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Adventitious Shoot Regeneration of Pear (Pyrus Spp.) Genotypes

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Adventitious Shoot Regeneration of Pear (Pyrus Spp.) Genotypes Plant Cell Tiss Organ Cult (2012) 108:229–236 DOI 10.1007/s11240-011-0034-4 ORIGINAL PAPER Adventitious shoot regeneration of pear (Pyrus spp.) genotypes Richard L. Bell • Ralph Scorza • Delores Lomberk Received: 29 September 2010 / Accepted: 24 June 2011 / Published online: 18 September 2011 Ó Springer Science+Business Media B.V. (outside the USA) 2011 Abstract Adventitious shoot regeneration of twenty-four cultures (0–70.7%). Maximum regeneration was observed pear genotypes was compared in a common in vitro shoot for ‘Conference’, followed by ‘Magness’, ‘Dr. Jules induction and development protocol. This study also Guyot’, and Packham’s Triumph’. The range of number of compared cultures newly established from scionwood with adventitious shoots was relatively narrow, with the mini- cultures that been in long-term cold storage. In vitro cul- mum of 1.0 for seven genotypes to 2.2 for ‘Conference’. tures of 13 Pyrus genotypes and budwood from 23 Pyrus genotypes were obtained from the National Clonal Germ- Keywords Biotechnology Á Morphogenesis Á Pyrus plasm Repository (NCGR) in Corvallis, Oregon. With the 9bretschneideri Á Pyrus pyrifolia var. sinensis Á Pyrus exception of one genotype of P. elaeagrifolia Pall., and communis Á Pyrus elaeagrifolia ‘Ya Li’ (P. pyrifolia var. sinensis Teng & Tanabe), all were P. communis L. cultivars. The basal shoot induction media consisted of Chevreau and Leblay (CL) basal nutrients, Introduction vitamins, and organics (Chevreau and Leblay in Acta Hortic 336: 263–268, 1993). The analysis of variance Tree fruit species are generally recalcitrant to in vitro indicated that differences among genotypes were highly regeneration. Pear is among these. Relatively high levels of significant and the main effect of culture origin was non- regeneration are required in order to recover plants fol- significant. However, there was a significant interaction lowing commonly employed genetic transformation pro- between genotype and culture origin, with percentage cedures such as the use of Agrobacterium tumefaciens.In regeneration of ‘Abate Fetel’ from new budwood signifi- terms of genomics research in fruit trees, the inability to cantly greater than that from long-term in vitro cultures, readily regenerate and transform a species relegates much while ‘Jesinji Vodenac’ cultures derived from the old of the testing of candidate genes and promoters to model NCGR cultures regenerated significantly more adventitious species. Testing genes directly on the recalcitrant species shoots. The ranges of mean regeneration frequency were and cultivars of interest may require a considerable allo- similar for both in vitro (0–87.7%) and scionwood-derived cation of time and resources with an uncertain outcome. Finally, recalcitrance to regeneration and transformation also precludes reverse genetics research strategies in the R. L. Bell (&) Á R. Scorza species of interest. Regeneration and/or transformation of US Department of Agriculture, Agricultural Research Service, several cultivars of Pyrus communis and genotypes of other 2217 Wiltshire Road, Kearneysville, WV 25430, USA Pyrus species have been reported. Regeneration frequen- e-mail: [email protected] cies are genotype-dependent (Abdollahi et al. 2006; URL: www.ars.usda.gov/main/site_main.htm?modecode = 19-31-00-00 Chevreau and Bell 2005; Chevreau et al. 1997; Hennayake et al. 2003; Lane et al. 1998; Leblay et al. 1991; Predieri D. Lomberk et al. 1989; Tang et al. 2008; Zhu and Welander 2000) and US Department of Agriculture, Agricultural Research Service, can vary depending on the protocol and even between EPCOT Science, 2013 North Avenue of the Stars, Lake Buena Vista, FL 32830, USA replicated experiments with the same protocol. Reported 123 230 Plant Cell Tiss Organ Cult (2012) 108:229–236 maximum regeneration frequencies range from 9% for Lepoivre shoot proliferation medium (Leblay et al. 1991) ‘Chojuro’ (Lane et al. 1998) to 100% for ‘Conference’ supplemented with 2.5 lM BA in MagentaTM GA-7 ves- (Chevreau et al. 1997). In most cases, rates of transgenic sels, with transfers to fresh medium every 4 weeks for plant regeneration are low, commonly less than 2% 6 months. During micropropagation, no evidence of inter- (Chevreau and Bell 2005), but with rates as high as nal bacterial contamination was observed. 