Application of a high-speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection

Breeding of apple (Malus × domestica) remains a slow process because of protracted generation cycles. Shortening the juvenile phase to achieve the introgression of traits from wild species into prebreeding material within a reasonable time frame is a great challenge. In this study, we evaluated early flowering transgenic apple lines overexpressing the BpMADS4 gene of silver birch with regard to tree morphology in glasshouse conditions. Based on the results obtained, line T1190 was selected for further analysis and application to fast breeding. The DNA sequences flanking the T-DNA were isolated and the T-DNA integration site was mapped on linkage group 4. The inheritance and correctness of the T-DNA integration were confirmed after meiosis. A crossbred breeding programme was initiated by crossing T1190 with the fire blight-resistant wild species Malus fusca. Transgenic early flowering F(1) seedlings were selected and backcrossed with 'Regia' and 98/6-10 in order to introgress the apple scab Rvi2, Rvi4 and powdery mildew Pl-1, Pl-2 resistance genes and the fire blight resistance quantitative trait locus FB-F7 present in 'Regia'. Three transgenic BC'1 seedlings pyramiding Rvi2, Rvi4 and FB-F7, as well as three other BC'1 seedlings combining Pl-1 and Pl-2, were identified. Thus, the first transgenic early flowering-based apple breeding programme combined with marker-assisted selection was established.     

Calcium oxalate crystals inOpuntia ficusindica (L.) mill.: Development and relation to mucilage cells — A stereological analysis

Summary In the succulent tissue ofOpuntia ficusindica development of calcium oxalate crystals was followed from the shoot apex to its base, using stereological analysis. The stereological parameters relating to the crystal cells were compared with those of mucilage cells, of the same tissue. A fairly monotonic relation between the stereological parameters of the two populations was noted. The possible physiological relation between the two cell populations is discussed.     

Molecular characterization of newS-RNases (‘S31’ and ‘S32’) in apple (Malus ×domestica Borkh.)’) in apple (Malus ×domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S₂S₃S₄), ‘Winesap’ (S₁S₂₈), ‘York Imperial’ (S₂S₃₁), ‘Stark Earliblaze’ (S₁S₂₈), and ‘Burgundy’ (S₂₀S₃₂), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S₃₁ and S₃₂, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S₃₁ showed high similarity to S₂₀ (94%), S₃₂ exhibited 58% (to S₂₄) to 76% (to S₂₅) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S₃₁- and S₃₂-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.     

The effects of external magnesium concentration on the growth and magnesium inflow rates of micropropagated cherry rootstocks 'F.12/1' (Prunus avium L.) and 'Colt' (Prunus avium L. × Prunus pseudocerasus L.)

The cherry rootstock 'F.12/1' is more susceptible to Mg deficiency than the cherry rootstock 'Colt'. The effects of different external concentrations (3000, 500, 50 and 10 µM) of Mg on the growth of micropropagated plants of 'F.12/1' and 'Colt' were investigated in a flowing solution culture system. The relative growth rates (RGR) and total dry weight of both cultivars decreased similarly with the reduction in the external concentrations of Mg. The decreases were caused by a lower net assimilation rate (unit leaf rate). 'F.12/1' had a greater RGR than 'Colt' at all external concentrations of Mg and this is ascribed to its greater leaf weight ratio (leafiness). 'Colt' partitions more dry matter to roots than 'F.12/1', resulting in a smaller shoot: root dry weight ratio. 'F.12/1' required a greater inflow rate of Mg than 'Colt' to maintain its maximum growth rate. When the external concentration of Mg fell below 500 µM the concentration of Mg in the leaves of 'F.12/1' fell well below the critical concentration whereas for 'Colt' this did not occur until the concentration fell below 50 µM.     

The effects of external potassium and magnesium concentrations on the magnesium and potassium inflow rates and growth of micropropagated cherry rootstocks, “F.12/1” (Prunus avium L.) and “Colt” (Prunus avium L.) × Prunus pseudocerasus L.)

The effects (and interaction) of two solution concentrations of Mg (50, 500, μM) and two of K (250, 4250 μM) on the growth of micropropagated plants of “F. 12/1” and “Colt” were investigated using a flowing solution culture system. Magnesium inflow and growth of “Colt” and “F. 12/1” were inhibited to a similar extent by an increased concentration of K in the nutrient solution. However, the consequences of this inhibition were different. Reduced inflow of Mg in “F. 12/1” caused Mg deficiency symptoms at high and low concentrations of K, whereas this only occurred with a combination of high K concentration and low Mg concentration in “Colt”. The distribution of dry matter within the plant was significant in determining susceptibility to Mg deficiency. Since “F. 12/1” has a smaller root:shoot ratio than Colt it is unable to sustain the same concentration of Mg in leaves as “Colt” irrespective of external K concentration. The molar ratio of K:Mg in soil solutions should remain <8.5:1 in order to ensure maximum growth of “F. 12/1” and “Colt”. This revised version was published online in June 2006 with corrections to the Cover Date.     

