Evaluating resistance of some rootstocks to grape phylloxera with in vitro and excised root testing systems

Summary The life table of a local strain of grape phylloxera was determined to evaluate the resistance of the most commonly used rootstocks: Rul40, R99, and 3309C, and one local variety ‘Helwani’. The study was carried out by applying both in vitro dual culture and small root pieces testing systems. The results showed that there was a great variation in percentage mortality of immature stages, number of eggs laid, adult fertility, oviposition period, and developmental time between all tested rootstocks and the local variety, regardless of the applied testing systems. Based on the population prediction, Helwani would be a suitable host as it would be susceptible to the destructive insects in the field and the rootstocks would be resistant. However, Rul 40 rootstock was more susceptible than R99 and 3309C rootstocks.     

Variation in performance on two grape cultivars within and among populations of grape Phylloxera from wild and cultivated habitats

When an indigenous insect becomes a pest, comparisons of performance of pest and non-pest populations on crop plants and of genetic variation in that performance may provide insight into the evolution of pest populations. To measure such genetic variation, 8–15 clones of the grape phylloxera (Daktulosphaira vitifoliae Fitch) were collected from wild grapevines in each of 3 geographically isolated sites (populations) and from commercial vineyards in northern California. A complete life table was made for clonal replicates from populations collected from wild grapevines on each of two commercial grape cultivars, the susceptibleVitis vinifera (L.) cultivar Cabernet Sauvignon, and the phylloxera-resistant rootstock ‘AxR # 1’. Variation in mean performance on these two hosts was partitioned among clones within collection sites and among sites. Performance measures included an individual analog to the intrinsic rate of increase (r), age at first oviposition, fecundity in the first ten days of reproduction, total fecundity, and longevity. The overall performance of phylloxera from the wild grapevines on the resistant cultivar AxR # 1 was greater than or equal to that on the susceptible cultivar Cabernet Sauvignon. There was significant variation among clones within populations from wild grapes in the rate of increase on ‘AxR # 1’ and marginally significant clonal variation in some of the component paramters. There was no significant variation among clones within populations on ‘Cabernet Sauvignon’ and no significant differences between populations on either crop in any trait. In a second experiment we compared the relative performance of 15–17 clones from wild grapevines and from commercial vineyards when reared on ‘Cabernet Sauvignon’ and ‘AxR # 1’. Phylloxera from commercial vineyards had much higher overall performance on ‘Cabernet Sauvignon’ than did phylloxera from the wild grapevines. Phylloxera from the commercial vineyard also had higher performance on ‘Cabernet Sauvignon’ than on ‘AxR′ 1’ but the performance of the phylloxera from wild and commercial grapes did not differ on ‘AxR # 1’. Our results show that there is genetic variation in traits related to performance on a resistant rootstock within these indigenous non-pest populations of phylloxera, but not among them. The pattern of performance of pest and non-pest populations on two commercial cultivars suggests that current levels of phylloxera performance on crop cultivars are the result of adaptation to those cultivars which has occurred while phylloxera has been associated with viticulture. Implications of these results for understanding the recent adaptation of phylloxera to ‘AxR # 1’ in California are also discussed.     

Construction of a dense genetic linkage map for apple rootstocks using SSRs developed from Malus ESTs and Pyrus genomic sequences

Marker-assisted selection (MAS) offers quick and reliable prediction of the phenotypes of seedlings in large populations and thus opens new approaches for selection to breeders of apple (Malus x domestica Borkh.). The development of framework maps enables the discovery of genetic markers linked to desired traits. Although genetic maps have been reported for apple scion cultivars, none has previously been constructed for apple rootstocks. We report the construction of framework genetic maps in a cross between ‘M.9’ (‘Malling 9’) and ‘R.5’ (‘Robusta 5’) apple rootstocks. The maps comprise 224 simple sequence repeat (SSR) markers, 18 sequence-characterised amplified regions, 14 single nucleotide polymorphisms and 42 random amplified polymorphic DNAs. A new set of 47 polymorphic SSRs was developed from apple EST sequences and used for construction of this rootstock map. All 17 linkage groups have been identified and aligned to existing apple genetic maps. The maps span 1,175.7 cM (‘M.9’) and 1,086.7 cM (‘R.5’). To improve the efficiency of mapping markers to this framework map, we developed a bin mapping set. Applications of these new genetic maps include the elucidation of the genetic basis of the dwarfing effect of the apple rootstock ‘M.9’ and the analysis of disease and insect resistance traits such as fire blight (Erwinia amylovora), apple scab (Venturia inaequalis) and woolly apple aphid (Eriosoma lanigerum). Markers for traits mapped in this population will be of direct use to apple breeders for MAS and for identification of causative genes by map-based cloning.     

