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Reduction to a haploid state followed by chromosome doubling is an established method for the production of inbred lines for diploid-hybrid true seed production. In potato, the focus on haploid induction has been to use haploid inducers to develop primary dihaploids from tetraploid breeding clones via maternal haploid induction. Further reduction of diploid breeding lines into true monoploids via haploid induction is also possible but has not been explored extensively. PL-4, a diploid potato line developed as an improved haploid inducer, was crossed as female in a limited number of crosses with a diploid breeding line derived from Solanum tuberosum Group Phureja-stenotomum. Embryo rescue resulted in two offspring below the guard cell count threshold, and SNP analysis identified one offspring as a monoploid derived from PL-4. [ABSTRACT FROM AUTHOR]

Embryo rescue is an effective method and has been widely applied in the breeding of seedless grape. In this study, we conducted eight cross combinations involving five stenospermocarpic seedless grape varieties, with the male parents being the variety ‘Beichun’ and a seedless variety ‘Venus Seedless’. The young fruits of the resulting hybrid plants were harvested at the optimal sampling time for each female parent and subjected to in vitro ovule culture. To enhance the efficiency of the seedless grape embryo rescue technology system, we conducted a comparative analysis on the impact of solid and solid-liquid diphasic MM3 embryo development medium, various parental genotypes, and the duration of ovule culture on embryo rescue. Additionally, marker assisted-selection (MAS), was employed on the hybrids using the seedless gene molecular marker SCF27-2000. Results revealed variability in the impact of medium phase on the successful rescue of embryos across different cross combination. Cultivars ‘Perlette’ and ‘Qinhong No.1’ emerged as suitable female parents for successful embryo rescue. Notably, the influence of the maternal genotype on embryo rescue exceeded that of the paternal genotype and medium phase. The rates of embryo development, growth, and seedling formation for the combinations ‘Perlette’ × ‘Venus Seedless’ and ‘Qinhong No.1’ × ‘Venus Seedless’ peaked at 10 weeks of ovule culture. A total of 894 hybrid strains resulting from eight cross combinations were obtained through embryo rescue, which 559 strains exhibiting a seedless marker, yielding an average seedless rate of 62.53%.Key message: The study investigated the influencing factors in embryo rescue for seedless grapes, resulting in the identification of 559 strains carrying the seedless gene molecular marker SCF27-2000. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicum L.) is the most important vegetable crop grown in India after potato. Root-knot nematodes (RKNs) (Meloidogyne spp.) cause yield loss between 11 and 35% in tomato. RKNs are also known to be involved in the tomato wilt disease complex in association with Fusarium oxysporum sp. lycopersici and Ralstonia solanacearum. The nematode resistance gene, Mi1 from Solanum peruvianum, is currently the only source of RKN resistance in modern tomato cultivars. Mi1 mediated resistance is temperature sensitive and ineffective above 28 °C soil temperature. Nematode resistance gene, Mi-9 found in S. arcanum accession LA2157 is heat-stable and is located on the short arm of chromosome 6. In our study, LA2157 was crossed with tomato cultivar Kashi Amrit. The inter-specific hybrid was successfully developed through embryo rescue. The hybridity of embryo rescued plants was confirmed by morphological features and molecular marker assay. LA2157 and F1 hybrid showed immune reaction to M. incognita under the high inoculation pressure of 2000s stage infective juveniles per plant, while cv. Kashi Amrit exhibited a highly susceptible reaction. Among hundred F3 plants from ten F2s, ten F3 plants each of two separate F3 families (43 and 119) have shown resistance and no segregation was observed within the family for resistance during April–May, 2020 (high temperature). All F4 plants derived from 43F3 also gave an immune reaction to M. incognita indicating homozygous nature of 43F2 plant for Mi-9 gene. We believe that this set of resistant plants can be utilized to transfer heat-stable nematode resistance in the background of cultivated tomatoes. Key message: Inter-specific tomato hybrid of Kashi Amrit and LA2157 was developed through embryo rescue from fruits of 33–34 days after pollination. The identified F4 family homozygous for Mi-9 can be used to transfer heat-stable nematode resistance to cultivated tomatoes. [ABSTRACT FROM AUTHOR]

