New types of major anthocyanins detected in Japanese garden iris and its wild forms

The anthocyanins of 130 cultivars, 13 lines and 3 wild forms of Iris ensata were analyzed by HPLC, and these plants were classified into 16 types of major anthocyanins. Among these types, 8 types such as petunidin 3RGac5G – delphinidin 3RGac5G, delphinidin 3RGac5G – petunidin 3RGac5G, cyanidin 3RGac5G – peonidin 3RGac5G, delphinidin 3RG – delphinidin 3RGac, petunidin 3RG5G – malvidin 3RG5G, malvidin 3RG5G – peonidin 3RG5G, peonidin 3RG5G – cyanidin 3RG5G and peonidin 3RG – cyanidin 3RG were obtained as new types. In these new types, peonidin 3RG – cyanidin 3RG and peonidin 3RG5G – cyanidin 3RG5G types were noteworthy because cyanidin 3RG and cyanidin 3RG5G are useful for the breeding of red flowers in I. ensata. This revised version was published online in July 2006 with corrections to the Cover Date.     

葡萄花色苷检测技术及抗氧化作用研究进展

为了进一步分析葡萄所含花色苷种类及功能,为相关研究者花色苷提纯鉴定及功能性研究等提供思路,本研究系统总结了葡萄花色苷提纯、定性定量鉴定及抗氧化作用,发现葡萄花色苷资源丰富,具有很强的抑制氧化修饰作用,可以从根本上清除DPPH、O2-·、·OH、H2O2等活性自由基,同时,对脂质过氧化有较好的抑制作用,部分种类可以有效减少动脉粥样硬化、心脑血管疾病、肝脏损伤、急性肺损伤等疾病的发生。而在提纯方式方面,得出超声波辅助溶剂提取法具有提取率高、时间短、能耗小等优点,提取工艺具有广阔的应用前景;HPLC-MS法是葡萄花色苷定性鉴定中使用最广泛的分析方法,可以准确检测出葡萄中所含花色苷的种类、含量和比例。因此,建议在提纯鉴定过程中要综合联用溶剂提取、超声波辅助溶剂提取、酶法提纯、HPLC-MS法等提纯鉴定方法。     

夏黑葡萄花色苷定性检测及组成成分分析

为研究夏黑葡萄花色苷的组成成分特点,本试验以夏黑葡萄皮为检测试材,以甲酸水溶液提取花色苷,利用高效液相色谱+飞行时间质谱和液质联用仪(UPLC-Q-TOF-MS)对夏黑葡萄未知花色苷的种类与含量进行测定。结果表明,夏黑葡萄成熟期果皮中检测到10种不同结构的花色苷,其糖苷主要以葡糖苷为主,但也存在鼠李糖苷、二葡糖苷。10种花色苷中有3种以锦葵色素为糖苷配基,有2种以芍药素为糖苷配基,有2种以飞燕草色素配基。其中锦葵色素-3-葡萄糖苷在总的花色苷含量中比例最大,为29%,其次是锦葵色素-3-(6-香豆基)-5-葡萄糖苷,所含比例为26%,芍药素-3-葡萄糖苷-5-葡萄糖苷第3位,为17%,飞燕草色素-３-葡糖苷第4位,为10%,锦葵素-3-葡萄糖苷-5-葡萄糖苷第5位,为9%。可见,夏黑葡萄含有花色苷资源相当丰富,自然界中常见的6种基本花色苷,夏黑葡萄占有3种。     

Preharvest application of abscisic acid promotes anthocyanins accumulation in pericarp of litchi fruit without adversely affecting postharvest quality

