Enhanced post-germinative growth of encapsulated somatic embryos of Siberian ginseng by carbohydrate addition to the encapsulation matrix

Based on a protocol for microspore culture in apple (Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of in vitro androgenesis, both methods, via anther and microspore culture, were investigated using the same bud material. A comparison of the efficiency of embryo induction in anther and microspore cultures showed that microspore culture resulted in an increase up to 10 times, depending on the genotype. The regeneration route in microspore culture is similar to that of androgenic embryos via anther culture and showed adventitious shoot formation in most cases after a long period of secondary embryogenesis.Communicated by H. Lörz     

SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Apple simple sequence repeats (SSRs) were intergenerically applied to the characterization of 36 pear accessions, including 19 Japanese pears (Pyrus pyrifolia), 7 Chinese pears (P. bretschneideri, P. ussuriensis), 5 European pears (P. communis), 3 wild relatives (P. calleryana), and 2 hybrids between P. pyrifolia and P. communis. All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear accessions. Nucleotide repeats were detected in the amplified bands by both Southern blot and sequencing analysis, and nucleotide sequences of pear were compared with those of apple. The differences in fragment size among pear or between pear and apple were, in many cases, due to the differences in repeat number. Interestingly, the DNA sequence of flanking regions in apple was highly conserved in pear. Hybrids from P. pyrifolia×P. communis showed one fragment inherited from each parent in all scorable cases, which suggested that each primer pair amplified fragments originating from the same locus. A total of 79 alleles were detected from seven SSR loci in pear, and all pear varieties except for the mutants could be differentiated. In conclusion, SSRs isolated from apple are highly conserved in pear and could be utilized as DNA markers in the latter genus. Received: 17 July 2000 / Accepted: 22 September 2000     

Effect of 1,2-benzisoxazole-3-acetic acid on adventitious shoot regeneration and in vitro rooting in apple

 The effect of 1,2-benzisoxazole-3-acetic acid (BOA), compared to 1-naphthaleneacetic acid (NAA), on adventitious shoot formation in leaf portions and compared to indolebutyric acid (IBA), on in vitro rooting in the apple (Malus domestica Borkh) cultivars McIntosh and Gala, and one rootstock, Jork 9, was investigated. BOA at 43.0 μm or 2.7 μm at NAA in combination with 17.8 μm benzyladenine (BA), induced the highest number of explants to produce adventitious shoots in Jork 9. In Gala, the combination of 21.5 μm BOA with 1.0 μm thidiazuron (TDZ) or with 22.0 μm BA induced the highest regeneration percentages, 58 and 54%, respectively, giving more satisfactory results than NAA (where only 42% of leaf explants exhibited shoot formation). In McIntosh, the highest percentage of regeneration was obtained with 1.3 μm NAA and 22.0 μm BA, while 51% was the highest response obtained with the BOA treatment. The combination of BOA with TDZ completely inhibited regeneration activity in leaf portions of this cultivar. The shoots of all the genotypes obtained with the most morphogenetic NAA or BOA treatments were excised, multiplied and successfully rooted and hardened. The results demonstrate that the synthetic auxin BOA is active in inducing shoot regeneration from leaf explants of apple and that the activity of BOA in plant regeneration is genotype dependent. When BOA was used to induce rooting in apple microcuttings, lower rooting percentages were obtained than with IBA, showing that the effect of BOA in inducing root formation is very low and that it cannot be used routinely to replace IBA in the in vitro rooting of microcuttings. Received: 18 June 1998 / Revision received: 4 January 1999 / Accepted: 29 January 1999     

Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

 The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo. Received: 27 December 1999 / Accepted: 11 May 2000     