12–43% for ‘Conference’ (Mourgues et al. 1996). The top 3 fully expanded leaves were excised from Shoot proliferation rates and adventitious regeneration 4-week old shoots. Each leaf was wounded by making 3 from leaf explants derived from continuous long-term in cuts with a scalpel transversely across the midrib and ex- vitro cultures of pear have been shown to decrease with planted abaxial side down onto shoot regeneration medium. time in culture (unpublished data). Cold treatments of up to The basal shoot induction media consisted of Chevreau and 6 months are sometimes used to reinvigorate shoot cul- Leblay (CL) basal nutrients, vitamins, and organics, 30 g/l TM tures, but initiation of new cultures from buds excised from sucrose, and 2.5 g/l Phytagel , with a pH of 5.8 (Chevreau scionwood is also used to maintain vigorous cultures for and Leblay 1993), but modified to contain 37.3 mg l-1 -1 experimentation (E. Chevreau, pers. comm.). Na2-EDTA and 27.85 mg l FeSO4Á7H2O instead of The objective of this study was to evaluate regeneration NaFe-EDTA. Each 100 mm 9 15 mm petri dish contained of a number of Pyrus scion and rootstock cultivars and 25 ml of medium. The initial shoot induction medium genotypes in order to select consistently high regeneration- (SIM) contained 12.5 lM indole-3-butyric acid (IBA) and competent genotypes that could be used in transformation 10 lM thidiazuron (TDZ). The cultures were incubated in studies leading to a model Pyrus system for candidate gene the dark for 4 days then transferred to similar medium and promoter testing, and for reverse genetic studies. This containing 10 lM thidiazuron (TDZ) and 1 lM IBA and study also compared cultures newly established from incubated in the dark for 4 weeks. At 4 and 8 weeks, the scionwood with cultures that been in long-term cold explants were transferred to fresh, but auxin-free, shoot storage. expression medium (SEM) and incubated in the dark. While regeneration was observed at 4 and 8 weeks, addi- tional regenerants were observed at 12 weeks, and those Materials and methods data were used for analysis. The CL medium, vitamins and organics were chosen as our basal medium because it has Shoot proliferation cultures been shown in several published studies and our own experience to result in better regeneration than other basal In vitro cultures of 13 Pyrus genotypes and budwood from media with which it has been compared. The TDZ and IBA 23 Pyrus genotypes were obtained from the National concentrations at the various stages of culture were those Clonal Germplasm Repository (NCGR) in Corvallis, Ore- which have been successfully used in our laboratory to gon. With the exception of one genotype of P. elaeagrifolia regenerated genetically transformed shoots (unpublished Pall., and ‘Ya Li’ (P. pyrifolia var. sinensis Teng & Tan- data). abe, formerly P. bretschneideri), the genotypes were P. communis L. cultivars selected for their commercial Experimental design importance or documented high levels of regeneration or in vitro adventitious rooting. In vitro shoot cultures were Ten of the 24 genotypes were represented by cultures established from the budwood by a standard shoot tip started from both in vitro and scionwood sources. Cultures culture initiation technique. Dormant budwood was washed of twelve of the genotypes were started from only scion- in soapy water and placed in beakers of water. After bud- wood, and five were from the NCGR long term in vitro break, 1–2 cm long shoots were excised and the largest (IV) source culture. Cultures from all sources were ana- unfurling leaves were removed. The shoots were surface- lyzed together to allow for comparisons among genotypes disinfected for 10 min in a 10% solution of ChloroxTM to and between culture sources. which a few drops of Tween 20 had been added, then Cultures were transferred to fresh medium every rinsed 3 times for 5 min in sterile Nano-Pure filtered water. 4 weeks. The apical three fully expanded leaves from Shoots from budwood and from NCGR in vitro cultures 4-week old shoots were excised for use as explants. Gen- were transferred to fresh Cheng’s medium (Cheng 1979) erally, ten leaf explants were explanted unto each of two supplemented with 2.5 lM 6-benzyladenine (BA), with pH petri plates. A set of 20 explants was considered one rep- adjusted to 5.2, and solidified with 1.75 g l-1 Bacto agar lication. There were a few cases of fewer than 10 explants and 0.75 g l-1 PhytagelTM (Sigma, St. Louis, MO) in per plate, depending on the number of high quality leaves MagentaTM GA-7 vessels every 4 weeks for 5 months. The available for use as explants, and therefore, the proportion cultures were then proliferated on a modified Quoirin and of regenerating explants was analyzed rather than the 123 Plant Cell Tiss Organ Cult (2012) 108:229–236 231 absolute number. There were five to eight replications per genotype for cultures originating from budwood and three to eight replications per genotype for cultures originating from the NCGR in vitro cultures. Statistical analyses Data for the ten genotypes for which cultures originated from old in vitro cultures and from younger cultures ini- tiated from scionwood were analyzed to determine whether origin of culture affected adventitious regeneration. The proportion of regenerating explants was initially analyzed according to a mixed effects factorial model with genotype and origin as fixed factors and set as a random effect nested within genotype 9 origin. The number of adventitious Fig. 1 Adventitious shoot regeneration of ‘Jesinji Vodenac’ after shoots per regenerating explant was also analyzed as a 4 weeks on shoot induction medium mixed effects model with plate within set as an additional random variable.
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