Background: Turkey is one of the major exporters of mandarins in the Mediterranean region. Seedlessness in citrus, which is one of the most desired fruit quality traits, especially in fresh mandarin export markets, can be obtained via triploidy as in many fruit species. Triploid plants can be recovered by 2x × 2x hybridizations in citrus, as well as 2x × 4x and 4x × 2x crosses. Hence, we aimed to develop local triploid hybrids by using the embryo rescue technique in five crosses using eight different citrus varieties in the present study. Methods and results: Embryos isolated from abortive seeds derived by 135 days after pollinations were cultured on modified Murashige and Tucker (MT) medium by adding different levels of GA3 to achieve a high germination rate. A population of 574 plants was developed as a result of embryo rescue. After screening the ploidy levels of this 574-plant population with the aid of flow cytometry, 4 triploids from 'Encore' × 'Murcott', 8 triploids from 'Fortune' × 'Willow leaf', 1 triploid from 'Kiyomi' × 'Murcott', and 1 triploid from 'Ortanique' × 'Murcott' hybridization were recovered. Triploid hybrid plants and related parents were analyzed with SSR markers heterozygotic for parental mandarin varieties. In addition, we evaluated stomatal characteristics of diploid and triploid hybrids obtained from different crosses. Stomatal traits of diploid and triploid hybrids in all crosses significantly differed except the stomata index. Conclusions: Genotyping of triploid plants confirmed using Simple Sequence Repeat (SSR) molecular markers and five SSRs were able to identify three alleles of triploid hybrids. Selected triploid mandarin hybrids have been grafted on several rootstocks for field trials and are in the process of yield and quality performances. [ABSTRACT FROM AUTHOR]

The development of salt‐tolerant genotypes is key to a better utilization of salinized irrigated lands. Given the relatively low genetic diversity within the cultivated wheats for salt tolerance, exploring the Aegilops cylindrica's genetic diversity for salt tolerance is thus crucial to breed wheat for saline environments. In the current study, wheat genotypes were hybridized with Ae. cylindrica (a hyper salt-tolerant genotype), and amphidiploid plants were produced using embryo rescue and chromosome doubling techniques. Crossability and cytological examinations of amphidiploids and BC1 were performed before sequencing the ITS4/5 and trnE/trnF DNAs to explore the phylogenetic relationships of the amphidiploids and their parents. Finally, amphidiploids were assessed for salt tolerance. Only two common wheat cultivars ('Chinese Spring' and 'Roshan') were crossable with Ae. cylindrica. The resultant intergeneric hybrids possessed 70 chromosomes, and morphologically either were similar to the male parent in 'Chinese Spring' × Ae. cylindrica or tended to be intermediate between parents in 'Roshan' × Ae. cylindrica. The phylogenetic tree divided the genotypes into two groups, in which Clade I contained Ae. cylindrica and three amphidiploids, and Clade II consisted of female parents and one amphidiploid. Amphidiploids exhibited significantly higher tolerance to salt stress compared to the female parents (wheat cultivars) in terms of a higher dry matter, lower accumulation of Na, higher K, and higher K/Na ratio in their root and leaf tissues. Taken together, the amphiploid plants might contain valuable salt tolerance factors. [ABSTRACT FROM AUTHOR]

Adequate knowledge, access and introgression of genetic diversity available in cultivated and wild relatives are pre-requisites for widening the genetic base of cultivars to sustain genetic improvement. An attempt was made to screen anthracnose resistance in 33 indigenous and exotic accessions of common bean (Phaseolus vulgaris L.) belonging to primary, secondary, tertiary and quaternary gene pools including three popular and widely cultivated landraces of North-Western Indian Himalayas. The cultivated and wild species of common bean were introgressed into the backgrounds of landraces, which resulted in the development of 22 crosses comprising 6 each of intra and interspecific crosses with P. vulgaris and P. coccineus, 3 with P. acutifolius, 2 with P. lunatus and 5 backcrosses. The hybridity of these crosses was confirmed using morphological and molecular markers. The interspecific crosses of P. vulgaris × P. coccineus, P. vulgaris × P. acutifolius and P. vulgaris × P. lunatus were attempted for the first time in India using in-vitro approaches. Further hybrid plants were grown to maturity following embryo rescue protocol, but all showed different developmental abnormalities. The overall establishment of successful hybrids and their complete development was the maximum in cross of Bhaderwah Local × Poonch Local (18.28%) and the minimum in Bhaderwah Local × IC258276 and Bhaderwah Local × IC328372 (0.5%). The results of the present study revealed that wild relatives of common bean viz. P. coccineus, P. acutifolius and P. lunatus are an excellent source of resistance against anthracnose, which can be exploited for diversification of cultivated gene pool. These resistant genetic resources could also be shared among common bean researchers in the country or elsewhere under Standard Material Transfer Agreement for strengthening ongoing crop improvement programmes. [ABSTRACT FROM AUTHOR]

The direct cross-hybridization approach between 16 Aegilops tauschii (AT) and 10 bread wheat (W) lines was carried out in reciprocal directions. W (♀) x AT (♂) yielded no embryo formation whereas embryo formation ranging from 34.09 to 43.33% was obtained in AT (♀) x W (♂) crosses in all 160 combinations. The major bottleneck of low plant regeneration in AT (♀) x W (♂) crosses was cleared by adopting detached tiller culture and embryo rescue medium adjuvants such as casein hydrolysate (200 mg/L) and activated charcoal (2 g/L). The regeneration frequency as a result of these two interventions increased from 3.61 to 19.29 per cent. Two innovative methods of chromosome doubling included in situ colchicine (0.05%) treatment with cotton swab method and pre-regeneration colchicine treatment (0.1 and 0.2%) to pollinated ovaries were found to be effective. The findings of the study make successful exploitation of direct cross approach (AT (♀) x W (♂)) possible in wheat improvement. [ABSTRACT FROM AUTHOR]