Pericarp colour of litchi fruit is an important quality attribute that determines its market value and consumer acceptance. Plant growth regulators (PGR) such as abscisic acid (ABA) and ethephon are known to play important roles in peel colour development during maturation and ripening of non-climacteric fruits (e.g. grape and litchi). Our aim was to investigate the effects of preharvest application of ABA, ethephon and their combination on pericarp colour and fruit quality of litchi (cv. Calcuttia) and also to assess the potential effects on postharvest performance of fruit. Exogenous application of ABA (150 or 300 mg L⁻¹) at the colour-break stage significantly increased the concentration of total anthocyanins and cyanidin-3-O-rutinoside, the major anthocyanin contributing ∼71–96% of the total anthocyanins, in litchi pericarp compared to ethephon (500 μL L⁻¹). Among different anthocyanins quantified, the relative contribution of cyanidin-3,5-diglucoside to the total anthocyanins was significantly higher in all PGR-treated fruit compared to the control, but the concentration of cyanidin-3-O-glucoside was specifically enhanced by ABA. No significant effect on the concentrations of epicatechin, and quercetin-3-O-rutinoside was observed in response to PGR treatments. Ethephon (500 μL L⁻¹) treatment did not significantly increase the anthocyanin levels in pericarp, but it caused more degradation of chlorophyll pigments than control. Aril quality with regard to firmness, soluble solids and acidity was not significantly affected by PGR treatments, except that ethephon-treated fruit showed significant softening and lower acidity. Postharvest changes in fruit quality attributes including pericarp browning during cold storage at 5 °C for 14 d were mainly related to the storage duration effect, rather than PGR treatment. In conclusion, ABA treatment (150 or 300 mg L⁻¹) at the colour-break stage enhanced anthocyanins accumulation in litchi pericarp without adversely affecting postharvest quality and storage stability for 14 d.     

Changes in anthocyanin and isoflavone concentrations in black seed-coated soybean at different planting locations

This study assessed the altitudinal variations in the anthocyanin and isoflavone contents of six black seed coated soybean [Glycine max (L.) Merrill] cultivars. The black soybean cultivars Heugcheong, Seonheuk, Geomjeong 1, Geomjeong 2, Cheongja 2, and Cheongja 3 were planted at Milyang (12 m above mean sea level — low altitude) and Muju (600 m — high altitude), Korea on 10 June 2005 and 2006. The total anthocyanin and isoflavone contents and individual components were investigated by reverse-phase high performance liquid chromatography (HPLC). All black soybean cultivars cultivated in high altitude possessed significantly higher total anthocyanin (p < 0.01) and isoflavone (p < 0.01) contents than those grown in low altitude. For anthocyanin composition, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and peonidin-3-O-glucoside contents were significantly higher while delphinidin-3-O-glucoside contents was significantly lower at high altitude. The composition of individual isoflavones, 6″-O-malonyldaidzin, and 6″-O-malonylgenistin contents significantly increased at high altitude.     

P-coumaroyl glycosides of cyanidin and peonidin in the flowers of Japanese garden iris,Iris ensata Thunb.

Summary The anthocyanins in outer perianths of seven varieties and one telotrisomic line of Japanese garden iris, Iris ensata Thunb., were analysed by high-performance liquid chromatography procedures. Two new anthocyanins, peonidin 3RGac5G and cyanidin 3RGac5G were putatively detected in this species, and peonidin 3RGac5G was the major anthocyanin for all the varieties except Telotri-1 and cyanidin 3RGac5G the major anthocyanin for Telotri-1. Moreover, cyanidin 3RGac5G and peonidin 3RGac5G are useful anthocyanins for the breeding of red and magenta flowers. Finally, the breeding strategy for new flower varieties using cyanidin 3RGac5G and peonidin 3RGac5G was discussed.Contribution from the Laboratory of Plant Breeding, Faculty of Agriculture, Miyazaki University, Japan, No. 84.     