Drought tolerance of apple rootstocks: Production and partitioning of dry matter

The drought tolerance of the commercial apple ( Malus domestica Borkh.) rootstocks M9, M26, M27 and MM111, and some new selections from the rootstock breeding programme at HRI-East Malling (AR69-7, AR295-6, AR360-19, AR486-1 and AR628-2), was assessed using potted, glasshouse-grown, unworked rootstocks. After an initial period of growth under well-watered conditions the amount of irrigation was gradually reduced, for some treatments, to simulate natural drying in the soil. At the end of a six-month growth period, the rootstocks were harvested and the production of dry matter and its partitioning to various plant parts determined. The rootstocks exhibited large differences in shoot and root dry matter, and root length but not all the rootstocks showed declines in root mass or length in response to the droughting treatment. The dwarfing rootstocks tended to have smaller amounts of both coarse (>2 mm diameter) and fine roots (<2 mm diameter), than the more vigorous rootstocks. Irrespective of rootstock or irrigation treatment there was a close linear relationship between coarse and fine root. There was also no change in the length/weight relationship for fine roots irrespective of rootstock or irrigation treatment, i.e. 42 m of fine root weighed 1 g dry weight. In some cases the amount of root produced could be directly correlated with the rootstock known potential to control scion vigour, but this was not true for all the rootstocks examined. The absence of this relationship was particularly evident in some of the new selections of rootstock. The possible causes for these differences, compared with commercially used rootstocks, is discussed in relation to the origin and parentage of the rootstock selections. Despite this lack of a root length/vigour relationship, the amount of dry matter partitioned to shoot growth reflected the rootstocks' known vigour. The different responses of these rootstocks to drought are discussed along with their implications for understanding the mechanisms by which rootstocks are thought to dwarf scion shoots.     

Analysis of the genetic diversity and structure of the Spanish apple genetic resources suggests the existence of an Iberian genepool

The nature and structure of genetic diversity in the Spanish apple germplasm preserved at the national level was widely unknown, since studies performed to date on this topic have been exclusively carried out at the regional scale. Here, 1453 accessions from Spanish collections of Malus × domestica were evaluated with a common set of 13 SSR (Simple Sequence Repeats) markers in order to estimate genetic diversity, to identify the underlying genetic structure and to unravel the relationships among them and among a wide set of international cultivars for reference. In total, 737 unique genotypes were identified, 581 diploids and 156 triploids. Using a model‐based Bayesian clustering procedure, two reconstructed populations were obtained for diploid genotypes; one retaining only Spanish cultivars (42% of genotypes), and a second containing all foreign cultivars the latter exhibiting evidence supporting the existence of a secondary sub‐structure. Similarly, analysis performed on the 156 triploid genotypes also revealed two reconstructed populations; one exclusively associated with local Spanish genotypes (44%). The Jaccard coefficient allowed clustering by UPGMA (Unweighted Pair Group Method) diploid and triploid genotypes, and remarkable differences in allelic composition among the different partitioning levels were found. AMOVA analyses showed moderate but significant differentiation among the main groups (0.08 ≤ F_ST ≤ 0.12). Our results highlight an important fraction of the Spanish apple germplasm that constitutes a differentiated genepool with respect to the international and commercial apple cultivars. Moreover, the extent of the Spanish genetic diversity was spatially distributed along the northern Iberian Peninsula, suggesting an extensive migration of genotypes along the country. This study is the first valuable action for genetic conservation of apple at the national scale, and constitutes a decisive step towards the definition of a Spanish core collection that will be useful for further studies in dissecting the genetic control of important horticultural traits through genome‐wide association analysis in apple.     