Using stenospermocarpic grapes as female parents could improve the seedless ratio of offspring via embryo rescue techniques, so they are widely used for breeding seedless grapes. Here, we report the variation in phenotype, endogenous hormones, and internal cellular structure of ovules during embryo abortion in 'Thompson Seedless'. Moreover, we used the stenospermocarpic grapes as female parents to breed new seedless grapes. Our result indicated that the seed coat of abnormal ovules turned brown from 38 to 42 days after flowering (DAF). In hormones level, two ratios of endogenous hormones' content [gibberellic acid (GA3) + indole-3-acetic acid (IAA)]/abscisic acid (ABA) and zeatin riboside (ZR) /abscisic acid (ABA) had decreased by 36 DAF. In paraffin sections of embryo sac, indicated that there were few or no embryos in ovules were observed from 37 to 42 DAF. Importantly, the globular embryos in 'Thompson Seedless' were significantly smaller than those in 'Ruby Seedless' at 40 DAF, indicating the former had a lower embryo rescue efficiency as a female parent; accordingly, the most suitable time for embryo cultured in vitro was 39 DAF for 'Thompson Seedless'. Result from embryo rescue, demonstrated that 5 mg/L exogenous brassinolide (BR) promoted embryo recovery, embryo germination, and polyembryony formation of 'Flame Seedless'. Concurrently, WPM medium containing 1 µM of 6-BA helped embryos germinate into normal plantlets and reduced the abnormal plantlet development percentage. A total of 3573 hybrid ovules and 402 new progeny plants were obtained using the stenospermocarpic grapes as female parents crossed with disease-resistant Chinese wild grapes, among which 212 progeny with seedless trait and 23 with disease-resistant were detected by molecular markers, respectively. Key message: By studying phenotype, hormone, and anatomy, the optimal time for embryo rescue in Vitis vinifera cv. 'Thompson Seedless' was found. In all, 402 new seedless and disease-resistant hybrids were obtained via embryo rescue when using stenospermocarpic grapevine cultivars as female parents. [ABSTRACT FROM AUTHOR]

Embryo rescue technique is widely utilized to save developing embryo from abortion in interspecific hybridization of plants. In Brassica species, success of an embryo rescue depends on the age of embryo, developmental stage of embryo, and media composition. In this study, ten crosses were conducted between diploid oilseed species B. rapa × tetraploid B. napus and B. rapa × tetraploid B. juncea along with their reciprocal crosses. The immature embryos from parents and crosses were cultured at 14 d after pollination in MS and ½ MS medium without any growth regulators for direct embryogenesis. Embryos were found at different stages of development during isolation, from globular to cotyledonary, indicating that rate of embryo development depends on genotype. Embryos isolated at the cotyledonary stage showed the highest regeneration percentage (av. 83.29%) and required the lowest time to regenerate (5.8 d) compared with another type of embryo, torpedo stage, cultured. Embryo rescued in ½ MS media accounted for 10–51%, 4.0–61%, 6.0–21%, and 5.0–43% greater shoot length, root length, number of leaves, and days to flowering, respectively, in different cross combinations compared with full-strength MS media. The results indicated that the rate of regeneration from embryo is higher in ½ MS media compared with full-strength MS media. Percent regeneration from the cultured embryos differed significantly in different cross combinations. Days required for regeneration from embryos, length of siliqua, and the number of ovules per siliqua accounted for high heterosis and broad sense heritability. The findings of this experiment would be useful in developing cost-effective embryo rescue protocol and desired interspecific hybrids through embryo rescue in Brassica species. [ABSTRACT FROM AUTHOR]

One of the major objectives of table grape breeding is to produce seedless grapes of Vitis vinifera L. Of high importance among these objectives is the development of new cold-resistant seedless grapes for the colder regions of the world. Embryo rescue is an effective tool in breeding seedless grapes. Here, we report on nine cross-combinations between seedless cultivars and cold-resistant seedless cultivars or seeded hybrids. We carried out embryo rescue and molecular marker-assisted selection to create new cold-resistant seedless grape germplasm. We also examined the effects of different parents and the use of exogenous hormones on the success of embryo rescue as well as the optimal sampling time for a new seedless cultivar Qinxiu. The results show that a total of 473 new grape genotypes were obtained by embryo rescue using double-phase MM3 (modified ER) as the medium for embryo development and solid phase WPM as the medium for embryo germination and plantlet formation. We found the seedless cultivars Perlette, Qinhong No.2, Ruby Seedless and Qinhong No.10 were the most suitable female parents for embryo rescue, and the cold-resistant seedless cultivar Jupiter (a V. vinifera × V. labrusca hybrid) as the male parent. This was better than the seeded hybrid 0-1-5 (V. vinifera × V. amurensis). The best embryo development medium was MM3 with 500 mg/L CH, 1 mmol/L serine, 0.5 mg/L GA3, 1.0 mg/L IAA and 0.5 mg/L 6-BA. The best embryo germination and plantlet formation medium was WPM with 0.2 mg/L 6-BA and 0.1 mg/L IAA. The best sampling time for Qinxiu for embryo rescue was 42 days after flowering. We obtained a total of 440 hybrids by embryo rescue using the seedless molecular marker SCF27-2000. Key message: Hybridization between grape seedless cultivars and cold-resistant cultivars and embryo rescue were performed. We obtained 473 new germplasms and performed marker assisted-selection using SCF27-2000. The embryo rescue system was optimized. [ABSTRACT FROM AUTHOR]