Identifying apomixis in matroclinal progeny from an interspecific crossing between <Emphasis Type="Italic">Iris domestica</Emphasis> and three different colors of <Emphasis Type="Italic">Iris dichotoma</Emphasis>

In a previous investigation on the reciprocal difference of interspecific hybridization between three different flower colors of Iris dichotoma and Iris domestica in the F₁ offspring from crosses where I. domestica was a maternal parent were similar in morphological and cytological characters to their maternal parent. This could be evidence of apomixis; however, matroclinal progeny with complete morphological similarity to the maternal parent could be attributed to the heterozygosity for these characters in the pollen parent. The F₁ plants were investigated in order to identify apomixis in I. domestica. Four matroclinal plants were randomly selected from each F₁ population produced from Iris domestica × Iris dichotoma that had three different colors of flowers and were allowed to self-pollinate to establish an F₂ population. All of the F₂ plants had no segregation to I. domestica in their morphological characters. In addition, 13 reciprocal F₁ plants were detected by 25,719 single nucleotide polymorphism (SNP) markers. When I. dichotoma plants with three different flower colors were used as maternal parents, all the progenies were genuine hybrids. When I. domestica were used as maternal parents, all the F₁ plants were apomictic progenies. Apomixis of I. domestica was successfully identified and SNP markers identified F₁ hybrids derived from six interspecific crosses between I. dichotoma and I. domestica, which provides a reference for authenticating offspring identity during Iris cross breeding in the future.     

Inheritance of anthocyanin content in the ripe berries of a tetraploid × diploid grape cross population

Variation patterns and inheritance of anthocyanin content in the ripe berries of a tetraploid × diploid table grape cross population were investigated in two successive years. The population segregated for three different ploid levels: dipolids, triploids, and tetraploids. A total of 28 different anthocyanins were detected and quantified in the progeny population. Transgressive segregation for the total anthocyanin content was observed in all the three ploid progeny populations. The total anthocyanin content increased as the ploid level increased. The broad sense heritabilities (H²) of the total anthocyanin content were all relatively high, ranging from 0.53 to 0.98, 0.57 to 0.97 and 0.43 to 0.94 in the diploid, triploid and tetraploid population, respectively. Our results suggested that the total anthocyanin content followed an additive inheritance model in this polyploid segregation population. We also observed that the relative contribution of individual anthocyanins to the total anthocyanin content varied significantly among different ploid populations, suggesting that genetic background has important impact on the accumulation of the individual anthocyanin compounds. These results will help develop better breeding strategies in a polyploid table grape breeding program for improving the content of anthocyanins, an important class of polyphenolics possessing antioxidant activities and many other health-related benefits.     

Stability of flower colors due to anthocyanin-flavone copigmentation in Japanese garden iris, Iris ensata Thunb.

The fading of flower color in bluish purple and reddish purple cultivars of Iris ensata and the in vitro stability of malvidin 3RGac5Gand petunidin 3RGac5G due to copigmentation with isovetixin under different pH conditions were examined. The bluish purple cultivars exhibited higher flower color stability than the reddish purple cultivars 2 days after anthesis. In the absence of isovitexin, malvidin 3RGac5G and petunidin 3RGac5G were not able to maintain color stability except at low pH. However, the color stability of malvidin 3RGac5G and petunidin 3RGac5G was increased by copigmentation with isovitexin under all pH conditions tested. Most remarkable was the stabilization of both anthocyanins due to the copigmentation at pH 4.2–6.2. Therefore, it can be concluded that the stability of flower color in the bluish purple cultivars of malvidin 3RGac5Gand petunidin 3RGac5G type of I. ensata is caused at least in part by the copigmentation between these anthocyanins and isovitexin. This revised version was published online in July 2006 with corrections to the Cover Date.     

The characterization of autoallotetraploid hybrids between Iris ensata Thunb. and I. laevigata Fisch.