S-RNases in apple are expressed in the pistil along the pollen tube growth path

 The molecular bases of self-incompatibility have been intensively studied in a restricted number of model species, but for most families the expression and distribution of S-proteins is unknown. In this work, pistil cryosections from apple were used for in situ detection of S-proteins. Two specific antibodies, one against the S3-protein and another against all apple S-proteins were used. S-proteins were shown to be localised in the intercellular space of the transmitting tissue, both in the stigma and style, which agrees with the proposed mechanism of action for S-RNases in gametophytic self-incompatibility. Some intracellular labelling was also observed in all ovary sections, confined to one layer of the nucellus surrounding the embryo sac, but this labelling was found to be non-S-allele-specific. Nevertheless, the signal in the ovary was tissue-specific, which may indicate that some component not encoded by the S-locus but similar to S-proteins was detected. To the best of our knowledge this is the first report on the precise distribution of S-RNases in a rosaceous species. Received: 15 July 1998 / Revision accepted: 29 December 1998     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

Induction of embryogenesis with colchicine instead of heat in microspores ofBrassica napus L. cv. Topas

Prior to this report, heat treatment (32.5°C, 24 h) was the method used to induce embryogenesis fromBrassica napus microspores. Continuous culture at 25°C results in pollen development. This study shows that colchicine alone, at the non-inductive temperature of 25°C, can induce embryogenesis, thus demonstrating that heat shock is not required for embryogenic induction inB. napus cv. Topas. Embryogenic frequencies of over 15% were obtained by culturing isolated microspores with 25 M colchicine for 42 h at 25°C. The microspore developmental stages responsive to colchicine were unicellular vacuolate and late unicellular, somewhat earlier stages than the population responsive to heat induction. Other groups have reported that heat-shock proteins are essential to the induction of embryogenesis. The present study offers a method of embryogenic induction without the use of heat which will allow discrimination between the factors associated with response to heat shock and those involved with changing cell development.Abbreviations LU Late-unicellular - PPB Preprophase band - UV unicellular-vacuolate The authors wish to thank C. Bornman for his interest and encouragement. We gratefully acknowledge support from the School of Graduate Studies and Research, Queen's University to J.-P. Z., from Hilleshog AB, Sweden to D.H.S., and from the Natural Sciences and Engineering Research Council of Canada to D.H.S. and W.N. Plant Research Centre contribution No. 1595.     

A protocol for repetitive somatic embryogenesis from mature peanut epicotyls

 The effects of 11 different auxins and one cytokinin-like compound were tested at four concentrations for their ability to induce primary and repetitive somatic embryos from mature, dry peanut (Arachis hypogaea L.) epicotyls of genotype AT120. Treatment with picloram and centrophenoxine at 83.0 and 124.4 μm resulted in the greatest number of embryos per explant and the highest percentage of explants responding. In a follow-up experiment, picloram, centrophenoxine, and dicamba were tested at 83.0 and 124.4 μm on four peanut genotypes (AT120, 59-4144, GK7, and VC1). Picloram and centrophenoxine induced similar numbers of globular-stage and total embryos from each genotype, while dicamba was less effective. Similar results were observed with percentage of responding axes. Genotypes AT120 and VC1 yielded more clusters of repetitive embryos than GK7 and 59-4144. After 5 months, embryos derived from repetitive embryogenic cultures were converted into mature plants. Received: 8 February 1999 / Revision received: 9 June 1999 / Accepted: 30 June 1999     

Differential localization of the LIM domain protein PLIM-1 in microspores and mature pollen grains from sunflower

 PLIM-1 is a LIM domain protein specifically expressed in pollen grains. Using two PLIM-1-specific monoclonal antibodies we studied its expression and intracellular location at various developmental stages of sunflower (Helianthus annuus L.) pollen. Our studies show that the protein appears at the microspore stage in a limited number of cytoplasmic bodies, becomes undetectable in bicellular pollen, and reappears in tricellular pollen grains in cortical patches particularly concentrated in the F-actin-enriched germination cones of the vegetative cell. The developmental stage-dependent, different location of the protein suggests a dual function during pollen development. While this function in microspore development remains obscure, the high concentration of PLIM-1 in the germination cones of mature pollen suggests that it participates in the germination process as well as in pollen tube growth. Received: 11 August 1998 / Revision accepted: 15 December 1998     

Re-examination of the self-incompatibility genotype of apple cultivars containing putative ’new’ S-alleles