In this study, the effect of spraying mother plants with various levels of putrescine, spermidine, and cycocel (each at 0, 50, 500, and 5000 mg/l) were assessed on the frequency of haploid embryos produced from unfertilized ovaries and subsequent regeneration of derived embryos. Significantly higher haploid embryos were obtained when mother plants were sprayed with putrescine at 500 mg/l (5.2 embryos/fruit), spermidine at 50 mg/l (4.8 embryos/fruit), and cycocel at 50 mg/l (5.2 embryos/fruit) as compared to the control (without spraying, 3.2 embryos/fruit). However, embryogenesis induction was decreased drastically as the concentration of all the three compounds tested was increased and the lowest haploid embryos were observed when 5000 mg/l of spermidine (0.4 embryos/fruit) or cycocel (2.0 embryos/fruit) were applied. Only spermidine at 50 mg/l led to 100% regeneration into fully developed plantlets. The seed setting and size of fruits were also affected by polyamines and cycocel applications. Ploidy analysis using a flow cytometer indicated that all regenerated plantlets contain the gametic chromosome number (n = x = 7) of parental plants and the results of chromosome counting also confirmed the haploid nature of regenerated plantlets. It can be concluded that the induction of haploid embryogenesis from unfertilized ovaries after pollination with irradiated pollen and subsequent conversion of derived embryos into the plantlets could be improved in Cucumis sativus L. by applying appropriate levels of putrescine, spermidine, and cycocel. [ABSTRACT FROM AUTHOR]

Production of seedless V. vinifera L. cultivars is one of the major goals of table grape breeding. Traditional methods result in only a low percentage of seedless progeny. Modern techniques, such as embryo rescue, are used to improve production efficiency of seedless cultivars. In this study, the effects of various putrescine concentrations (0, 1, 2, 3, 4, and 5 mmol L−1) and different stages of embryo development (globular-, heart-, torpedo-, and cotyledon-shaped embryo) on the success rate of embryo rescue were assessed in three lines of the F1Vitis vinifera L., cross ‘Delight’ × ‘Ruby Seedless,’ ‘DR2,’ ‘DR3,’ and ‘DR6’ (with small seed traces). The plant development rates of ‘DR2,’ ‘DR3,’ and ‘DR6’, which contain small seed traces, as female parents, were significantly higher than those of seedless varieties. The rates of embryo development on woody plant medium (WPM) of ‘DR2’ × ‘Thompson Seedless,’ ‘DR3’ × ‘Thompson Seedless,’ and ‘DR6’ × ‘Monukka’ were highest with 2, 2, and 4 mmol L−1 putrescine, respectively. The germination rates of torpedo- and cotyledon-shaped embryos were significantly higher than those of globular- and heart-shaped embryos. Characteristics of the embryo germination progress of ‘DR2’ × ‘Thompson Seedless’ at different developmental stages were recorded. The Grape Seedless gene Probe 1 (GSLP1) marker was used to help identify the seedless descendants; 14 plantlets showed a corresponding electrophoresis band at 569 bp and were preliminarily defined as seedless. [ABSTRACT FROM AUTHOR]

Background: Lilium callosum is native to Taiwan, but little is known about it since it has been considered extinct since 1915. After the rediscovery of this rare species after a fire in 2011 in Tunghsiao Township, intensive work has been conducted to count the number in the wild population, to develop a conservation strategy, and to understand its reproductive characteristics and even economic potential. Results: To conserve the germplasm of this population, three scales from a wild L. callosum plant were collected to establish a mass propagation system. Flowers from two regenerated plants were crossed by hand-pollination, the ovules were rescued and cultured in vitro, and 10 offspring were obtained. The karyotype was determined to be 2n = 2x = 24 = 2m + 2m(sat) + 2sm + 8st + 10t. The phylogenetic analysis using ITS sequences revealed that the sample of L. callosum from Taiwan was not grouped with the other accessions of L. callosum from other regions. The native habitat is classified as grass-dominated vegetation at the early successional stage and a subtropical monsoon-type climate. To clarify the causes of population scarcity in the native environment, reproductive characteristics of regenerated plants were investigated. Conclusions: Based on the information from this study, it is possible that factors intrinsic to L. callosum could combine to limit pollination and seed formation. The L. callosum pollen only germinated at a temperature that was higher than the native environment, the plants are self-incompatibile, there was a and scarce population, scattered flowering time and dichogamy. Through the culture of these wild harvested parts, the diversity of the germplasm has been broadened and is now available to preserve this rare and valuable species for the future. [ABSTRACT FROM AUTHOR]

