Anthers from more than 17000 flowers of 19 bell pepper Capsicum annuum L. hybrids (provided by `Semillas Fitó S.A.') were cultured in a double layer modified H medium (Nitsch and Nitsch, 1969) supplemented with 0.5 % activated charcoal and 0.26 % Gelrite in the solid phase. Significant differences between genotypes were observed on embryogenesis (472.3 to 9.7 embryos / 100 flowers) and number of plants rescued (4.0 to 0.3 plants / 100 flowers). Trying out maltose, malt extract, and sucrose. as carbohydrates, at 20, 40, 60 or 80 g/l, gave significantly better results for maltose (20 or 40 g/l). In addition, maintaining the anther cultures in an atmosphere enriched with 600 ppm CO2 was beneficial for embryo number, embryo development and number of rescued plants. Isocitrate dehydrogenase zymograms from leaf extracts indicate the microspore origin of the acclimated plants. Flow citometry of nuclei was used to determined an early diploidization of 70 % of the acclimated plants.
Ramon Dolcet-Sanjuan and Elisabet Claveria
Agustin Huerta and Ramon Dolcet-Sanjuan
Adventitious shoots and viable plants were regenerated from bell pepper (Capsicum annuum L.) cultivars and dihaploid lines (DHLs) obtained from F1 hybrids via androgenesis (Dolcet-Sanjuan et al., in press). Hypocotil and cotyledon sections from in vitro-germinated seeds were used as explants. A modified MS medium (Murashige and Skoog, 1962) supplemented with IAA (0 to 3.2 μM) and BAP (0 to 100 μM) was used in a 3-week-long shoot primordia induction phase. Shoot elongation was best performed in the same basal medium, but supplemented with silver thiosulfate and GA3. Shoots were regenerated from eight selected DHLs (`C213', `C215', `C218', `C2123', `C2125', `C3111', `C3113', and `P493') and two cultivars (`Padrón' and `Yolo Wonder'). The percentage of cotyledon sections with shoot primordia after the induction phase was not genotype-dependent and always higher than with hypocotil sections (93.4% and 17.9%, respectively). The number of shoot primordia per responsive cotyledon section was also higher than with hypocotil sections (3.3 and 1.7, respectively). The genotype had a significant effect on the number of shoots regenerated per responsive cotyledon (1.1 to 5.5) or hypocotil (0.5 to 3.5) section. All adventitiously regenerated plants were fertile. This adventitious shoot regeneration protocol is being used to obtain transgenic plants from sweet bell pepper genotypes.
Elisabet Claveria and Ramon Dolcet-Sanjuan
Micropropagation of Pistacia vera L. `Mateur' was improved with the addition of methyl jasmonate (MeJA) to the multiplication and rooting media. Shoot tip cultures established from grafted trees were maintained on a modified Murashige and Skoog medium containing 5μM BA and 0.05μM IBA. Addition of 1μM MeJA improved the multiplication rate but inhibited shoot growth when present at higher concentrations. Rooting experiments comparing the effects of IAA, NAA, or IBA at 0, 1, 3.2, 10, or 31.6 μM demonstrated a significant effect of temperature on auxin root induction for shoots maintained at 25 or 28°C. At 25°C NAA was better than IAA or IBA, whereas no differences among auxins were observed at 28°C. Addition of MeJA (0, 0.3, 1, 3.2, or 10 μM) to the best rooting media significantly improved the rooting percentage and root number. Greater than 80% rooting was obtained when 1 μM MeJA was added to both the root induction medium, containing 31.6 μM NAA, and the auxin-free medium. In addition, transfer to soil and acclimation was easier for plantlets rooted in MeJA-containing medium.
