Plant Cell Tiss Organ Cult (2012) 108:229–236 DOI 10.1007/s11240-011-0034-4

ORIGINAL PAPER

Adventitious shoot regeneration of (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 var. sinensis Á Pyrus exception of one genotype of P. elaeagrifolia Pall., and communis Á ‘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 , and organics (Chevreau and Leblay in Acta Hortic 336: 263–268, 1993). The analysis of variance Tree 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 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 ‘’ 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 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 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. Residuals were computed using SAS Preliminary analysis of 10 genotypes derived PROC MIXED (Littell et al. 1996) and analyzed for nor- from both culture sources mality by the Shapiro–Wilk test and normal probability plots using SAS PROC UNIVARIATE (SAS Institute The Shapiro–Wilk W statistic for proportion of regenerat- 1990). To assess equality of variances, the relationship ing explants (W = 0.96, Pr \ W = \0.0001) indicated between the absolute value of the residual and the predicted that the distribution was non-normal, as did the box plot values was assessed by calculating Spearman’s correlation and normal probability plot. Spearman’s correlation coef- coefficient and examining a plot for the two variables. ficient (r = 0.58, Pr [ r = 0.0001) indicated a slightly After performing the appropriate transformation, the data negative relationship between mean and variance. Arcsin were analyzed to determine whether normality and heter- square root transformation slightly improved normality and ogeneity of variances were improved and the assumptions homogeneity of variances (W = 0.98, Pr \ W = 0.003; for parametric analysis of variance were met. Least squares r = 0.38, Pr [ r = 0.0001). Therefore, arcsin square root means were computed for the main effects of genotype and transformed data was used for the analysis of variance. culture origin for all analyses except when culture origins were combined, in which case marginal means were computed. The association between percentage of explants regen- erating and number of shoots per regenerating explant was analyzed by computing Pearson’s correlation coefficient using SAS PROC CORR (SAS Institute 1990).

Results

During the initial 4 week incubation period, all explants expanded considerably and white nodular callus developed. While some adventitious shoot regeneration was observed at both 4 (Fig. 1) and 8 weeks, data for analysis was recorded at 12 weeks. Most shoot development appeared to arise from callus (Fig. 2) rather than by direct organo- genesis (Fig. 3). The adventitious shoots varied somewhat in size, depending on time of development. Uniform hy- perhydricity occurred in only 11 of the genotype by origin combinations, and was associated with poor regeneration Fig. 2 Adventitious shoot regeneration of ‘Bartlett’ of in vitro (\30%). culture origin regenerated from callus 123 232 Plant Cell Tiss Organ Cult (2012) 108:229–236