Interspecific hybrids were developed between Trifolium alexandrinum cultivar Wardan × Trifolium vesiculosum and T. alexandrinum cultivar BL1 × T. vesiculosum through embryo rescue, as the crosses failed to set seed under natural conditions. Trifolium vesiculosum was used as a donor/male parent in this study as it is reported to possess tolerance to stem rot and high forage yield. Fertilization in crossed florets of the crosses was manifested from the recovery of swollen ovaries (< 7.80%) and confirmed from the presence of one degenerated ovule in most (> 93.00%) of the swollen ovaries. The hybrid embryos at various developmental stages (heart, torpedo and cotyledonary) were rescued at a frequency of 2.56% from Wardan × T. vesiculosum and 6.12% from BL1 × T. vesiculosum. Differentiation occurred only in the cotyledonary stage embryos, resulting in 17 putative interspecific hybrid plantlets. The assessment of plantlet hybridity through SSR markers (for the alleles inherited from the donor parent), micromorphological leaf traits (leaf texture and stomata) and morphological characters (plant height, leaflet length and width) confirmed production of two interspecific hybrids designated as AV1 and BV3 representing both the crosses. AV1 displayed moderate resistance and BV3 was resistant to stem rot. [ABSTRACT FROM AUTHOR]

Main conclusion: The changes in the reproductive barrier between hexaploid wheat ( Triticum aestivum L.) and rye ( Secale cereale L.) can be induced using in situ embryo rescue of abnormal embryos, yielding stable fertile amphidiploid plants. In intergeneric crosses between hexaploid wheat ( Triticum aestivum L.) and rye ( Secale cereale L.), postzygotic barriers may occur at different stages of hybrid development. One such mechanism is embryo lethality, which is genetically determined by the interaction and expression of two incompatible genes in wheat ( Eml-A1) and rye ( Eml-R1). Using in vitro culture methods as stressors, we overcame this hybrid lethality. Normal and abnormal embryos were observed to build embryogenic calli and produce regenerated plantlets in a similar manner. The high regenerative capacity of the abnormal embryos led us to conclude that the reproductive barrier in these intergeneric hybrids may have an epigenetic origin that can be easily overcome by culturing immature embryos via callus induction. After colchicine treatment during callus culture, amphidiploid plants were obtained. However, most of these plants did not produce seeds, due mainly to sterility of the pollen but also of the embryo sacs. These findings demonstrate that hybrid sterility affects both male and female gametophytes in plants obtained from abnormal embryos. The key roles of double fertilization and stress factors in the implementation of the apical meristem formation program in embryos from incompatible intergeneric crosses between hexaploid wheat and rye during in vitro culture are discussed. We also propose a hypothetical model for a wheat-rye lethality system involving differential expression of incompatible wheat Eml-A1 and rye Eml-R1b alleles in an identical genetic background. [ABSTRACT FROM AUTHOR]

Oryza brachyantha, the African wild rice which is resistant to yellow stem borer (YSB), was exploited as a source for introgression of YSB resistance trait into cultivated rice ( O. sativa), through development of monosomic alien addition lines (MAALs). BCF backcross hybrids ( O. sativa cv Savitri/ O. brachyantha// O. sativa cv Savitri/// O. sativa cv Savitri) were produced employing embryo rescue, with a crossability of 0.12 %. Hybrid embryos that were collected at 10 and 12 days after pollination showed highest percentage of survival in culture with a germination percentage of 35.8 %. Cytological analysis of pollen mother cells (PMCs) of the BCF hybrids revealed 15 hybrids with 2n + 1(2n = 25) chromosome number that exhibited typical trisomic chromosomal configurations. These aneuploids were putatively designated as MAALs and characterized morphologically and cytologically. Based on their morphological similarity to primary trisomics of the cultivated rice ( O. sativa), 8 MAALs viz., MAALs-4 (Sterile), 5 (Twisted leaf), 7 (Narrow leaf), 8 (Rolled leaf), 9 (Stout), 10 (Short grain), 11 (Pseudonormal) and 12 (Tall) were isolated, each of which exhibited several distinct morphological features. Among the 505 PMCs analysed, 79.80 % had 12, 5.45 % had 11 and 1.78 % PMCs had 1 configurations. The size of the extra chromosome was found to be smaller than those of O. sativa chromosomes and in 97.25 % of PMCs it remained unpaired and at a distance from the rest of the chromosomes. Of the 8 MAALs screened for YSB resistance, MAAL 11 was found to be moderately resistant. Undesirable morphological traits of O. brachyantha were found to be eliminated in the MAALs. [ABSTRACT FROM AUTHOR]