Light quality affectsin Vitro growth of grape ‘Teleki 5BB’

We investigated the effects of various qualities of light-emitting diode (LED) light sources on the growth and carbohydrate accumulation of grape rootstock ‘Teleki 5BB’ culturedin vitro. Shoot fresh and dry weights and net photosynthetic rates were increased when plants were exposed to fluorescent lighting (control), red light, or a mixture of blue plus red, but were unaffected by blueonly radiation. Shoot elongation was significantly stimulated by red light whereas the combination of blue and red light was associated with the shortest shoots. However, the number of nodes did not differ among these treatments. Under monochromic blue or red light, sugar content and starch accumulation increased under the mixed-radiation treatment.     

Regeneration of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch) cultivars and <Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">Prunus dulcis</Emphasis> rootstocks via organogenesis

Somatic peach plants were regenerated from callus derived from the base of stem explants of the scion cultivars ‘UFO-3’, ‘Maruja’, ‘Flariba’ and ‘Alice Bigi’, and the peach × almond rootstocks ‘Garnem’ and ‘GF677’. A protocol for organogenic plant regeneration was developed using three culture media containing different concentrations of 6-benzyladenine (BA) and indolebutyric acid to produce organogenic calli. Shoots were obtained from sliced calli after their transfer to a differentiation culture medium containing 2 mg l⁻¹ BA and 1 mg l⁻¹ α-naphthalene acetic acid. Using this procedure, up to 29 regenerated plants per callus were obtained. The highest regeneration rate was obtained with the peach × almond rootstocks. This work provides an effective protocol that could be utilized for peach transformation research.     

Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of Fiesta scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml^–1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of Fiesta scion grafted onto the rootstocks. Cytokinin content (ng shoot^–1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Early detection of grapevine leafroll virus in <Emphasis Type="Italic">Vitis vinifera</Emphasis> using <Emphasis Type="Italic">in vitro</Emphasis> micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l^–1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.     

Development and characterization of a codominant marker linked to root-knot nematode resistance, and its application to peach rootstock breeding

 The presence of a codominant AFLP marker, EAA/MCAT10, correlates with the primary source of resistance to root-knot nematodes (Meloidogyne incognita and M. javanica) in rootstock cultivars of peach [Prunus persica (L.) Batsch]. Two allelic DNA fragments of this AFLP marker were cloned, sequenced and converted to sequence tagged sites (STS). Four nucleotide differences (i.e. one addition and three substitutions) were observed between the two clones. Furthermore, there was a diagnostic Sau3 AI cleavage site (GATC) in the large fragment that was absent from the small fragment (GTTC at this site). The applicability of this STS marker system to peach germplasm improvement was evaluated: genomic DNAs of cross parents (i.e. ‘Lovell’ and ‘Nemared’), four F₁ hybrids (K62-67, K62-68, P101-40 and P101-41) and two F₂ populations (from K62-68 and P101-41), as well as DNA from a test panel of 18 rootstock cultivars or selections, were PCR-amplified with the Mij3F/Mij1R primer pair and then digested with Sau3 AI. The banding patterns showed that the EAA/MCAT10 STS markers can clearly distinguish the three genotypes – homozygous resistant, heterozygous resistant and homozygous susceptible – in the ‘Lovell’בNemared’ cross. In addition, results from the rootstock survey were consistent with each rootstock’s phenotypic response to nematode infection, except for ‘Okinawa’, ‘Flordaguard’ and ‘Yunnan’ where root-knot resistance may have arisen independently. Therefore, the EAA/MCAT10 STS markers will be a useful tool to initiate marker assisted selection studies in peach rootstock breeding for root-knot nematode resistance. Received: 4 January 1999 / Accepted: 4 January 1999     

Effect of γ-radiation on development,yield and quality of microtubers <Emphasis Type="Italic">in vitro</Emphasis> in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Explants obtained from in vitro-propagated plantlets of two potato cultivars, Shepody and Atlantic, were treated with five doses of γ-radiation (0, 2, 4, 6 and 8 Gy) to investigate the stimulating effects of low irradiation on the production and quality of microtubers in vitro. Microtubers of both cultivars treated with γ-radiation initiated 5 d earlier than in the non-irradiated control. The whole period of microtuberization was prolonged by 10 – 15 d with 4, 6 and 8 Gy irradiation treatment for cv. Atlantic. Irradiation of the plantlets (4 Gy) led to a significant increase not only in the microtuber number (116.7 and 34.5 % over the control) but also in the fresh mass (77.6 and 23.2 % in Shepody and Atlantic, respectively). Low dose irradiation (2 – 4 Gy) increased the starch content of microtubers. High doses (6 – 8 Gy) enhanced ascorbic acid and reducing sugar contents. 4 – 6 Gy doses also effectively increased the protein contents of microtubers.     