differences between culture origin for mean number of regenerating explants, these data on all genotypes were also analyzed after deleting culture origin from the model. In both these analyses, arcsin square root transformation was found to be necessary and appropriate. Data for number of adventitious shoots per regenerating explant exhibited deviations from normal distributions and inequality of variances for each culture origin separately and for both origins combined. Square root transformed data were therefore analyzed. The analyses indicated that genotype differences were significant within culture origin as well as for the combined data. The ranges of mean regeneration frequency were similar for both in vitro (0–87.7%) and scionwood-derived cultures (0–70.7%). The range of number of adventitious shoots was relatively narrow, with the minimum of 1.0 for Fig. 3 Adventitious shoot regeneration of ‘Magness’ of budwood origin regenerated directly from cut surface of leaf explant seven cultivars to 2.2 for ‘Conference’. The ranges for in vitro and scionwood-derived cultures were similar (1.0–2.2 and 1.0–1.9, respectively). The correlation between The analysis of variance indicated that differences the two traits was also highly significant (r = 0.91, among cultivars were highly significant (Pr [ F = Pr [ r \0.0001), that is, the cultivars which exhibited the \0.001), while the main effect of culture origin was non- highest regeneration frequency also tended to produce the significant (Pr [ F = 0.30). However, there was a signifi- largest number of adventitious shoots per explant. Scion- cant interaction between cultivar and culture origin wood-derived cultures of ‘Beurre d’Anjou’ did not differ (Pr [ F = \0.01). Investigation of the interaction effect significantly from its putative mutant, ‘Naumes d’Anjou’, using the SLICE option and plots of the interaction means indicating that any genetic and phenotypic differences in (graphs not shown) indicated that percentage regeneration other traits did not affect adventitious regeneration. Vari- from leaf explants ‘Abate Fetel’ from shoot proliferation ability among sets resulted in relatively large standard errors cultures derived from new budwood was significantly of the least square means and much overlap in separation greater than that from older in vitro NCGR cultures classes. (Pr [ F = 0.001; Table 1), while leaf explants from ‘Jesinji Vodenac’ cultures derived from the long-term NCGR cultures regenerated significantly more adventitious Discussion and conclusions shoots than explants derived cultures started from new budwood (Pr [ F = 0.01; Table 1). In addition, explants The range of percentage regeneration was quite large, of ‘Clara Frijs’ from the long-term in vitro shoot cultures varying from 0% for ‘Ya Li’, ‘Old Home’ and ‘Fondante regenerated more adventitious shoots than explants from de Charneuse’ to nearly 88% for ‘Conference’. This range new cultures with a significance of Pr [ F = 0.1. is similar to that indicated by a survey of the literature on Data on the number of adventitious shoots per regen- pear regeneration, albeit with a variety of basal shoot erating explant were non-normal (W = 0.90, Pr \ W = induction media and other techniques, which ranged from 6 0.0001), approximating a Poisson distribution, and Spear- to 36% for ‘Beurre Bosc’ (Abdollahi et al. 2006) and 9% man’s correlation (r = 0.36, Pr [ r = 0.001) and the for ‘Chojuro’ and ‘Hosui’ (Lane et al. 1998) to 100% for residual versus predicted value plots indicated a significant ‘Conference’ (Chevreau et al. 1997). Seven genotypes in relationship between residuals and the mean. After square our study had been the subject of previous regeneration root transformation, the analysis of variance indicated no experiments. Our results were similar to the values previ- significant differences due to genotype, culture origin, or ously reported for ‘Conference’ (Abdollahi et al. 2006; their interaction (Table 2). Chevreau et al. 1997; Leblay et al. 1991) and ‘Beurre´ Bosc’ (Abdollahi et al. 2006; Bell et al. 1999), slightly lower Analysis of all genotypes for ‘Abate Fetel’ (Abdollahi et al. 2006) and ‘Bartlett’ (Abdollahi et al. 2006; Chevreau et al. 1997; Leblay et al. Data for all genotypes of each culture origin, budwood and 1991), but considerably lower for ‘Beurre´ d’Anjou’ (Sun old in vitro cultures, were initially analyzed separately. et al. 2003), ‘Doyenne´ du Comice’ (Chevreau et al. 1997; Given that only two genotypes exhibited significant Leblay et al. 1991) and ‘Old Home’ (Chevreau et al. 1997; 123 Plant Cell Tiss Organ Cult (2012) 108:229–236 233

Table 1 Mean percentage of regenerating explants derived from new in vitro cultures started from budwood and/or cold-stored in vitro cultures of 24 Pyrus genotypes Genotype Budwood In vitro cultures Combined No. sets Mean ± SEZ No. sets Mean ± SE No. sets LS Mean ± SE

Conference 8 7.7 ± 7.1 a 8 87.7 ± 7.1 a Magness 8 70.7 ± 7.2 a 8 70.7 ± 7.2 ab Dr. Jules Guyot 8 69.8 ± 7.4 ab 8 69.8 ± 7.4 abc Packham’s Triumph 8 53.0 ± 7.2 ab 8 53.0 ± 7.2 a–d Bartlett 8 44.3 ± 7.4 a–d 3 50.0 ± 13.1 abc 11 47.2 ± 7.8 b–e Clara Frijs 8 29.9 ± 7.4 abc 8 46.5 ± 7.4 cY 16 38.2 ± 5.4 b–f Abate Fetel 7 54.4 ± 7.4 abY 8 14.4 ± 8.1 c 15 34.4 ± 5.7 b–g Beurre d’Anjou 8 30.4 ± 7.5 b–e 8 30.4 ± 7.5 c–h P. elaeagrifolia 8 28.8 ± 7.3 b–e 8 28.8 ± 7.3 d–h Clapp’s Favorite 5 27.2 ± 9.6 b–e 5 20.0 ± 10.2 bc 10 23.6 ± 7.3 d–h Naumes d’Anjou 8 22.4 ± 7.6 b–e 8 23.2 ± 7.8 c 16 22.8 ± 5.7 d–h Jesinji Vodenac 8 6.1 ± 7.4 e 8 34.6 ± 7.6 bcY 16 20.4 ± 5.5 d–h Beurre Bosc 8 23.8 ± 7.5 b–e 8 7.7 ± 7.6 c 16 15.8 ± 5.5 d–h OH 9 F 97 8 9.8 ± 7.4 cde 8 9.8 ± 7.4 e–h Beurre Hardy 8 8.9 ± 7.4 de 8 8.9 ± 7.4 e–h Doyenne du Comice 8 8.6 ± 7.6 de 8 8.9 ± 7.4 e–h Blakeney Red 7 11.6 ± 7.8 cde 8 4.1 ± 8.4 c 15 7.9 ± 6.0 e–h Rocha 3 6.7 ± 13.1 c 3 16.7 ± 13.1 d–h Admiral Gervais 8 5.5 ± 7.4 e 8 5.5 ± 7.4 e–h Akca 5 3.2 ± 9.1 de 6 4.4 ± 9.4 c 11 3.8 ± 6.8 f–h Mustafabey 8 6.4 ± 7.8 de 8 0.0 ± 0.0 c 8 3.3 ± 5.6 gh Ya Li 7 0.0 ± 0.0 e 7 0.0 ± 0.0 f–h Old Home 6 0.0 ± 0.0 c 6 0.0 ± 0.0 f–h Fondante de Charneuse 8 0.0 ± 0.0 e 8 0.0 ± 0.0 h Z Means within columns followed by the same letter are not statistically different; means followed by different letters are statistically different at the P = 95% level Y Means by culture origin in bold font are statistically different from the mean of the other origin at the P = 95% level. The mean ‘Clara Frijs’ in italics (from new in vitro culture) was different from the mean of the budwood-derived cultures at the P = 88% level. All other means for each genotype 9 culture origin combination were not statistically different