Ramon Dolcet-Sanjuan and Elisabet Claveria
Micropropagation of Pistacia vera `Mateur' was improved by adding MeJA to the multiplication and rooting media. Shoot-tip cultures established from grafted trees were maintained on a modified Murashige and Skoog medium containing 5 μm BA and 0.05 μm IBA. Adding 0.3, 1, or 3.2 μm MeJA improved shoot multiplication rates 2.5, 3.0, and 2.3, respectively. There was a significant interaction between the effects of auxin and temperature on the percentage of shoots forming roots. At 25C, the percentage of shoots forming roots was higher in the presence of NAA than IAA or IBA, whereas, at 28C, there was no difference among the auxins. Adding MeJA to the best auxin treatments-31.6 μm NAA at 25C and 31.6 μm IAA at 28C-increased the percentage of shoots forming roots and number of roots per shoot but decreased root length. More than 80% of the shoots rooted at 25C when 1 μM MeJA was added to the root induction medium, which contained 31.6 μm NAA, and the root elongation medium, without auxin. The large number of short roots induced by MeJA facilitated plantlet transfer to soil and acclimation. Chemical names used: methyl jasmonate (MeJA); N6-benzyladenine (BA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), indole3-acetic acid (IAA).
Ramon Dolcet-Sanjuan, Elisabet Claveria, and Agustin Huerta
A new and simple protocol for androgenesis in bell pepper is described. The initial medium, a modification of Nitsch and Nitsch's H medium, consisted of a two-phase system of semi-solid and liquid medium and contained maltose as carbon source. The total number of embryos formed was greater with maltose at 40 g·L-1, but embryos developed better at 10 to 20 g·L-1. Depending on the genotype, the number of embryos and plants recovered ranged from 3 to 750 and 0.25 to 8, respectively, per 100 flowers. Further increases in the number of embryos (up to 3561 per 100 flowers) and plants (up to 23 per 100 flowers) could be attained by flushing cultures with air enriched with CO2 at 900 μL·L-1. The ploidy level and the microspore origin of the recovered plants were determined by flow cytometry and zymograms for isocitrate dehydrogenase. Nearly 65% of the acclimated plants had undergone spontaneous doubling of the chromosome number, as confirmed by flow cytometry of leaf nuclei. Isocitrate dehydrogenase zymograms demonstrated that plants originated from microspores and that the two parental alleles were equally represented among the haploid and dihaploid plants.
Ramon Dolcet-Sanjuan, Elisabet Clavería, Alfonso J. Rodríguez, and Marta Llaurado
Callus and shoot organogenesis were obtained from anthers of Dianthus caryophyllus L. `Manon', `Amapola', `Elsy', and `IB212', harboring mid-uninucleated microspores. Significant differences between genotypes were observed on number of responsive anthers (10.4% to 72.1%) and rescued plants per responsive anthers (1.2% to 4.8%). A modified H medium (Nitsch and Nitsch, 1969) with 20 g/L maltose and 0.25% Gelrite, supplemented with 10 μM 2,4-D and 1 μM TDZ, was most appropriate for callus induction. Plants were regenerated after subsequent subculture to the same medium, but amended with 0.1 μM TDZ. Zymogram types for aminopeptidase (AAP) in polyacrilamide gel electrophoresis proved that all 40 plants regenerated from `Amapola', `Elsy', or `IB212' where heterozygous, and consequently not originated from the microspores but from somatic tissue. Alternatively, in situ-induced parthenogenesis through pollination with gamma-irradiated pollen and in vitro embryo rescue was tested. A total of 92 embryos, including normal and no cotyledonary embryos, were rescued from 38 fruits harvested out of 70 crosses between `Scania' and `Amapola'. Embryos were rescued 21 to 28 days after pollination by culture in a modified E20A (Sauton and Vaulx, 1987) medium. Phosphogluco isomerase (PGI) and Shikimic dehydrogenase (SDH) zymograms in starch gel electrophoresis, and AAP in polyacrilamide gel electrophoresis, indicated the parthenogenic origin of three of the regenerated plants. Flow cytometry of nuclei proved the early diploidization, during in-vitro micropropagation, of the parthenogenic carnation haploid plantlets.
Elisabet Claveria, Jordi Garcia-Mas, and Ramon Dolcet-Sanjuan
Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of selected lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μm colchicine. Rescue of diploid or chimeric plants was shown by flow cytometry, prior to their acclimation and planting in the greenhouse. Selfing of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.
