaluate the effects of the com- ing water-saturated planting medium (Mich- TDZ + 1.97 mM IBA + 100 mM acetosyrin- bination and concentration of hormones igan Grower Products, Inc., Galesburg, MI) gone) to an optical density, with a wave- (Table 1). Leaf explants were cultured with that was autoclaved at 121 °C for 10 min length of 600 nm (OD600), of 0.1, 0.5, and 1.0 the abaxial surfaces either up or down on before use.
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