Habitat fragmentation and invasive alien species contribute to genetic bottlenecks and the threat of extinction of many endemic species of Mauritius, part of the Madagascan and Indian Ocean biodiversity hotspot. The genus Cylindrocline has two species, C. commersonii (critically endangered) and C. lorencei (extinct in the wild). The last living specimens of C. lorencei disappeared in the wild after the recorded collecting of seeds in 1982 by Conservatoire Botanique National de Brest (CBN Brest). Embryo rescue was used as a method to germinate these seeds and the seedlings raised this way were shared by CBN Brest with Royal Botanic Gardens Kew (RBG Kew) as part of an exchange programme in 2001. Mature plants both at CBN Brest and RBG Kew stopped producing viable seeds and this has made the long-term conservation of C. lorencei even more difficult. Seeds of C. commersonii collected from the wild in 2010 have a very low viability while ex situ grown C. commersonii produce non-viable seeds. Molecular studies conducted in C. lorencei using amplified fragment length polymorphisms (AFLP) showed no genetic variability among remaining individuals. Two samples of C. commersonii showed a very small amount of genetic variability. The variability between the two species was well within the limits commonly found within species or between closely related species and the long-term conservation of the genus requires a radical (to a degree) approach to avoid its extinction. The importance of novel approaches for restoration and long-term conservation are discussed. [ABSTRACT FROM AUTHOR]

Brassica is a key agricultural genus, containing oilseed and vegetable crops sharing three major genomes (A, B and C) distributed in diploid (genome: AA, BB and CC) and allotetraploid (genome: AABB, AACC and BBCC) species. Interspecific hybridisation between Brassica species is an important way to combine valuable characters of different species. In the present study, interspecific hybrids between tuber mustard (AABB) and red cabbage (CC) were obtained with the aid of embryo culture. The results showed that ovules at five different maturity levels (7, 9, 10, 13 and 14 days after pollination), which were cultured on Murashige and Skoog medium supplemented with 3 % sucrose, 0.2 % active carbon and 400 mg/L glutamine, showed varying seed survival rates. A total of 15 (5 %) seeds were obtained, with ovules cultured at 14 days after pollination yielding the most seeds. Subsequently, eight (53 %) of the seeds did not germinate into normal plants. Furthermore, the putative hybrids were confirmed by random amplified polymorphic DNA (RAPD) and flow cytometry analysis, and only one (14 %) of the seven plants was confirmed to be a true tri-genomic hybrid (genome: ABC). Then the nodal segments of the true hybrid were treated with liquid medium supplemented with colchicine to obtain an allohexaploid, as confirmed by flow cytometry analysis and morphological observation. The synthesized hybrid showed unique phenotypes in comparison with the parents, such as in stem colour and leaf margin. The synthesized Brassica tri-genomic vegetable germplasm with A, B and C genomes has potential as a source of new crops for agriculture as well as a basis for further investigation of the mechanisms of Brassica evolution and adaptation. [ABSTRACT FROM AUTHOR]

The Campanula genus comprises several economically important ornamental plants species. Wide hybridisation is a method to increase phenotypic variability, but is limited due to interspecies hybridisation barriers. In this study we investigated whether ovule culture could be used to increase the success rate of interspecific hybridisation between C. portenschlagiana × C. poscharskyana and C. medium × C. formanekiana. The effect of different ovule isolation times on ovule germination in vitro was examined. In general, the number of collectible ovules and ovule germination was low. Interspecific hybrids between C. medium and C. formanekiana exhibited an increased number of viable ovules with later isolation time, but with different ovule germination rates. A parent-of-origin effect on both the number of collectible ovules and ovule germination was observed for C. medium × C. formanekiana. Histological analysis of embryo and endosperm development in collectible ovules isolated at different time points from interspecific crosses showed that the vast majority of ovules did not contain an embryo. When present, embryo development only progressed with ovule collection time in the C. medium and C. formanekiana crosses. The occurrence of miscoloured seedlings in interspecific crosses indicated incompatibilities between the parental lines that could not be prevented by reciprocal crossing. The low number of collectible ovules and germination rates might be inhibited due to fertilisation barriers. With this study, a protocol for ovule culture was established and the usefulness of ovule culture to obtain interspecific hybrids of selected Campanula species was demonstrated. [ABSTRACT FROM AUTHOR]

A program for producing new seedless table and raisin grape cultivars was initiated using embryo culture methods. F descendants of the cross Delight × Ruby Seedless (DRs) were used as the female parent and crossed with various seedless cultivars. The berries of DRs have excellent table traits but they retain small seed traces at maturity. Blush Seedless and DRs were used as female parents, and 354 ovules from five crosses were tested. The percentage of embryos that developed using DRs as the female parent was higher (26.7-35.8%) than when a seedless cultivar was the female parent (5.9-6.7%). The best ovule excision time [days after flowering (DAF)] for ovule inoculation was tested in another set of crosses and found to be DAF 48, 50, and 50 for DR1 × Monukka, DR6 × Thompson Seedless, and DR7 × Zhengguo Seedless, respectively. A total of 569 hybrid progeny were obtained from 13 hybrid combinations; 311 of these survived and were established in soil after hardening. We used three molecular markers (SCC8, SCF27, and GSLP1) to analyze 15 parents used in the hybrid combinations. SCC8 had a 1018-bp band in the seedless parents and in some of the DR parents, while SCF27 had a 2000-bp band in all seedless and DR parents. GSLP1 had a 569-bp band in all of the seedless parents tested, whereas the seeded and DR parents had no bands of this size. Therefore, GSLP1 was used to screen the progeny for possible seedlessness. Eight strains had the 569-bp band; these were preliminarily identified as being seedless since they are in juvenile phase, but they have not yet been evaluated for fruiting characteristics. If the strains identified by using GSLP1 are confirmed to be seedless, this marker will be a valuable tool in breeding for seedlessness in grape. [ABSTRACT FROM AUTHOR]

