Somaclonal and chromosomal effects of genotype, ploidy and culture duration in Asparagus officinalis L.

Chromosome and morphological variations of embryogenic calli-derived plants of gynogenic haploid, diploid, triploid and tetraploid asparagus (Asparagus officinalis L.) were investigated. Artificial tetraploids were produced using colchicine treatment of seeds of diploid cultivar, ‘Poultom’. ‘Haidel’ (2X) was crossed with the artificial tetraploids, from which one gynogenic haploid, one diploid, 6 triploid, 3 mixoploids were obtained. Embryogenic calli were first obtained from crown buds, subsequently induced to form somatic embryos, and after 30 days, induced to germinate. Chromosome variation in embryogenic calli-derived plants increased with increasing duration of subculture, particularly when low ploidy levels of plants such as haploid and diploid were used as explants. Approximately 80% of haploid-derived plants showed morphological variations such as dwarfness and abnormal morphological characteristics, although no differences were observed in cladodes and stem characteristics between other polyploid-derived plants and their parents. The data presented here would supply important fundamental information for commercial mass-propagation using somatic embryogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomal regions associated with green plant regeneration in wheat (Triticum aestivum L.) anther culture

Genetic capacity for green plant regeneration in anther culture were mapped in a population comprising 50 doubled haploid lines from a cross between two wheat varieties ‘Ciano’ and ‘Walter’ with widely different capacity for green plant regeneration. Bulked segregant analysis with AFLP markers and composite interval mapping detected four QTLs for green plant percentage on chromosomes 2AL (QGpp.kvl-2A), 2BL (QGpp.kvl-2B.1 and QGpp.kvl-2B.2) and 5BL (QGpp.kvl-5B).The three QTLs detected on chromosome 2AL and 2BL all derived their alleles favouring green plant formation from the responsive parent ‘Ciano’.The remaining QTL on chromosome 5BL had the allele favouring green plants from the low responding parent ‘Walter’. In a multiple regression analysis the four QTLs could explain a total of 80% of the genotypic variation for green plant percentage. None of the chromosomal regions with QTLs for green plant percentage showed significant influence on either embryo formation or regeneration frequencies from the anther culture. The three major QTLs located on group two chromosomes were fixed in a second DH population derived from two parents ‘Ciano’ and ‘Benoist’,both with high capacity to produce green plants. A QTL explaining31.5% of the genetic variation for green plant formation were detected on chromosome 5BL in this cross as well. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro induction of tetraploid plants from callus cultures of diploid bananas (<Emphasis Type="Italic">Musa acuminata</Emphasis>, AA group) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’

Tetraploid plants were induced successfully from diploid bananas Musa acuminata (AA genome) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’ (2n = 2x = 22) with in vitro oryzalin treatment. Calluses from in vitro-grown shoot tips of both cultivars were treated with oryzalin at concentrations of 1.5 or 3 mg l⁻¹ for 24, 48 and 72 h, respectively. The oryzalin treatments produced tetraploids at a frequency of 15.6% in ‘Kluai Leb Mu Nang’ and 16.7% in ‘Kluai Sa’ as detected by flow cytometry. Chromosome counting showed that the tetraploid plant chromosome number was (2n = 4x = 44). The selected tetraploid plants were transplanted in the field and variations in the morphological characteristic of leaf shape and fruit bunch compared to normal diploid plants were found under the same growing condition even after 3 years of cultivation.     

Potato Virus Y Resistance Gene, Ry chc, Mapped to the Distal End of Potato Chromosome 9

Summary A Japanese leading cultivar ‘Konafubuki’ has an extreme resistance gene Ry _chc to Potato virus Y (PVY) in a simplex fashion. To reduce complexity of the highly heterozygous autotetraploid genome and map the Ry _chc gene, haploid plants were parthenogenetically induced from ‘Konafubuki’. The ratio of 7 resistant: 5 susceptible haploids confirmed the single dominant, simplex genotype of ‘Konafubuki’. A selected resistant haploid 98H20-5 was crossed with a susceptible diploid, resulting in 119 F ₁ plants. Using RFLP and RAPD markers, Ry _chc was mapped to the most distal end of the chromosome 9 where the recovery of recombinant genotypes was extremely reduced in this mapping population possibly due to the linked inferiority factor. The location of Ry _chc is different from those of the other extreme resistance genes to PVY, but possibly resides in one of resistance gene clusters.     