A 9 × 9 diallel analysis in peanuts (A. hypogaea L.): Flowering time,tops' weight,pod yield per plant and pod weight

Summary Four quantitative traits were studied by analysing F₂ data derived from a 9 × 9 diallel cross utilizing widely divergent, inbred, erect cultivars of peanuts, A. hypogaea. Bidirectional dominance was found in the traits total pod yield per plant and number of days from planting to first flower; in pod size, the alleles giving small pods were consistently dominant and for high tops' weight, dominance and overdominance were found. The high heritability of pod yield/plant (0.79) indicates that breeding for higher yield/plant can succeed if large F₂ populations are grown and rigorous visual selection combined with progeny testing are employed. The genetic correlations of pod yield/plant with other traits were low. Breeding for plants with large (jumbo) pods can be aided by the fact that they are homozygous recessive, or nearly so. Simultaneous breeding for high yields and large pods is possible: there was a positive (but low) genetic correlation between the two (0.16). A modification by which less biased estimates of the number of effective factors can be obtained and a possible relationship between bidirectional dominance and genic interaction were proposed.     

Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’ production from Bacopa monnieri (L.) Wettst.

Stable lines of hairy roots were established from leaf explants of Bacopa monnieri using different strains (A4, R1000, SA79, MTCC 532 and MTCC 2364) of Agrobacterium rhizogenes. The efficiency of hairy roots induction of these strains varied significantly and the maximum transformation frequency (75 %) was observed in case of strain SA79 using leaf explants followed by internode (55 %) in the presence of acetosyringone. Different parameters such as cell density of Agrobacterium suspension, co-cultivation period and infection time influenced the root induction frequency. Maximum frequency of root induction was obtained with bacterial density of 0.6 OD₆₀₀, 2 days of co-cultivation period and 10 min of infection time. Integration of T-DNA in the genome of hairy roots was confirmed by PCR amplification of rolB gene. Elimination of Agrobacterium from the established root cultures was ascertained by amplifying the DNA fragment specific to 16S rDNA and virD gene. All lines of hairy roots except strain A4 induced showed higher growth rate and accumulated higher levels of ‘bacoside A’ than the untransformed roots. Maximum biomass accumulation (6.8 g l^?1) and ‘bacoside A’ content (10.02 mg g^?1 DW) were recorded in case of the hairy root line induced by strain MTCC 2364.     

The effects of IAA and tetcyclacis on tuberization in potato (Solanum tuberosum L.) shoot cultures in vitro

Potato (Solanum tuberosum cv. Désirée) shoots grown in vitro in continuous darkness or in long days (LDs), were used to investigate indole-3-acetic acid (IAA) effects on stolon initiation and tuber formation, combining IAA with increased or decreased gibberellin levels. An increased gibberellin (GA) level was achieved by the applying 1 μM GA₃, while decreased gibberellin level was presumably realized by the adding 3 μM tetcyclacis (Tc). About 15% of potato shoots developed stolons both in LDs and in darkness. Stolon initiation was stimulated by GA₃ in darkness and by Tc in LDs. Tuber formation was strongly inhibited in LDs and by GA₃ both in light and darkness, but stimulated in darkness at low GA level. Exceptionally, tuber formation occurred in LDs at the highest Tc concentrations, in about 25% of explants. Indole-3-acetic acid alone stimulated stolon formation in LDs, both in the presence or absence of GA₃. IAA alone also stimulated tuber formation in dark-grown shoots, but could not overcome the inhibitory effect of LDs. Indications that, depending on their concentration ratio, IAA may interact with GA₃ in different tuberization phases, have been discussed. Radomir Konjević—Deceased in July 2006.     

Joint Insecticidal Action of Bacillus thuringiensis kurstaki with Annona squamosa L. and Prosopis juliflora Swartz DC on Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The efficacy of methanolic seed extracts of Annona squamosa (As) and Prosopis juliflora (Pj) individually and in combination with spore-crystal mixture of Bacillus thuringiensis kurstaki (Btk) was tested against third instar larvae of Helicoverpa armigera. The combination of botanicals with Btk significantly decreased LC₅₀ values in less time as compared to individual treatments. As and Pj in combination with Btk significantly affected feeding and dietary utilization of H. armigera significantly than in individual treatments. Physiological effects found in larvae fed on Btk and botanicals included extended larval and pupal durations, decreased adult longevity and fecundity, difficulty in moulting between various instars, formation of larval–pupal intermediates and abnormal pupae. As was found to have a higher interaction with Btk than Pj. Further, Btk showed strong synergistic action with As and Pj, which are the promising candidates for development of alternative bioinsecticide in IPM programs.     

Growth,development and yield of pawpaw (Carica papaya L.) ‘Homestead selection’ in response to soil moisture stress

Summary The effects of four soil water potential regimes under greenhouse conditions on growth, development, and yield of ‘Homestead selection’ pawpaw were investigated. The objectives were to determine the critical soil water potential regime and the moisture sensitive stages of growth of pawpaw. Repeated cycles of stress to −6.0 bar soil water potential imposed as from vegetative phase prevented fruit formation by constantly causing flower abscission. The stressed plants were stunted in size. The −2.0 bar soil water potential value was considered the critical level for normal growth and reproductive development of pawpaw. The mid-vegetative, flowering and fruit enlargement phases were moisture sensitive.     