Sun et al. 2006). Our result for ‘Old Home’ is considered Martinelli et al. 2009; Quoirin and Lepoivre 1977), B5 or anomalous, since in a previous experiment (data not pre- close derivatives (Gamborg et al. 1968; Lane et al. 1998; sented), high frequency regeneration was observed. Dif- Yancheva et al. 2006), or Linsmaier and Skoog (Linsmaier ferences between our results and those previously and Skoog 1965; Predieri et al. 1989). published could be due to differences in the regeneration In many cases, lower levels of TDZ have been used media. The plant growth regulator types and concentrations when the objective was simply regeneration. However, our are the most likely cause, since the CL basal medium has purpose was to use a protocol and media which had been consistently resulted in a high level of regeneration of successfully employed in transformation (Bell et al. 1999; several cultivars (Chevreau et al. 1997; Malnoy et al. 2003; Phat Dang, unpublished data). A higher concentration of Zhu and Welander 2000). Reported regeneration frequen- TDZ for transformation (Mourgues et al. 1996) than had cies are comparable to those reported for protocols using previously found successful for adventitious shoot regen- MS (Abdollahi et al. 2006; Nacheva et al. 2009; Shibli eration (Leblay et al. 1991) has sometimes been used. The et al. 2000), Nitsch and Nitsch (Abu-Qaoud et al. 1991; concentration of TDZ we employed (10 lM) did not Hennayake et al. 2003; Matsuda et al. 2005; Nitsch and appear to be detrimental, as severe hyperhydricity was not Nitsch 1969; Sun and Sun 1999; Sun et al. 2011; Tang et al. observed in the majority of genotypes, in contrast to some 2008), Quoirin and Lepovire (Caboni et al. 1999; reports (Caboni et al. 1999; Kadota and Niima 2003). That

123 234 Plant Cell Tiss Organ Cult (2012) 108:229–236

Table 2 Mean number of adventitious shoots per regenerating explant either from new in vitro cultures started from budwood and/or cold-stored in vitro cultures for 24 Pyrus genotypes Genotype Budwood In vitro cultures Combined No. sets Mean ± SEZ No. sets Mean ± SE No. sets LS Mean ± SE