Assessment of the origin of new citrus tetraploid hybrids (2<Emphasis Type="Italic">n</Emphasis> = 4x) by means of SSR markers and PCR based dosage effects

We report the accurate determination of the allelic configurations of a total of eight new citrus tetraploid hybrids by means of SSR analysis, coupled with capillary electrophoresis, and PCR based dosage effects. Tetraploid hybrids were spontaneously obtained from different interploid crosses (2x × 4x) between diploid ‘Femminello’ lemon and the allotetraploid somatic hybrid (2n = 4x = 36) ‘Key’ lime + ‘Valencia’ orange, and between diploid ‘Wilking’ and ‘Fortune’ mandarins and an autotetraploid ‘Dancy’ mandarin (2n = 4x = 36). To understand the opportunity to employ them in further backcross programs, the cytological mechanisms underlying their ploidy level were unambiguously determined using six SSR primers. PCR conditions were optimized and skewness in template/product ratios were verified. Tetraploid allelic configurations were determined from PCR based dosage effects using electropherogram peak heights to estimate the copy number per allele. In all the tetraploid hybrids we found out that diploginy (2n eggs) has occurred, contributing the extra haploid genome in the tetraploids. According to the marker genotypes, it was further inferred that the 2n eggs in ‘Femminello’ lemon resulted from first division restitution (FDR), while in ‘Wilking’ and ‘Fortune’ mandarins 2n eggs occurred in second division restitution (SDR). These new genotypes, with their improved genetic female background, can be therefore considered very valuable in our citrus genetic improvement program as pollen donors in backcrosses suitable to eliminate negative traits.     

Trueness-To-Type and Yield Components of the Banana Hybrid Cultivar FHIA-18 Plants Regenerated Via Somatic Embryogenesis in a Bioreactor

Summary A population of 1,500 plants of the banana hybrid ‘FHIA-18’ (AAAB), regenerated from somatic embryos, which were multiplied in bioreactors, showed similar characteristics to plants propagated from shoot tip cultures both in the acclimatization stage and in field experiments carried out in Cuba. The plants originating from somatic embryos were similar to the plants obtained from shoot tips with respect to plant height, diameter of the pseudostem and number of suckers. Both groups of plants obtained from in vitro cultures were significantly different to the plants obtained from suckers during the flowering period of the mother plants, which was shortened by two months. The greater plant height and diameter of the pseudostem in the plants coming from somatic embryos and shoot tip is due to the effect of in vitro culture, and this was observed in different banana and plantain cultivars. During the second cycle of evaluation, the plants coming from the three propagation methods studied in this work had similar growth habits without significant differences in the majority of the morphological parameters evaluated. These results confirm that the difference obtained during the first cycle between the distinct populations is attributed to temporary changes. The original characteristics of the cultivar were evident from the second cycle of culture. Only 0.13% somaclonal variant was observed in the plants coming from somatic embryogenesis. These percentages are low taking into consideration that other propagated methods accept up to 5% variants in field conditions.     

Introgression of unique characteristics of floral initiation under 24 hour day-length of Fragaria chiloensis ‘CHI-24-1’ into F. × ananassa