Recently, the self-incompatibility (S-) genotypes of 56 apple cultivars were examined by protein analysis, which led to the identification by Boskovic and Tobutt of 14 putative ’new’ S-alleles, S12 to S25. This paper reports a re-examination of the S-genotypes of some of these cultivars through S-allele ’specific’ PCR and sequence analysis. The results obtained by this analysis indicated that the number of S-alleles that are present in apple is probably smaller than the number proposed by Boskovic and Tobutt. The existence of three ’new’ S-alleles (S20, S22 and S24) was confirmed. The existence of two other putative ’new’ S-alleles (S23 and S25) was, however, contradicted. The coding sequences of the S-alleles that correspond to the S10 and the S25 ribonuclease bands as well as those corresponding to the S22 and the S23 ribonuclease bands were shown to be identical in sequence. Interestingly, the S-allele corresponding to the S22 and the S23 ribonuclease bands shared a high sequence identity (99% identity) with S27, which was previously cloned and sequenced from Baskatong, but which was not included in the analysis conducted by Boskovic and Tobutt. Both S-alleles only differ in point mutations, which are not translated into differences in amino-acid sequence. To our knowledge, this is the first report of two S-alleles that differ at the nucleotide level but still encode for identical S-RNases. The implications of these observations for determining the S-genotypes of plants by PCR analysis or protein analysis are discussed. Received: 10 January 2001 / Accepted: 19 January 2001     

Induction of atrazine resistance and somatic embryogenesis in Solanum melongena

 The effects of atrazine on cotyledon cultures of Solanum melongena were investigated with a view to establishing a system for in vitro selection of resistant mutants. At herbicide levels producing little growth inhibition some chlorophyll loss occurred associated with the production of albino shoots. At 15 mg/l bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenesis with EMS resulted in herbicide-resistant mutants based on the embryogenic ability of mutagenised explants placed on medium containing selective levels of sucrose (0.2%) and atrazine (15 mg/l). Different morphogenetic responses were observed when the levels of sucrose (0.2–5%) were altered. Somatic embryogenesis was observed at low sucrose concentrations (0.2–0.5%). Both embryogenesis and shoot regeneration occurred in 1% sucrose. Shoot regeneration was maximum in 2% sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3–5%). However, lowering of sucrose concentration from 2% to 0.2% caused complete bleaching, permitting the selection of herbicide-resistant mutants. Received: 26 November 1996 / Accepted: 20 December 1996     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

Use of the multi-allelic self-incompatibility gene in apple to assess homozygocity in shoots obtained through haploid induction

 To obtain homozygous genotypes of apple, we have induced haploid development of either the female or the male gametes by parthenogenesis in situ and anther culture, respectively. Of the shoots obtained, which were mainly of a non-haploid nature, some could be derived from fertilised egg cells or from sporophytic anther tissue. In order to select the shoots having a true haploid origin, and thus homozygotes, we decided to use the single multi-allelic self-incompatibility gene as a molecular marker to discriminate homozygous from heterozygous individuals. The rationale behind this approach was that diploid apple cultivars contain 2 different alleles of the S-gene and therefore the haploid induced shoots obtained from them should have only one of the alleles of the single parent. The parental cultivars used were ‘Idared’ (parthenogenesis in situ) and ‘Braeburn’ (androgenesis), and their S-genotypes were known, except for 1 of the ‘Braeburn’S-alleles. To stimulate parthenogenetic development ‘Idared’ styles were pollinated with irradiated ‘Baskatong’ pollen, the S-alleles of the latter (2n) cultivar were also unknown. The cloning and sequence analysis of these 3 unidentified S-alleles, 1 from ‘Braeburn’ and 2 from ‘Baskatong’ is described, and we show that they correspond to the S ₂₄ -, S ₂₆ - and S ₂₇ -alleles. We have optimised a method for analysis of the S-alleles of ‘Idared/Baskatong’- or ‘Braeburn’-derived in vitro plant tissues and have shown that this approach can be applied for the screening of the in vitro shoots for their haploid origin. Received: 18 August 1997 / Accepted: 10 September 1997     