Widening the genetic base of minor grain legume crops such as lentil ( Lens culinaris Medik.) is important for achieving future gains in productivity. In order to access genes from wild lentil species embryo rescue techniques are required to overcome reproductive barriers. Removing the seed coat from developing 14-day old immature hybrid seeds and culturing the interspecific zygotic embryos in medium containing auxin 4-chloroindole-3 acetic acid (4-Cl-IAA) improved successful hybrid recovery. Addition of 4-Cl-IAA to media also increased shoot proliferation when combined with a low concentration of zeatin. No significant difference in shoot elongation was observed between 4-Cl-IAA or IAA treatments. Hybrid shoots were then successfully grafted in vivo onto faba bean rootstocks. Hybrids were obtained from crosses of L. culinaris with L. tomentosus Ladiz., L. lamottei Czef., and L. odemensis Ladiz. This efficient and simple embryo rescue protocol resulted in seed production of large interspecific F populations from inherently weak zygotic embryos produced from wide hybridization. [ABSTRACT FROM AUTHOR]

A highly efficient technique of embryo rescue is critical when using stenospermocarpic Vitis vinifera cultivars (female parents) to breed novel, disease-resistant, seedless grape cultivars by hybridizing with wild Chinese Vitis species (male parents) having many disease-resistance alleles. The effects of various factors on the improvement of embryo formation, germination, and plantlet development for seven hybrid combinations were studied. The results indicated that Beichun and Shuangyou were the best male parents. The best sampling time for ovule inoculation differed among the female parents. When hybrid ovules were cultured on a double-phase medium with five different solid medium types, percent embryo formation was highest (11.3-28.3%) on a modified MM3 medium. Percentages of embryo germination (15.4-55.4%) and plantlet development (11.15-44.6%) were all highest when embryos were cultured on Woody Plant Medium + 5.7 μM indole-3-acetic acid + 4.4 μM 6-benzylaminopurine + 1.4 μM gibberellic acid + 2% sucrose + 0.05% casein hydrolysate + 0.3% activated charcoal + 0.7% agar. In the absence of other amino acids, the addition of proline significantly increased embryo formation (36.1%), embryo germination (64.6%), and plantlet development (90.5%). A highly efficient protocol has been developed for hybrid embryo rescue from seedless V. vinifera grapes × wild Chinese Vitis species that results in a significant improvement in breeding efficiency for new disease-resistant seedless grapes. [ABSTRACT FROM AUTHOR]

Resistance to Tomato yellow leaf curl virus (TYLCV) and Tomato spotted wilt virus (TSWV), among other diseases, has been reported in Solanum peruvianum PI 126944. Introgression lines (ILs) from S. peruvianum PI 126944 into the genetic background of cultivated tomato ( S. lycopersicum) are being developed. Several generations were derived from three interspecific hybrids previously obtained. A lot of crosses and embryo rescue were required until the third backcross, due to the high degree of incompatibility existing between tomato and PI 126944. Crosses between F plants were made to obtain a pseudo-F generation. The same procedure was followed up to the pseudo-F generation. Additional crosses between plants of different generations were made in order to increase progeny. Of 263 molecular markers tested, 105 were polymorphic between tomato and PI 126944. This set of polymorphic markers consisted of 90 simple sequence repeats (SSR) and 15 cleaved amplified polymorphic sequences (CAPS). The amount of the S. peruvianum genome was reduced in advancing generations and this was coupled in some cases with a reduction of incompatibility. However, the S. peruvianum genome was almost completely represented among the different plants of the most advanced generations. ILs will be basically developed from them. Some of the generations developed were resistant to TYLCV and TSWV. [ABSTRACT FROM AUTHOR]