Production,morphology, and cytogenetics of Triticum aestivum (L.) Thell × Elymus scabrus (R. Br.) Love intergeneric hybrids obtained by in ovulo embryo culture

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD), and an apomictic Triticeae species, Elymus scabrus (syn. Agropyron scabrum) (2n=6x=42, HHSSSS), the first successful report of this cross. Nine tiny, underdeveloped, and structureless embryos were obtained in vitro only by in ovulo embryo culture at 4 days after pollination, which gave rise to five mature hybrid plants. All the hybrid plants were vigorous and possessed a phenotype intermediate to the two parents. There were 2n=6x=42 (ABDHSS) somatic chromosomes in the hybrids. There was little or no homology between the parental genomes, as shown by an overall meiotic chromosome association of 32.83 I + 4.08 rod II + 0.21 ring II + 0.18 III + 0.02 IV. The hybrids were completely sterile and so far backcrosses to wheat parent have not been successful. Alternate approaches to induce gene transfer(s) from E. scabrus to wheat are being attempted.Agriculture Canada Contribution No. 398.     

Photosynthesis studies on European evergreen and deciduous oaks grown under Central European climate conditions. I: a case study of leaf development and seasonal variation of photosynthetic capacity in Quercus robur (L.), Q. ilex (L.) and their semideciduous hybrid, Q. × turneri (Willd.)

Leaf development of Quercus robur (deciduous), Q. ilex (evergreen) and of their hybrid Q. × turneri was assessed under Central European climate conditions. In all three taxa, development of maximum photosynthetic capacity was slow, although whole chain electron transport, grana development, the xanthophyll cycle and the biochemical capacity for photosynthesis were already completely present at day 7 after budbreak (DAB 7). In the course of the following 30 days, shifts in the levels of metabolites of the photosynthetic dark reactions were observed, indicating a change from ribulose bisphosphate (RubP) regeneration limitation towards RubP consumption limitation of photosynthesis. At the same time, electron transport rates had strongly increased and the capacity of the light reactions did not seem to limit photosynthesis. Sucrose levels in the leaves increased strongly, indicating sink limitation of photosynthesis, which might be responsible for the observed slow development of maximum photosynthetic capacity in all three taxa.     

Investigations on the effect of „light break” on the nature of the endogenous rhythm in the flowering response of Biloxi soybean (Glycine,Max, L. merr.)

Zusammenfassung Biloxi-Soja-Bohnen (Glycine max. L. Merr) erhielten photoperiodische Cyclen, die aus 8-oder 9stündigen Lichtperioden und unterschiedlich langen Dunkelperioden bestanden. Die Dunkelperioden wurden zu verschiedenen Zeitpunkten durch halbstündiges Störlicht unterbrochen. Die Wirkung dieser unterschiedlichen Behandlungen wurde durch Messung der Blühreaktionen bestimmt.Ein Störlicht, welches in frühen Abschnitten der langen Dunkelperiode geboten wird, verhindert die Blütenbildung unabhängig von der Länge des Gesamtcyclus, dem die Pflanzen ausgesetzt werden. Diese Unterdrückung erreicht ihr Maximum mit einem Störlicht, welches 8 Std nach dem Beginn der Dunkelperiode wirkt; die Hemmung ist schwächer, wenn das Störlicht früher geboten wird. Ein Störlicht, das gegen Ende der Dunkelperiode geboten wird, reduziert die Blütenbildung ebenfalls. Dieser Effekt erreicht sein Maximum mit einem Störlicht, welches ungefähr 8 Std vor dem Ende der Dunkelperiode geboten wird, er wird mit abnehmender Länge der nach dem Störlicht folgenden Dunkelperiode kleiner. Die Wirkung von Störlicht, das zu anderen Zeiten während der Dunkelperiode geboten wird, hängt von der Länge des Gesamtcyclus ab. Bei Cycluslängen, die für Blütenbildung ungünstig sind, kann solches Störlicht blühfördernd wirken.

---

---

Wit 7 Figures in the Text

---

Supported in part by a National Science Foundation grant toKarl C. Hamner.     

“Stress” reactions in teleosts: Effects of temperature and activity on anaerobic energy production in roach (Rutilus rutilus L.)

• 1.1. In roach any kind of excitement due to swimming, handling, or to unfavourable environmental conditions, is reflected in an increase of lactate above 1.0 μ mol and a decrease of phosphocreatine (PCr) below 15.0 μ mol g⁻¹ in the muscles.
• 2.2. In fish acclimated to and tested at 4°C lactate accumulation and PCr hydrolysis after activity were only half those in fish tested at 12 and 20°C.
• 3.3. Fish swimming at submaximal speed for 60 min at 20°C had much less lactate in the anterior body musculature than fish swimming for the same length of time at 12°C.
• 4.4. It is suggested that the “sudden death syndrome” of severely exercised fish is due to rapid hydrolysis of PCr, rather than to acidosis, in sensitive tissues.