A unique flower initiation of the wild Junebearing strawberry strain ‘CHI-24-1’ in Fragaria chiloensis occurs under 24 h DL and high temperature conditions. To introduce the floral initiation characteristics of ‘CHI-24-1’ into cultivated strawberries of F. × ananassa, a cross pollination was conducted between ‘CHI-24-1’ and the Japanese short-day type strawberry cultivar ‘Nyoho’. The floral initiation of ‘CHI-24-1’ was induced in both parent and daughter plants linked with runners under a 24 h DL and 23/20 ^∘C, but not 8 and 16 h DLs at the same temperatures. Of the 21 F1 hybrids grown under the 24 h DL, 12 showed flower truss production in the parent and/or daughter plants linked with runners. Among 64 F1 hybrids, 26 exposed to 8, 16 and 24 h DLs for 30 days produced no flower trusses. However, 32, one and five F1 hybrids produced flower trusses under the 24 h DL alone, 8 h DL alone and both the 8 and 24 h DLs, respectively. The results of the experiments indicated that none of the F1 hybrids were day-neutral plants, but approximately 60% had the characteristics of floral initiation under 24 h DL, which was inherited from the pollen parent of ‘CHI-24-1’. The importance of the unique floral initiation characteristics under 24 h DL with high temperature for strawberry breeding was discussed.     

Isolation of wheat mutants with increased resistance to powdery mildew from small induced variant populations

Small variant wheat populations created by induced mutagenesis (n = 69) or adventitious regeneration (n = 66) were intensively screened for an altered response (compared to the parent variety ‘Guardian’) to the causal pathogen of powdery mildew in wheat, Blumeria graminis f. sp. tritici. Intensive field screening following natural infection of replicated plots of wheat lines over two years revealed a total of 13 mutants exhibiting significantly greater resistance than ‘Guardian’: eight from induced mutagenesis (11.6%) of the M2 population and five from adventitious regeneration (7.6%). Complete resistance was identified in two lines, (one (M66) developed following induced mutagenesis, and the other (SC240) by adventitious regeneration). The complete resistance in the induced mutant was stable over two generations and was associated with a high frequency of leaf flecking, and consequently a low grain yield. Resistance in SC240 proved to be unstable; SC240 exhibited complete resistance to powdery mildew in the SC₂ and SC₃ generations, but only 20% of the SC₄ plants were completely resistant, while the remainder were indistinguishable in mildew response to ‘Guardian’. The mildew response of all the SC₅ generation of SC240 was not significantly different from ‘Guardian’. Yield analysis of the thirteen mutants with increased resistance in the presence of powdery mildew indicated that eleven exhibitedgrain yields at least as high as that of ‘Guardian’, while the mutant M19 exhibited a yield significantly higher than that of ‘Guardian’. This revised version was published online in July 2006 with corrections to the Cover Date.     

High frequency direct plant regeneration from leaf and petals of Cape Primrose (<Emphasis Type="Italic">Streptocarpus</Emphasis>)

High frequency direct plant regeneration from leaf and petal explants was accomplished for the first time in Streptocarpus varieties. The shoot induction frequency varied with respect to the benzylaminopurine (BAP) concentration added to the Murashige and Skoog (MS) medium. MS medium with 0.5 mg l⁻¹ BAP exhibited the highest (69.9%) plant regeneration frequency with an average of 186 shoots per explant. A higher concentration of BAP inhibited shoot bud induction and plant regeneration along with necrosis of explants. Petal explants derived from the varieties ‘Branwen’ (pink and white) and ‘Chorus Line’ (violet and white) displayed plant regeneration frequency of 22.2–47.4% (within a total of 12 weeks) on MS medium containing 2.0 mg l⁻¹ α-naphthaleneacetic acid and 0.5 mg l⁻¹ BAP for 8 weeks followed by 4 weeks on MS medium with 1.0 mg l⁻¹ BAP. Scanning electron microscopy confirmed direct plant regeneration without callus. Regenerated plants from leaf explants with well-developed leaves and roots were hardened and successfully transferred to pots in glasshouse exhibiting 86% survival at the end of 4–6 weeks. Whereas, regenerated plants from flower petal explants upon transfer to pots in glasshouse exhibited 75–82% survival at the end of 4–6 weeks.     

Obtention of haploid embryos and plants through irradiated pollen technique in squash (Cucurbita pepo L.)