The characteristics of autoallotetraploid hybrids obtained from the cross between Iris ensata cv. Raspberry Rimmed (4X) and amphidiploids of I. laevigata × I. ensata were examined and compared with those of their parents. The color of inner and outer perianths in the autoallotetraploids were bluish purple and similar to those of the amphidiploid parent. However, the autoallotetraploids exhibited low pollen fertility. In addition, the autoallotetraploids were characterized by 17 or 19 anthocyanins and had high resemblance to their parents in the anthocyanin expression. Among these anthocyanins, malvidin 3RGac5G and petunidin 3RGac5G were regarded as major anthocyanins in the autoallotetraploids and their parents, but the differences in the ratios of malvidin 3RGac5G:petunidin 3RGac5G between the autoallotetraploids and their parents were ca. 2:1 for the former and ca. 1:1 for the latter. No viable hybrid seeds were obtained from the reciprocal crosses between I. ensata (2X and 4X) and the autoallotetraploids. Finally, the interspecific cross-breeding of I. ensata using the autoallotetraploids is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Girdling and gibberellic acid effects on yield and quality of a seedless red table grape for saving irrigation water supply

Table grapes need of substantial water supply for achieving commercial requirements. Viticulture practices such as girdling (G) and gibberellic acid (GA) application, as well as water supply, can improve table grape quality. The study, which was conducted in two consecutive seasons (2010–2011), aimed to assess the counterbalance effect of these viticulture practices on yield and quality parameters and flavonoids composition in case of a significant and unusual irrigation water reduction (40%) applied to a table grape variety. The data from the two-way ANOVA and PCA analyses indicated that viticulture practices were clearly related to anthocyanins and flavonols variations whereas water management appeared mainly involved in the yield parameters variability. Specifically, the reduced water supply (RWS) decreased the grape yield (−20%) with respect to full water supply (FWS); by contrast GA, G, and G × GA treatments determined an increment of grape production ranging from 10 to 23%, independently from irrigation strategy. Moreover, G, in particular applied to RWS grapes, was able to improve the total soluble solids over titrable acidity (TSS/TA), a ratio strictly related to the quality perception by the consumer. Total anthocyanins were found positively linked to FWS whereas flavanols content was indifferent to water management. Instead viticulture practices seemed to have a greater impact on anthocyanin composition, considering that in RWS grapes under GA condition, higher contents of malvidin and peonidin (mainly responsible for the color stability of the skins) were revealed. Furtheremore, catechins and rutin appeared significantly enhanced by G and G × GA, and GA, respectively. From gathered findings, it can be concluded that suitable viticulture practices can allow a sensible reduction of water supply during table grape growth cycle without detrimental effects on yield and quality.     

Individual anthocyanins and their contribution to total antioxidant capacity in response to low temperature and high CO2 in stored Cardinal table grapes

In this study, we have analyzed the profiles of individual anthocyanins in the skin of Cardinal table grapes and their contribution to the total antioxidant capacity (TAC) in response to low temperature (0 °C) and high CO₂ levels (20% for 3 days). An analysis of the representative colour parameters of this red-violet variety was also determined. The anthocyanin composition was determined using high-performance liquid chromatography coupled to diode array detector and mass spectrometry (HPLC–DAD–MS). The contribution of individual anthocyanins to the TAC value of table grapes was calculated on the basis of their concentration and antioxidant capacity measured as the TEAC value (slope of the anthocyanin/slope of Trolox). Chromatographic analysis identified six anthocyanins, including pelargonidin 3-glucoside (Pg-3-G), in the skin of Cardinal table grapes. Short-term storage at 0 °C in air had an increasing effect on the concentration of each of the identified anthocyanins. After 3 days at 0 °C, untreated grapes had the highest anthocyanin content (27.55 mg/100 g FW) and displayed the largest TAC value (52.45 mM TE/100 g FW). Peonidin 3-glucoside (Pn-3-G) was the predominant anthocyanin, and it was mainly responsible for the rise in the calculated TAC value in untreated grapes. Pn-3-G had a low average TEAC value (1.73 mM), but its contribution could be explained by the sharp increase in the content of this anthocyanin the first days of storage at 0 °C. In contrast, the Pn-3-G content in grapes at the end of the 3-day CO₂ treatment did not change, and both the total anthocyanin content and the calculated TAC value remained significantly constant in comparison to the levels in pre-stored grapes. In addition CO₂ treatment had a positive effect on the amount of Pg-3-G concomitant with a pronounced decline in delphinidin 3-glucoside (Dp-3-G) and smaller decreases in petunidin 3-glucoside (Pt-3-G) and malvidin 3-glucoside (Mv-3-G). These effects of high CO₂ levels on the profile of individual anthocyanins were progressively lost when grapes were transferred to air. Indeed, after prolonged low-temperature storage when the colour of the berries become dark violet, similar calculated TAC values were quantified in untreated grapes and in those exposed to 20% CO₂. These data provide new insights about the effect of low temperature and high CO₂ levels on the concentration of the individual anthocyanins identified in cardinal table grapes.     