Conference 8 2.2 ± 0.1 a 8 2.2 ± 0.1 a Jules Guyot 8 1.8 ± 0.1 ab 8 1.8 ± 0.1 ab Magness 8 1.8 ± 0.1 ab 8 1.8 ± 0.1 ab Bartlett 8 1.9 ± 0.2 a 3 1.5 ± 0.3 ab 11 1.7 ± 0.1 ab Beurre d’Anjou 8 1.6 ± 0.2 ab 8 1.6 ± 0.2 ab Clara Frijs 8 1.5 ± 0.1 ab 8 1.4 ± 0.2 ab 16 1.5 ± 0.1 ab Beurre Bosc 8 1.4 ± 0.2 ab 8 1.5 ± 0.3 ab 16 1.5 ± 0.2 ab Packham’s Triumph 8 1.4 ± 0.1 ab 8 1.4 ± 0.1 b Clapp’s Favorite 5 1.3 ± 0.3 ab 5 1.4 ± 0.2 ab 10 1.4 ± 0.2 b Naumes d’Anjou 8 1.4 ± 0.2 ab 8 1.3 ± 0.2 ab 16 1.4 ± 0.2 b Abate´ Fetel 7 1.6 ± 0.1 ab 8 1.1 ± 0.3 ab 15 1.4 ± 0.1 b Jesinji Vodenac 8 1.1 ± 0.3 b 8 1.4 ± 0.2 ab 16 1.3 ± 0.2 b Admiral Gervais 8 1.2 ± 0.3 ab 8 1.2 ± 0.3 ab P. elaeagrifolia 8 1.2 ± 0.2 ab 8 1.2 ± 0.2 b Mustafabey 8 1.2 ± 0.3 ab 8 1.2 ± 0.3 ab Blakeney Red 7 1.2 ± 0.3 ab 8 1.0 ± 0.6 b 15 1.1 ± 0.3 b OH 9 F 97 8 1.1 ± 0.3 b 8 1.1 ± 0.3 b Beurre Hardy 8 1.1 ± 0.3 b 8 1.1 ± 0.3 b Doyenne du Comice 8 1.0 ± 0.3 b 8 1.0 ± 0.3 b Rocha 3 1.0 ± 0.6 b 3 1.0 ± 0.6 b Akca 5 1.0 ± 0.6 b 6 1.0 ± 0.5 b 11 1.0 ± 0.4 b Z Means within columns followed by the same letter are not statistically different; means follwed by different letters are statistically different at the P= 95% level concentration is similar to the 9.1 lM that used by Mour- pear cultivars (Bell 2003). IBA has also been used suc- gues et al. (1996) to transform ‘Conference’ and ‘Doyenne cessfully for regeneration of ‘Niitaka’ (Lee et al. 2002). For du Comice’. The concentration is lower than the 22.7 lM the OH 9 F 333 pear rootstock, Nacheva et al. (2009) used used in transformation experiments with ‘Passe Crassane’ 2.5 lM IBA in combination with 20 lM indole acetic acid (Mourgues et al. 1996), with ‘Silver Bell’ (Matsuda et al. (IAA). However, in a direct comparison of 0.54 lM NAA 2005), with ‘La ’ (Gao et al. (2007), with an addi- versus IBA, albeit with Murashige and Skoog (MS) med- tional eight P. communis and three P. pyrifolia cultivars ium (Murashige and Skoog 1962) and 26.6 lM BA as the (Hennayake et al. (2003), and with the 13.6 lM used for cytokinin, Tang et al. (2008) observed a reduction in the pear rootstock GP217 (Lebedev and Dolgov 2000) and regeneration frequency from 63 to 24%. Such marked ‘Zaosu Li’ (Tang et al. 2008). However, our concentration differences were not observed in the other three cultivars of was higher than the 5 lM that has been reported used for Asian species in the experiment. In contrast, 98% regen- ‘Passe Crassane’, Bartlett’, ‘Doyenne´ du Comice’ and eration of leaf explants of ‘Topred’ apple was achieved, ‘Conference’ to achieve regeneration frequencies of with up to 10 shoots per explant, using 4.9 lM IBA, 43–100% (Chevreau et al. 1997; Leblay et al. 1991). although it was not compared with NAA (Yancheva et al. Another difference was that the auxin most frequently 2003). used in the shoot induction media by other authors has been Another difference with other published protocols was naphthalene acetic acid (NAA) (Chevreau and Bell 2005; our use of an initial 4 day incubation ono SIM containing Chevreau and Skirvin 1992). However, we have previously 12.5 lM IBA. This use of an initially high concentration of used IBA successfully (Bell et al. 1999; Phat Dang, IBA may have resulted in a reduction of adventitious shoot unpublished data) in transformation experiments, and have regeneration by influencing acquisition of organogenic demonstrated that it is equally effective in inducing competence or determination for shoot development adventitious regeneration of ‘Bartlett’ and ‘Beurre´ Bosc’ (Christianson and Warnick 1983, 1984, 1985; Yancheva

123 Plant Cell Tiss Organ Cult (2012) 108:229–236 235 et al. 2003). Further experimentation would be necessary to Gamborg OL, Mioller RA, Ojima K (1968) Nutrient requirements of assess this hypothesis. Improving the regeneration of the suspension cultures of soybean root cells. Exp Cell Res 50:150–158 poorly regenerating genotypes will require the adoption of Gao M, Matsuda N, Murayama H, Toyomasu T, Mitsuhashi W, more efficient protocols or the development of genotype- Dandekar AM, Tao R, Nishimura K (2007) Gene expression and specific protocols with examination of factors such as ethylene production in transgenic pear (Pyrus communis cv. ‘La concentrations and timing of growth regulators. However, France’) with sense or antisense cDNA encoding ACC oxidase. 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