The influence of gamma ray doses (25, 50, 75, 100, 200, 300 and 400 Gy) and genotypes (Eskenderany F₁, Acceste F₁, Sakiz, Urfa Yerli) on the induction of haploid embryos obtained by irradiated pollen technique was studied in squash (Cucurbita pepo L.). Different shapes and stages of embryos were derived from seeds extracted from fruits harvested 4–5 weeks after pollination. As a result of the present study, haploid embryos and haploid plants were obtained, with haploid production strongly influenced by gamma ray doses, embryo stages and genotypes. Gamma ray doses of 25 and 50 Gy gave the highest parthenogenetical response. All of the point shape, globular shape, arrow tips and stick-shaped embryos developed into haploid plants. However, only 53.8% of torpedo and 23.1% of heart-shaped embryos gave haploid plants. In contrast, cotyledon-shaped and amorphous-shaped embryos produced only diploid plantlets. The number of embryos per 100 seeds was the highest in ‘Eskenderany F₁’ and ‘Sakiz’ genotypes. After in vitro culture, a total of 93 haploid plantlets were obtained. This revised version was published online in August 2006 with corrections to the Cover Date.     

High regeneration potential in vitro of sunflower (Helianthus annuus L.) lines derived from interspecific hybridization

Successful selection of interspecific hybrid progenies with superior ability to regenerate shoots from apical meristems was performed in sunflower which now allows for the development of lines for improved biotechnological applications. Early generations of interspecific hybrids originating from crosses between the two H. annuus CMS lines ‘HA89’ and ‘Baso’, and 9 wild species were screened for their ability to regenerate in vitro. Evaluation of 36 progenies allowed to identify seven progenies from crosses involving H. mollis, H. giganteus, H. strumosus, and H. decapetalus which showed a significantly higher regeneration potential than the commercial hybrid ‘Albena’ regarding the number of shoots per explant. Among these progenies, 47.2 to 62.4% of explants produced shoots with an average of 2.3 to 3.5 shoots per cultured explant. Regeneration in vitro was significantly determined by the genotype. More than half of the investigated interspecific hybrids performed better than the inbred ‘HA89’ demonstrating that the high regeneration potential available in the wild species can be efficiently transferred to cultivated sunflower. The seven progenies with high regeneration potential in vitro were characterised by agronomic performance in the field. Two of the interspecific hybrids derived from H. strumosus and H. decapetalus not only showed a superior regeneration potential but also proved to be competitive to commercial hybrids with regard to important agronomic traits, e.g. fat content and TGW. This revised version was published online in July 2006 with corrections to the Cover Date.     

Yield and disease resistance of plantain (Musa spp., AAB group) somaclones in Nigeria

This study examined the potential of somaclonal variation for the improvement of plantain. Approximately 500 somaclones each of ‘Agbagba’ (False Horn plantain) and ‘Bise Egome’ (French plantain) were field evaluated for their agronomic performance and response to the black Sigatoka disease. The micropropagated populations were independently generated from a number of suckers from each accession. Significant differences between micropropagated accessions and crop cycles were observed. Differences between plants derived from suckers of the same accession were also expressed, indicating the chimerical nature of variation in the traits studied. None of the plants of the micropropagated populations from ‘Bise Egome’ exhibited significantly better disease tolerance and agronomic performance than the source accession. However,one somaclonal variant of ‘Agbagba’ (‘AO 2B2-2’) expressed lower susceptibility to the black Sigatoka disease. Compared to ‘Agbagba’, ‘AO 2B2-2’ had a higher bunch weight, more fruits per bunch with higher average weight, greater average length, and greater average girth. These data clearly show that, in contrast to previous reports, it is possible to recover superior somaclonal mutants in Musa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pear mutagenesis: In vitro treatment with gamma-rays and field selection for productivity and fruit traits