Sub-arm location of prolamin and EST-SSR loci on chromosome 1H<Superscript>ch</Superscript> from <Emphasis Type="Italic">Hordeum chilense</Emphasis>

Hordeum chilense Roem. et Schult. is a diploid wild South American barley that contains genes of interest for cereal breeding, many of them located on chromosome 1H^ch. In the current study, two H. chilense-wheat addition lines with deletions in the 1H^ch chromosome were used for sub-arm localization of five prolamin (glutenin and gliadin) loci and 33 EST-SSR marker loci on chromosome 1H^ch. The two sets of markers were distributed across five sub-arm chromosome regions. Three glutenin loci (Glu-H ^ch 2, Glu-H ^ch 3, Glu-H ^ch 4) together with the gliadin locus Gli-H ^ch 1 were located on the distal 20% of the 1H^chS arm, whereas the glutenin locus Glu-H ^ch 1 was on the proximal 88% region of 1H^chL. Among 33 EST-SSR marker loci, 7 (21.2%) were on the 1H^chS arm and, of them, 3 (9.1%) were on the distal 20% end and 4 (12.1%) on the proximal 80% region. The 26 loci (78.8%) on 1H^chL were distributed across three different regions: 18 (78.8%) in the proximal 88%, 3 (9.1%) in the distal 12% and 5 (15.2%) in a region less than 12% from the distal end. The deletions in the 1H^ch chromosome added to the common wheat background were thus shown to be useful for determining the sub-arm location of EST-SSR and prolamin loci. This could facilitate the identification of molecular markers linked to genes of agronomic interest and the isolation of such genes for use in common wheat improvement.     

Genetic analysis of resistance to <Emphasis Type="Italic">Fusarium</Emphasis> root rot in common bean

Fusarium root rot (FRR) is a major disease of common bean worldwide. Knowledge of the inheritance of resistance to FRR would be important in devising strategies to breed resistant varieties. Therefore, a 12 × 12 full diallel mating scheme with reciprocal crosses was performed to generate 132 F₁ progenies, which were then advanced to the F₃. The progenies were evaluated for resistance to FRR under green house conditions in Uganda. General combining ability (GCA) effects were highly significant (P ≤ 0.01) for disease scores. Specific combining ability effects were not significant (P > 0.05) in the F₁, but were highly significant (P < 0.01) in the F₃ generation. These results indicate that resistance to FRR was governed by genes with additive effects in combination with genes with non-additive effects. Reciprocal differences were also significant (P = 0.01) at F₁ and F₃, primarily reflecting a large influence of maternal effects in both these generations. In fact, susceptible parents did not differ significantly (P > 0.05) for disease scores when used as paternal parents in the F₃, but differed strongly as maternal parents (P = 0.0002). Generally, the progenies were distinctly more resistant when the resistant parent was used as the female in crosses, especially as observed in the F₃. The maternal effects were strong in the F₃ generation, suggesting a complex form of cytoplasmic–genetic interaction. The non-maternal reciprocal effects in the F₃ were significant (P < 0.05) in both the resistant × resistant diallel, and in the resistant × susceptible crosses. Mid-parent heterosis (MPH) occurred in most crosses, with average heterosis approximately equal in each of the three generations, indicating that epistasis was probably more influential than dominance of individual genes. Gene-number formulas indicated that several genes were involved in resistant × susceptible crosses. Among resistant × resistant crosses, many produced continuous distributions of F₁ progeny scores, suggesting polygenic inheritance, while bi-modal distributions were characteristic of the F₃ distributions, and fit expected ratios for two or three loci segregating in each cross. Dominant forms of epistasis favoring resistance were strongly indicated. Parent–offspring heritability estimates were moderate. Overall, the results indicate that resistant parents contain a number of different resistance genes that can be combined with the expectation of producing strong and durable resistance. The lines MLB-49-89A, MLB-48-89, RWR719 and Vuninkingi, with large and negative GCA effects, contributed high levels of resistance in crosses and would be recommended for use in breeding programs.     