Alterations in the photosynthetic pigments and antioxidant machineries of red pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) seedlings from gamma-irradiated seeds

To characterize the stimulatory effects of low-dose gamma radiation on early plant growth, we investigated alterations in the photosynthesis and antioxidant capacity of red pepper (Capsicum annuum L.) seedlings produced from gamma-irradiated seeds. For two cultivars (Yeomyung and Joheung), three irradiation groups (2, 4, and 8 Gy, but not 16 Gy) showed enhanced development, although Fv/Fm, the maximum photochemical efficiency of Photosystem II (PSII), did not differ significantly among any of the four groups. In contrast, values for 1/Fo — 1/Fm, i.e., a measure of functional PSII content, decreased in the irradiated groups of ‘Yeomyung’ but increased in those of ‘Joheung’. Pigment analyses and enzyme activity assays revealed that irradiation altered the compositions of photosynthetic pigments (chlorophylls and carotenoids) as well as the activities of antioxidant enzymes (superoxide dismutase and glutathione reductase). However, these shifts were not directly related to the increase in early growth, although they were cultivar-and developmental stage-dependent In addition, the effects of irradiation on the enzymatic activities measured here were at opposition between the two cultivars.     

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

 The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid. Received: 6 January 1998 / Accepted: 1 April 1998     

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (<Emphasis Type="Italic">Vitis</Emphasis> sp.) genotypes

Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.     

A consensus map of rye integrating mapping data from five mapping populations

A consensus map of rye (Secale cereale L.) was constructed using JoinMap 2.0 based on mapping data from five different mapping populations, including ‘UC90’ × ‘E-line’, ‘P87’ × ‘P105’, ‘I_0.1-line’ × ‘I_0.1-line’, ‘E-line’ × ‘R-line’, and ‘Ds2’ × ‘RxL10’. The integration of the five mapping populations resulted in a 779-cM map containing 501 markers with the number of markers per chromosome ranging from 57 on 1R to 86 on 4R. The linkage sizes ranged from 71.5 cM on 2R to 148.7 cM on 4R. A comparison of the individual maps to the consensus map revealed that the linear locus order was generally in good agreement between the various populations, but the 4R orientations were not consistent among the five individual maps. The 4R short arm and long arm assignments were switched between the two population maps involving the ‘E-line’ parent and the other three individual maps. Map comparisons also indicated that marker order variations exist among the five individual maps. However, the chromosome 5R showed very little marker order variation among the five maps. The consensus map not only integrated the linkage data from different maps, but also greatly increased the map resolution, thus, facilitating molecular breeding activities involving rye and triticale.     

Effects of exogenous B supply on growth, B accumulation and distribution of two navel orange cultivars

To investigate the effects of boron (B) on growth, B concentration and distribution of two navel orange cultivars, ‘Newhall’ (Citrus sinensis Osbeck) and ‘Skagg’s Bonanza’ (Citrus sinensis Osbeck) grafted on the rootstock trifoliate orange [Poncirus trifoliata (L.) Raf.], B at five levels was exogenously supplied to 1-year-old grafted plants of both cultivars under greenhouse conditions. Plants were grown in sand:perlite (1:1, v/v) medium and were irrigated every 2 days with half-strength Hoagland’s No. 2 nutrient solutions containing different B, 0.01, 0.05, 0.10, 0.25 and 2.50 mg l⁻¹ (0.25 and 2.50 mg l⁻¹ were considered as control and excess B treatment, respectively, and the other three B levels were considered as low B treatments). After treatments for 183 days, leaves (from basal, middle, upper parts of the shoots), stem of scion, stem of rootstock and root were separately sampled. Our results showed that plant growth (plant height, root volume and dry weights of various parts) was inhibited in response to low or excess B supplies in both cultivars. It was found that B concentrations in the upper leaves of both cultivars were substantially higher than those in the basal leaves when low concentrations (≤0.05 mg l⁻¹) of exogenous B were applied, suggesting that B was preferentially translocated to the upper-younger leaves to support their growth. Analysis of B distribution in different parts indicated that translocation of B from the root to the scion’s shoots (stems and leaves of scion) may be restricted upon exposure to low B conditions. When B was inadequately supplied, growth of ‘Skagg’s Bonanza’ was better than ‘Newhall’, implying that the former cultivar was more tolerant to low B status, which may be due to the higher efficiency of B translocation from the root to the scion’s shoots. However, when the plants were treated with excess B (2.50 mg l⁻¹), both cultivars showed a similar degree of B toxicity. The probability of scion–rootstock interactions in relation to the differential responses of growth and different efficiency of B translocation involved in the two orange cultivars following the long-term low B stress were discussed.     