In vitro shoots of six pear (Pyrus communis L.) cultivars, ‘Conference’, ‘Doyennéd'Hiver’, ‘Passe Crassane’, ‘Bartlett’, ‘AbbéFetel’ and ‘Butirra Precoce Morettini’ were irradiated with gamma rays (3.5 Gy). After three subcultures, microcuttings from the irradiated shoots and from additional non-irradiated microcuttings were rooted to establish plants for survey orchards. All trees were individually observed for variation in fruit traits and for productivity. Trees were selected for improved characters related to production such as early bearing and consistent productivity. Variations observed in fruit appearance concerned degree of russeting, fruit shape and size. The frequencies of the observed variations in fruit traits depended on the cultivar, ranging from 0.81% in ‘Doyennéd'Hiver’ to 3.64% in ‘Passe Crassane’. Of the 97 variants selected, only two showed chimeral behavior. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD markers linked to microspore embryogenic ability in Brassica crops

In order to identify the markers linked to microspore embryogenic ability in Brassica crops, RAPD segregation analyses were performed in a microspore-derived (MD) population and a F₂ population derived from F₁between ‘Ho Mei’ (high responsive parent in microspore embryogenesis) and ‘269’ (low responsive parent) in Chinese cabbage, and between ‘Lisandra’ (high responsive parent) and ‘Kamikita’ (low responsive parent) in oil seed rape. After 230 and 143 primers were screened, a total of 148 and 52markers were detected to be polymorphic between the parents in Chinese cabbage and oilseed rape, respectively. Twenty-seven percent of the markers in the MD population showed a significant segregation distortion in both crops. Of the markers showing segregation distortion in the MD population, 71–75% of the markers followed the expected Mendelian segregation ratio in the F₂ population. When the relationships between such distorted markers and microspore embryogenesis of the F₂ population were examined, 7 and 3 markers were identified to be associated with embryogenic ability in Chinese cabbage and oilseed rape, respectively. These markers showed additive effects on embryo yields, and the plants having more alleles of the high responsive parent produced higher embryo yields. These markers maybe useful in marker-assisted selection for improving microspore responsiveness straits in Brassica crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance and relationships of important agronomic traits in almond

The inheritance of 16 important agronomic traits and its relationship were studied for four years in a population of 167 almond [P. dulcis (Mill.) D.A. Webb] seedlings obtained from a cross between the French selection ‘R1000’ (‘Tardy Nonpareil’ × ‘Tuono’) and the Spanish cultivar ‘Desmayo Largueta’. For some traits (blooming and leafing date) descendants segregated between the value of both progenitors, meanwhile for others the mean of the offspring was lower (bloom density, productivity and ripening date) or higher (in-shell/kernel ratio and double kernels). As expected, kernel bitterness and self-incompatibility behaved as monogenic traits. Some important correlations between traits were detected. The implications of the transmission and the correlation of these traits in the breeding programmes are discussed.     

Mapping QTLs for pre-harvest sprouting traits in the spring wheat cross ‘RL4452/AC Domain’

Pre-harvest sprouting (PHS) in spring wheat (Triticum aestivum L.) is a major downgrading factors for grain producers and can significantly reduce end-use quality. PHS resistance is a complex trait influenced by genotype, environment and plant morphological factors. A population of 185 doubled haploid (DH) lines from the spring wheat cross ‘RL4452/AC Domain’ were used as the mapping population to detect quantitative trait loci (QTLs) associated with three PHS traits, germination index (GI), sprouting index (SI) and falling number (FN). Six major QTLs linked with PHS traits were mapped on bread wheat chromosomes 3A, 3D, 4A (2 loci), 4B and 7D. ‘AC Domain’ alleles contributed to PHS resistance on 3A, 4A (locus-2) and 4B, and ‘RL4452’ alleles contributed resistance on 3D, 4A (locus-1) and 7D. QTLs detected on chromosome 4B controlling FN (QFN.crc-4B), GI (QGI.crc-4B) and SI (QSI.crc-4B) were coincident, and explained the largest amount of phenotypic variation in FN (22%), GI (67%) and SI (26%), respectively.     

Culture of unpollinated ovules,ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Allium cepa L.)