High-performance liquid chromatographic analysis of anthocyanins in induced amphidiploids of Iris laevigata Fisch. × I. ensata Thunb.

Summary Anthocyanins of colchicine-induced amphidiploids of I. laevigata × I. ensata were analyzed by the HPLC procedure and compared with those of parental species and F₁ hybrids of I. laevigata × I. ensata. The amphidiploids were characterized by seventeen anthocyanins, and exhibited all anthocyanins of the parental species and the F₁ hybrids. Malvidin 3RGac5G and petunidin 3RGac5G in anthocyanins detected were major pigments in the amphidiploids, which exhibited strongly the malvidin 3RGac5G-petunidin 3RGac5G type of I. ensata rather than the petunidin 3RGac5G-malvidin 3RGac5G type of I. laevigata. Moreover, a higher degree of anthocyanin content was observed in the amphidiploids and the F₁ hybrids. Namely, the anthocyanin content of the amphidiploids and the F₁ hybrids were 2.81 and 2.45 times as much as that of the mid-parent. This implies that there is a complementary effect on anthocyanin synthesis of parental genes in them. Finally, the utility of the amphidiploids was discussed.Contribution from the Laboratory of Plant Breeding, Faculty of Agriculture, Miyazaki Univ., No. 64.     

Phenotypic segregation and relationships of agronomic traits in Monastrell × Syrah wine grape progeny

The aim of this work was to study the phenotypic segregation of different agronomic and fruit quality traits, and their relationships, in Monastrell × Syrah wine grape progeny. Twenty-two agronomic traits were evaluated and compared for three consecutive years in this progeny. The results show the phenotypic diversity existing in a cross between two different wine grape cultivars. Most of the phenological, productive, morphological, and enological parameters evaluated displayed continuous variation within the progeny, suggesting a polygenic inheritance. Some correlations between traits were detected by the Spearman correlation test, although high coefficients were not found for most of them. Cluster analysis of the progeny grouped the hybrids based on criteria with significance for wine grape breeding. Also, we investigated the relationship between the skin color and total content of anthocyanins with the VvmybA genotype, using the CAPS (Cleaved Amplified Polymorphic Sequence) marker 20D18CB9. The results show that hybrids with two copies of the functional color allele tend to have increased anthocyanins content. Based on this study, 14 genotypes were pre-selected from the breeding population for additional quality studies.     