In vitro propagation of Gladiolus hybridus Hort.: Synergistic effect of heat shock and sucrose on morphogenesis

Three cultivars (cvs.) of Gladiolus hybridus Hort., namely ‘Her Majesty’, ‘Aldebaran’ and ‘Bright Eye’ were successfully micropropagated. The cultures were established using intact cormels or segments of cormels and inflorescence axes on Murashige and Skoog (1962; MS) medium. The response depended on media supplements; both callus formation or direct induction of shoot buds was observed. Shoot differentiation from callus could be obtained on MS medium containing 1.0 μM BA (6–benzyladenine) and 10.0 μM NAA (α-naphthalene acetic acid) in all three cultivars. The same could be achieved by giving a heat shock (HS; 50 °C, 1h) to callus cultures (in case of ‘Her Majesty’ and ‘Aldebaran’ only) maintained on the basal medium. In these two cultivars, high sucrose concentration (0.232, 0.290 or 0.348 M) also favoured growth and proliferation of shoot cultures on a plant growth regulator-free medium at 20 °C in comparison to the cultures kept at 25 °C. On the other hand, shoot cultures maintained on the basal medium at 25 °C containing normal (0.058 M, i.e., 2.0%, w/v) sucrose concentration responded similar to those maintained at 20 °C on a high sucrose medium; reduced response was observed on normal sucrose containing medium at 20 °C. Heat shock enhanced shoot proliferation in the cultures maintained on basal medium, but induced prolific rooting in shoot cultures, within 5 days of HS, on high sucrose (optimum 0.232 M) medium. While the number of roots increased at higher sucrose concentrations in the medium in case of cvs. ‘Her Majesty’ and ‘Aldebaran’, the same was found to be independent of sucrose concentration in cv. ‘Bright Eye’. Generally the rooted plants produced on high sucrose (0.232 M) medium in comparison to medium with normal sucrose concentration showed better survival (ca. 90% as against 40%) in the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Scion and Rootstock Effects on ABA-mediated Plant Growth Regulation and Salt Tolerance of Acclimated and Unacclimated Potato Genotypes

Tolerance of salt stress in potato (Solanum tuberosum L.) increased when the plants were pre-exposed to low concentrations of salt (salt acclimation). This acclimation was accompanied by increased levels of abscisic acid (ABA) in the shoot. To further study the role of roots and shoots in this acclimation process, reciprocal grafts were made between a salt-tolerant (9506) and salt-sensitive ABA(−) mutant and its ABA(+) normal sibling potato genotype. The grafted plants were acclimated with 75 or 100 mM NaCl for 3 weeks and then exposed to 150–180 mM NaCl, depending on the salt tolerance of the rootstock. After 2 weeks of exposure to the salt stress, the acclimated and unacclimated plants were compared for physiologic and morphologic parameters. The response to the salt stress was strongly influenced by the rootstock. The salt-tolerant 9506 rootstock increased the salt tolerance of scions of both the ABA-deficient mutant and its ABA(+) sibling. This salt tolerance induced by the rootstock was primarily modulated by salt acclimation and manifested in the scion via increased plant water content, stem diameter, dry matter accumulation, stomatal conductivity, and osmotic potential, and is associated with a reduction in leaf necrosis. There was also a pronounced scion effect on the rootstock. Using 9506 as a scion significantly increased root fresh and dry weights, stem diameter, and root water content of ABA(−) mutant rootstocks. Specific evidence was found of the role of exogenous ABA in the enhancement of water status in grafted plants under salt stress beyond that of grafting alone. This was verified by more positive stomatal conductivity and upward water flow in ABA-treated grafted and nongrafted plants and the absence of upward water flow in nontreated grafted plants through NMR imaging. Grafting using either salt-tolerant scions or rootstocks with inherently high ABA levels may positively modify subsequent responses of the plant under salt stress.