Freddi A. Hammerschlag

RAPD analysis of somaclonal variants derived from embryo callus cultures of peach

Peach [Prunus persica (L.) Batsch] regenerants from cv ‘Sunhigh’ embryo no. 156, regenerants obtained from cv ‘Redhaven’ embryo no. 30, and two peach cultivars ‘Sunhigh’ and ‘Redhaven’, were screened for polymorphic RAPD (Random Amplified Polymorphic DNA) markers with up to 60 10-mer primers. Although 35 primers produced results with scoreable bands, only 10 of the primers revealed polymorphism for regenerants of embryo no. 156 and cv ‘Sunhigh’, and 1 revealed a low level of polymorphism for regenerants of embryo no. 30 and cv ‘Redhaven’. This study demonstrates the feasibility of using RAPD markers to identify somaclonal variants of peach and provides evidence for the existence of genetic differences among these variants.

Factors influencing in vitro multiplication and rooting of peach cultivars

Success at propagating peach (Prunus persica (L.) Batsch) scion cultivars in vitro has been limited. This study describes factors influencing in vitro multiplication and rooting of 8 peach scion cultivars and one rootstock, as well as acclimatization and genetic stability of these cultivars. Shoot multiplication was best when 8.8 μM 6-benzylamino purine (BA) was added to the shoot proliferation medium. Maximum rooting occurred when shoots were placed on 1/2-strength Murashige and Skoog (MS) medium, stored in the dark at 4°C for 35 to 40 days and then incubated on rooting medium in the dark at 26°C for 14 days. All cultivars exposed to 1/2-strength MS medium supplemented with 28.5μM of either indoleacetic acid (IAA), indolebutyric acid (IBA) or α-napthaleneacetic acid (NAA) rooted best on NAA medium. A 5-fold reduction in NAA concentration to 5.8 μM, the use of IAA plus phenolic rooting cofactors, and length of time shoots were in vitro prior to rooting each increased the percentage of rooting for most cultivars. No plant loss occurred during acclimatization. Cytogenetic analysis of micropropagated plants indicated that all plants were diploid, 2n=2x=16. Examination of the performance of in vitro propagated plants under field conditions is now in progress.

Evidence for the breakdown of cecropin B by proteinases in the intercellular fluid of peach leaves

Abstract Peach ( Prunus persica (L.) Batsch) leaves were found to tolerate relatively high levels of the bacteriocidal polypeptide cecropin B as determined by infiltration tests. Detached leaves showed no symptoms when infiltrated with 25 μM cecropin B, and only infrequent necrotic symptoms with 50 μM. Incubation of cecropin B with intercellular fluid (ICF) extracted from peach leaves reduced its biotoxicity toward the peach pathogen Pseudomonas syringae pv. syringae . The reduction in toxicity was lessened by adding proteinase inhibitors to ICF — and prevented by boiling ICF prior to incubation with cecropin B. Electrophoretic evidence suggested that ICF constituents caused endopeptidase cleavage of cecropin during the first hour of incubation followed by complete digestion of the cleavage products during the following 8 h. Incubations of various levels of cecropin B and ICF indicated that as much as 90% of the activity could be destroyed in 10 min, but that levels adequate for lethality against P. syringae pv. syringae would remain. We conclude that peach trees transgenic for cecropin B could feasibly produce a wide range of cecropin levels sufficient to control pathogenic bacteria without damaging the leaf tissue.

GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency

Abstract Several factors were investigated for their influence on the transfer of an intron-containing β-glucuronidase (GUS) gene into blueberry (Vaccinium spp.) leaf explants during the early stages of Agrobacterium-mediated gene transfer, including days of cocultivation, strain of Agrobacterium tumefaciens, explant age and genotype. The number of GUS-expressing leaf zones and calli were counted immediately and 2 weeks after cocultivation, respectively, to evaluate the gene transfer process. Agrobacterium tumefaciens strain EHA105 (pEHA105/p35SGUS-int) was significantly more effective for transformation than strain LBA4404 (pAL4404/p35SGUSint). Four days of cocultivation with A. tumefaciens strain EHA105 yielded about 50-fold more GUS-expressing zones than 2 days of cocultivation. Significant differences among cultivars were observed for both GUS-expressing leaf zones and calli. For some cultivars, explant age influenced the number of GUS-expressing leaf zones and calli. In most cases, the number of GUS-expressing calli was highest in those cultivars where GUS expression in the leaves was high.