Pyramiding of two bacterial blight resistance and a semidwarfing gene in Type 3 Basmati using marker-assisted selection

A traditional Type 3 Basmati rice cultivar grown in India is tall and lodges even under low nitrogen fertilizer dose. In addition to lodging, it is highly susceptible to several diseases and pests including bacterial blight (BB). BB resistance genes (Xa21 and xa13) and a semidwarfing gene (sd-1) were pyramided in Type 3 Basmati from a rice cultivar PR106-P2 using marker-assisted selection (MAS). Foreground selection for BB resistance genes, Xa21 and xa13 and reduced plant height gene, sd-1 was carried on the basis of linked molecular markers pTA248, RG136 and ‘h’, respectively. The BC₂F₃ progenies with both the BB resistance genes were highly resistant with lower lesion length than either of the genes individually. Background profiling of the selected 16 BC₂F₃ progenies was done using 95 anchored SSR and 12 ISSR markers. Among the selected 16 BC₂F₃ progenies, 38-5-2 and 38-5-36 closely clustered along with the recipient parent Type 3 Basmati showing above 85% genetic similarity with the same. Further selection was continued till F₅ generation for higher recovery for Type 3 Basmati characteristics. The desirable alleles of intermediate amylose content (wx) and aroma (fgr) loci of Type 3 Basmati were also tracked using the linked SSR markers. The BC₂F₅ pyramid lines T3-4, T3-5, T3-6 and T3-7 homozygous for the three target genes Xa21, xa13 and sd-1 from the donor parent with wx and fgr alleles of Type 3 Basmati had excellent cooking quality and strong aroma.     

Application of C-banding and fluorescence in situ hybridization for the identification of the trisomics of Hordeum chilense

Hordeum chilense is a wild barley species that has a high degree of genetic variability and significant potential for use in plant breeding. To establish a series of trisomics in H. chilense (2n = 14), plants with 2n + 1 chromosome numbers were isolated from the progenies of selfed triploid plants. Based on both fluorescent in situ hybridization with pAs1 and pTa71 repetitive DNA probes and C-banding patterns, seven different trisomics were tentatively identified. Primary trisomic plants were for chromosomes 1H^ch, 4H^ch, 5H^ch, 6H^ch and 7H^ch. A secondary trisomic carrying a 5H^chS-5H^chS isochromosome as the extra chromosome and a trisomic for chromosome 3H^ch heterozygous for the 3H^chS-4H^chL and 4H^chS–3H^chL interchange were identified. The trisomic for chromosome 1H^ch cannot be phenotypically distinguished from the diploid. The rest of the trisomic types were distinguishable from the diploid by their morphological characteristics (relatively poor vigour, decreased size and shorter spikes) but they were morphologically indistinguishable from each other. The frequencies of trisomics among the progenies derived from self-fertilization of these aneuploids ranged from 10.7% to 37.5%, with an average frequency of 26.1%. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis

Intergeneric sexual hybridizations were conducted between two genera of Rutaceae, with Satsuma mandarin (Citrus unshiu Marc) and Red tangerine (C. reticulata Blanco) as maternal parents, and Poncirus trifoliata (L.) Raf as the paternal parent, in an effort to generate hybrid populations for both molecular mapping and rootstock breeding. Embryo rescue is important for citrus sexual breeding because polyembryony can interfere with hybrid embryo recovery. Immature embryos of 80, 85, 90 days after pollination (DAP) from the Satsuma mandarin (S) × trifoliate orange (P) cross, 80 and 85 DAP from the Red tangerine (R) × trifoliate orange (P) cross, were cultured on MG1.0 medium consisting of MT basal medium supplemented with 1.0 mg l⁻¹ GA₃ and 4% sucrose. The results showed that 80 DAP was the optimal time for embryo rescue of the tested crosses, as evidenced by embryos at this stage exhibiting high germination rates, 37.3% for S × P and 51.3% for R × P. Among the eight tested media, MT medium supplemented with 0.5 mg l⁻¹ GA₃ was the best one for Satsuma mandarin, and MT plus 1.0 mg l⁻¹ GA₃ for Red tangerine. A total of 85 plants were obtained from S × P, and 340 from R × P. Out of them, 44 progenies from S × P and 111 from R × P were sufficiently confirmed to be hybrids by morphological characterization and SSR analysis. In addition, two hybrid callus lines were obtained from S × P and R × P respectively.