Segregation distortion at marker loci: variation during microspore embryogenesis in maize

In the present study, we analyzed the segregation distortions of markers during in vitro androgenesis in maize. This was based on four segregating populations derived from the A188×DH7 one-way-cross. These populations consisted of very young androgenetic embryos, well-developed calluses, haploid regenerated plantlets and spontaneous diploid plantlets. These structures all represented different developmental stages, from that of microspores to the regenerated plantlets. This study complemented a previous one by Murigneux et al. 1994, where distorted segregations of RFLP markers were detected in a single-seed-descent population and in a doubled-haploid population derived from the same cross. The weakly biased SSD maize genetic map was used as a reference to locate 145 AFLP loci whose allelic segregations were also analyzed in the androgenetic segregating populations. Segregation distortions were determined based on chi-square analysis (P<0.01 and P<0.001). Regions on chromosomes 2 and 8 showed distortions from the beginning of embryo formation, with large effects throughout the process. Regions on chromosomes 3, 4, 6 and 10 could control callus formation from microspores. Other deviations of marker genotypes on chromosomes 1, 4, 6 and 10 could be associated with the regeneration phase. Moreover, the statistical method of Cheng et al. for mapping a lethal factor locus inside segments of linked distorted markers was used to estimate the position of seven partial lethal androgenetic factors on chromosomes 1, 2, 8 and 10. These factors could represent selective genes actively involved in maize androgenesis. Received: 31 July 2000 / Accepted: 2 January 2001     

Factors affecting the transformation of 'Marshall McIntosh' apple by Agrobacterium tumefaciens

The goal of this research was to develop an efficient transformation system for 'Marshall McIntosh' apple. To determine the optimum combination of agar and Gelrite gelling agents in the media to maximize regeneration and minimize hyperhydicity (vitrification), the following combinations of agar (A)+Gelrite (G) in g l-1 were tested: 7.0 A+0 G; 5.2 A+0.6 G; 3.5 A+2.5 G; 1.7 A+1.8 G; and 0 A+2.5 G. Both 5.2 A+0.6 G and 3.5 A+1.2 G provided greatest regeneration of healthy non-hyperhydric shoots. To determine the optimal concentration of aminoglycoside for the selection and regeneration of transgenic 'Marshall McIntosh' on agar-Gelrite-based media, kanamycin was tested at 0, 10, 25, 50, 75 and 100 mg l-1, and paromomycin was tested at 0, 50, 100, 150, 200 and 250 mg l-1. Kanamycin was more effective than paromomycin in the initial selection of transgenics. For selection of transformants of 'Marshall McIntosh', the use of kanamycin at 25 mg l^-1on 5.2 A+0.6 G solidified medium is suggested. By optimizing the medium and selection conditions, a protocol was developed that resulted in four transgenic lines as confirmed by a GUS assay, NPT II ELISA, PCR, and Southern analysis. In repeated experiments with this protocol, transformation efficiencies of 3.1 and 2.6% were obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Empirical models of the conductance of leaves in apple orchards

Abstract. Seasonal data on leaf conductance (g_l) for three different apple cultivars grown in four separate orchards with different aged trees was studied between 1979 and 1985. A number of empirical models for predicting leaf conductance from environmental measurements were compared using this data and a general method for adapting such models for the prediction of different data sets is proposed. Although stepwise multiple regression identified relative humidity or vapour pressure as important variables, it frequently did not identify the optimal set of independent variables, which often did not include either of these. There was no advantage in regressing g_l against principal components of the environment, rather than against the raw environmental variables. A simple model involving air vapour pressure deficit, air temperature and a hyperbolic function of irradiance was found to explain between 32 and 62% of the variance in g_l for the different data sets. Parameters fitted for one data set led to the effective prediction of g_l in other years or plots. The model fit could generally be improved significantly by including soil moisture deficit among the independent variables.