Changes in β-1,3-glucanase mRNA levels in peach in response to treatment with pathogen culture filtrates, wounding, and other elicitors

Summary The response of three different peach, Prunus persica (L.) Batsch, genotypes to bacterial and fungal culture filtrates (CFs), wounding, and sterile nutrient broth (NB) treatments were studied by evaluating β-1,3-glucanase mRNA levels. Northern blot analysis was conducted using the 3′ end of a peach β-1,3-glucanase gene, PpGns1 , as a probe. Autoradiographs were analyzed using a Stratagene Eagle Eye II gel documentation system. Analysis of the accumulation of mRNAs encoded by β-1,3-glucanase demonstrated that activation trends were different among the three peach genotypes. All genotypes, ‘Evergreen’, ‘Starks Earliglo’, and ‘White Lady’, showed an increase in β-1,3-glucanase mRNA following treatment with CF of the bacterial pathogen Xanthomonas campestris pv. pruni. Two genotypes, ‘Evergreen’ and ‘White Lady’, showed an increase in mRNA levels following treatment with CF of the bacterial pathogen Pseudomonas syringae pv. syringae , and two genotypes, ‘Evergreen’ and ‘Starks Earliglo’, showed an increase in mRNA levels following treatment with CF of the fungal pathogen Monilinia fructicola . Differences in induction patterns were observed between bacterial and fungal culture filtrate treatments. Wounding induced high levels of β-1,3-glucanase mRNA in one genotype, ‘White Lady’; while, treatment with a sterile nutrient broth showed an increase in mRNA in another genotype, ‘Evergreen’. The use of gene-specific primers in RT-PCR indicated that PpGns1 and a second closely-related gene family member, PpGns2 , were transcriptionally active, and were differentially regulated.

Effect of cecropin B on peach pathogens, protoplasts, and cells

Abstract The aim of this study was to determine the effect of cecropin B on peach cells and bacterial pathogens of peach for assessing the feasibility of introducing the cecropin gene into peach for pathogen protection. Cecropin B, a lytic peptide, caused depolarization of the plasmalemma, as determined by the change in fluorescence of cells and protoplasts stained with merocyanine 540, and impaired the integrity of the plasmalemma, as determined by fluorescein diacetate staining. Both protoplasts and cells were more affected by cecropin B when isolated in highly osmotic solutions of the non-penetrating osmoticum mannitol or in solutions containing low calcium. Cells were more sensitive than protoplasts to cecropin B under increased osmoticum, but less sensitive under low calcium. Under conditions of high calcium and low osmotic potential, cells were unaffected by 10 μM cecropin B but were rendered non-viable by about 100 μM. Lethal concentrations of cecropin B on peach pathogens Xanthomonas campestris pv. pruni and Pseudomonas syringae pv. syringae were at 0.1–0.5 μM. These findings suggest that it will be feasible to introduce the gene for cecropin B into peach cells for enhancing the resistance against bacterial pathogens.

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.

Merocyanine 540 as an optical probe to monitor the effects of culture filtrates of Phytophthora cactorum on apple cell membranes

Abstract The response of apple ( Malus domestica ) cells to valinomycin, gramicidin and culture filtrates (CF) from an avirulent and from virulent strains of Phytophthora cactorum was monitored by measuring changes in fluorescence of cells stained with Merocyanine 540, an optical probe for changes in transmembrane electrical potential (PD). Cells of MM. 106 rootstock (susceptible to P. cactorum ) exposed to either valinomycin or to CF from virulent strains of P. cactorum showed a decrease in fluorescence, whereas cells exposed to gramicidin showed an increase in fluorescence. The response of cells to fungal culture medium (FCM) was similar to the response to CF from an avirulent strain of the fungus. These results support the feasibility of using Merocyanine 540 as a probe of PD changes in apple cells and suggest a role for P. cactorum toxin in disease development. This research opens up the possibility of using fluorescence measurements as a screening system for identifying resistance in apple germplasm to P. cactorum and for using the toxin as an in vitro selective agent.

Stability of bacterial leaf spot resistance in peach regenerants under in vitro, greenhouse and field conditions

SummaryPhenotypic stability of bacterial leaf spot resistance in peach (Prunus persica (L.) Batsch) regenerants, either selected at the cellular level for insensitivity to a toxic culture filtrate of Xanthomonas campestris pv. pruni or screened at the whole plant level for resistance to X. campestris pv. pruni, was investigated. A detached-leaf bioassay was used to evaluate the original regenerants again after three years in the greenhouse and also after a two to three year cycle of tissue culture propagation. Peach trees derived through micropropagation from the original regenerants were also evaluated after one to three years growth in the field. Although leaf spot resistance was retained in some regenerants over time in the greenhouse, following in vitro propagation, and under field conditions, resistance was either lost or not expressed in others. Regenerants # 19-1 and #156-6, derived from embryo callus of bacterial spot susceptible ‘Sunhigh’, were significantly more resistant than ‘Sunhigh’. High levels of resistance were exhibited in greenhouse plants and field-grown trees of regenerant #122-1, derived from embryo callus of moderately resistant ‘Redhaven’. This research provides additional evidence that selecting or screening for somaclonal variants with disease resistance is a feasible approach to obtaining peach trees with increased levels of bacterial spot resistance.

Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants

Yields of 106–108 peach mesophyll cells and protoplasts · gfw-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml-1. Purification of protoplasts on a sucrose gradient yielded about 107 protoplasts · gfw-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.