Ignasi Iglesias, Pere Vilardell, Joan Bonany, Elisabet Claveria, and Ramon Dolcet-Sanjuan
A new spontaneous mutation of the pear variety Dr. Jules Guyot, named `IGE 2002', was selected from a pear growing area in Catalonia. The clone was established in vitro from a 40-year-old tree, a highly recalcitrant material unable to root by cuttings. An in vitro micropropagation protocol, with an average multiplication rate of 5, a 90% rooting, and an acclimation of 79% of the plantlets, was defined. Self-rooted plants were grown in two experimental stations, covering two distinct fruit growing areas. The main agronomic characteristics of the clone `IGE 2002' were evaluated during six seasons, 1997 to 2002. Blooming and harvest period were at a similar time than `Dr. Jules Guyot'. Soluble solids concentration and acidity are also similar to `Dr. Jules Guyot'. However, at the same harvest time, a lower fruit firmness of `IGE 2002' in comparison to `Dr. Jules Guyot' indicated an advanced ripening. In addition, a finer flesh texture of `IGE 2002' than `Dr. Jules Guyot', distinguished the former from the later variety. Important differences between both plot sites were found on cumulative fruit yield, fruit size, and fruit size distribution, of `IGE2002' grown on its own roots. However, the site did not affect the fruit quality parameters. Superior fruit yields were associated with higher vigor and yield efficiency of the self-rooted variety.
Sumonthip Bunnag, Ramon Dolcet-Sanjuan, David W.S. Mok, and Machteld C. Mok
Somaclonal quince (Cydonia oblonga Mill.) variants IE-1 and IE-2 (Dolcet-Sanjuan et al., 1992) were more tolerant to Fe-deficient conditions in vitro than the original clone Quince A. The tolerance was evidenced by higher chlorophyll concentrations in leaves, higher Fe(III) reduction in roots, and increased acidification of the medium. In Summers 1993 and 1994, the two variants and Quince A were compared in the greenhouse, grown in normal potting soil (pH 5) and in soil obtained from an Fe-deficient orchard, with and without the addition of lime and KHCO3 solution to increase the pH (up to pH 8.3) and intensify Fe stress. In both years, the variants had significantly higher leaf chlorophyll concentrations than Quince A when grown in high-pH soils. The Fe+2 concentrations in leaves were also higher in the variants. Under field conditions at IRTA Reus, Spain, IE-1 and IE-2 had higher chlorophyll concentrations than the controls at some, but not all, analysis dates. These results suggest that IE-1 and IE-2, when grown as young plants in the greenhouse, have higher tolerance to Fe deficiency than Quince A, as observed in vitro, but that the tolerance is more variable under uncontrolled conditions in the field.
Ramon Dolcet-Sanjuan, Elisabet Claveria, Robert Gruselle, Adreas Meier-Dinkel, Christian Jay-Allemand, and Thomas Gaspar
Various factors were found to influence the in vitro induction and elongation of adventitious roots from walnut shoot microcuttings. Diverse walnut genotypes (Juglans regia, J. nigra × J. regia hybrids) and selected elite J. regia clones were micropropagated throughout the establishment of in vitro shoot-tip cultures. New evidence is presented here that demonstrates the importance of the genotype and juvenility of the plant material on the in vitro rooting ability. Selection of the best adapted genotypes to multiplication and rooting, and rejuvenation of mature clones through repetitive subcultures or micrografting were examined. Adult J. regia clones were rejuvenated through subsequent subcultures and their rooting was consequently improved. The same results were not accomplished by micrografting on juvenile shoots. A differential response to auxin type and concentration was observed for Juglans regia or J. nigra × J. regia clones. A short prerooting culture in multiplication medium, lowering the sucrose concentration in the root elongation medium and increasing the atmospheric carbon dioxide during the root elongation phase affected the number of shoots forming roots as well as the quality of plantlets and roots.