Summary Induction of haploid plants is of great importance for breeding purposes because of the possibility to obtain from haploids homozygous material by artificial chromosome doubling in relatively short times. The present study reports the first evidence of successfull haploid induction in onion. Isolated ovules, ovaries, or whole flower buds of different Allium species were cultured on BDS agar medium. Testa browning in the ovules and an extensive growth of the latter were observed. In cultures of ovaries and flower buds, development of callus and subsequent regeneration of plantlets from the region of the nectaries were observed. In leek, sometimes supernumerary flower organs like ovules were formed in this callus. In onion (Allium cepa L.), plantlets developed from the ovules in all culture methods. Chromosome numbers of these plantlets were counted in root tip squash preparations. They were found to be haploid. Haploid plants were significantly smaller than diploid ones. They were transferred to soil and developed until bulb formation. Because of their importance for breeding, haploid plants obtained by gynogenesis are further stored in vitro.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Selection of a mutant from adventitious shoots formed in X ray treated cherry leaves and differentiation of standard and mutant with RAPDs

Summary An obstacle when using scions orin vitro shoots for mutation induction is the occurrence of chimeras. When adventitious shoots are formed from irradiated material these usually are derived from single cells, this leading to homohistont mutants. SincePrunus avium regenerates adventitious shoots from leaves at a low rate only (Yang & Schmidt, 1992), leaves of the interspecific cherry rootstock ‘209/1’ (P. cerasus ×P. canescens) were irradiated. ‘209/1’ regenerates adventitious shoots readily. Dosages applied were 5, 10, 20 and 40 Gy. Shoot production following 5 Gy irradiation was similar to the control. The application of 40 Gy resulted in strong damage with only few leaves regenerating. Among the adventitious shoots from leaves irradiated with 20 Gy one shoot was evident alreadyin vitro with thicker and smaller leaves having a serrate margin. It was cloned as ‘209/1-20m’. The clone stayed stable since 1990in vitro, in the greenhouse and the field. Compared with standard ‘209/1’ the mutant is very dwarf. Research to differentiate between standard ‘209/1’ and ‘209/1-20m’ was done using RAPDs. Among the decamer primer kits D, J, and T from Operon Technologies, Calif. Only primer OPJ05 (5‘CTCCATGGGG3’) differentiated between ‘209/1’ and ‘209/1-20m’. Rootstock ‘209/1’ showed one band of 2 kb additionally. This band is missing in the mutant.     

Heritability and gain from selection for field resistance against multiple root-knot nematode species (Meloidogyne incognita race 1 and M. javanica) in carrot

Heritability and gain from selection of traits associated with field resistance to multiple root-knot nematode species were estimated in carrot (Daucus carota L.). Experimental plots were uniformly and simultaneously infested with Meloidogyne incognita race 1 and M. javanica. Forty-seven half-sib families derived from the cv. ‘Brasília’ were evaluated for: (1) percentage of commercial roots with non-galling symptoms (%RNG); (2) percentage of commercial roots with gall symptoms (%RGS) and (3) percentage of non-commercial roots (%NCR). The cultivars ‘Brasília’ (resistant), ‘Carandaí’ (intermediate) and ‘Nova Kuroda’ (susceptible) were employed as standards. Broad-sense heritability estimates were 61.9 % for %RNG; 30.6 % for %RGS and 67.9 % for %NCR. However, the gains from selection were very small for all traits. The genotypic correlation between %RGS and %NCR was –0.38 and the correlation between %RNG and %RGS was 0.13. Selection on increasing %RNG resulted in a negative correlated response on %NCR (genotypic correlation = –0.99) indicating repulsion phase linkage(s) between the genetic factor(s) controlling these traits. The phenotypic correlation of –0.88 between %RNG and %NCR suggested that incomplete penetrance and dosage effects might be involved in the expression of these traits. Selection of superior genotypes (i.e. high %RNG, low %RGS, and low %NCR) can be achieved. ‘Brasília’ was among the genotypes selected for the following cycle of recombination reinforcing the notion that this cultivar is one of the most promising sources of stable, wide-spectrum field resistance to Meloidogyne species in D. carota. This revised version was published online in July 2006 with corrections to the Cover Date.