Auxinic herbicides are widely used in agriculture to selectively control broadleaf weeds. Prolonged use of auxinic herbicides has resulted in the evolution of resistance to these herbicides in some biotypes of Brassica kaber (wild mustard), a common weed in agricultural crops. In this study, auxinic herbicide resistance from B. kaber was transferred to Brassica juncea and Brassica rapa, two commercially important Brassica crops, by traditional breeding coupled with in vitro embryo rescue. A high frequency of embryo regeneration and hybrid plant establishment was achieved. Transfer of auxinic herbicide resistance from B. kaber to the hybrids was assessed by whole-plant screening of hybrids with dicamba, a widely used auxinic herbicide. Furthermore, the hybrids were tested for fertility (both pollen and pistil) and their ability to produce backcross progeny. The auxinic herbicide-resistant trait was introgressed into B. juncea by backcross breeding. DNA ploidy of the hybrids as well as of the backcross progeny was estimated by flow cytometry. Creation of auxinic herbicide-resistant Brassica crops by non-transgenic approaches should facilitate effective weed control, encourage less tillage, provide herbicide rotation options, minimize occurrence of herbicide resistance, and increase acceptance of these crops. [ABSTRACT FROM AUTHOR]

The present study reports a protocol for germination of Strychnos potatorum (ver. Tel. Chilla) using zygotic embryo culture as an embryo rescue method. A 100% germination rate was obtained by culturing the embryos on full-strength Murashige and Skoog's medium (MS) containing 20 g/L sucrose in comparison to McCown and Lloyd's Woody Plant Medium (WPM). Germination rates decreased when the sucrose concentration was lower or higher than 20 g·L. WPM/MS medium containing glucose at levels 30, 20, 15 g·L showed a smaller percentage of germination and at quarter strength, WPM/MS medium with glucose did not respond. Multiple shoot formation was found at 1.0-2.0 mg/L BAP; 3.0 mg/L Kn; 2.0 mg/L TDZ on MS medium with 20 g·L sucrose. Germination rates improved when the embryos were placed upright (vertically) in the medium. The in vitro germinated seedlings were acclimatized in a walk-in-chamber and maintained in the green house with the survival rate of 65%-75%. These plants were transferred to the field and were found to be phenotypically normal, healthy and similar to donor plants. This protocol will be useful to overcome seed dormancy and for rapid multiplication and conservation of S. potatorum using zygotic embryo culture. [ABSTRACT FROM AUTHOR]

Using embryo rescue, we generated an intergeneric hybrid between Chrysanthemum × morifolium 'Maoyan' and Artemisia japonica Thunb. Cytological tests confirmed that regenerated plantlets were all genuine hybrids possessing 45 chromosomes, with 27 chromosomes inherited from C. × morifolium (2 n = 6 x = 54) and the other 18 derived from A. japonica (2 n = 4 x = 36). Hybrid plant flowered normally. The shape and color of the hybrid flowers and leaves resembled those of chrysanthemum, while leaf width, leaf length, plant height, and inflorescence diameter were intermediate between those of the parents. Hybrid plant had higher levels of chlorophyll and free proline, and lower concentrations of malondialdehyde and Na, than the maternal parent ( C. × morifolium), and these levels were correlated with the hybrid's enhanced salt tolerance. These results clearly demonstrate that intergeneric hybridization is an effective method of cultivar improvement in chrysanthemum. [ABSTRACT FROM AUTHOR]

Oat ( Avena sativa L.) and pearl millet ( Pennisetum glaucum L.) belong to different subfamilies of Poaceae. When emasculated oat was pollinated by millet, fertilization took place and all seven millet chromosomes were retained along the complete haploid oat complement during early stages of embryogenesis. Fourteen days after pollination, we cultured 170 embryos onto rescue medium, of which 99 were attached with endosperm tissue. Twenty-one embryos germinated and showed shoot growth. One of them also developed roots. The shoots of the rootless embryos elongated, but rolled to the scutellum side and eventually died in light conditions. Chromosome observations and marker analyses indicated that the seedling plants were true hybrids that retained all of the oat and millet chromosomes. One exceptional embryo with shoot and root grew under light conditions. This was a haploid of oat and developed to a fertile adult plant. One embryo generated a callus after 6 months cultivation, and it was found to harbor four out of the seven millet chromosomes corresponding to linkage groups 2, 4, 6, and 7. The callus grew vigorously but did not develop shoots or roots. [ABSTRACT FROM AUTHOR]

Mungbean ( Vigna radiata) and rice bean ( V. umbellata) (both species 2n = 2x = 22) have desirable traits that complement each other. In this study, we rescued embryos from a cross between mungbean cv. 'Kamphaeng Saen 2' and rice bean cv. 'Miyazaki' and resolved the hybrid sterility problem by colchicine treatment. The interspecific hybrids were obtained when Kamphaeng Saen 2 was used as the female parent. Four out of 80 immature seeds at 12 days old were able to germinate on an MS medium supplemented with 1 mg L IAA, 0.2 mg L kinetin, and 500 mg L casein hydrolysate. Forty random amplified polymorphic DNA (RAPD) primers were screened for polymorphism among the parents, and two specific primers were finally chosen for testing of hybridity. Using the two primers, all putative F hybrids were confirmed as the interspecific hybrids. To observe their fertility, some of the hybrid seedlings were transplanted. The hybrid produced flowers profusely but failed to set pods. To overcome the sterility, plants were induced to become tetraploid by colchicine treatment in vitro. The ploidy level of the regenerated seedlings was confirmed from leaf DNA using a flow cytometer. Three out of 20 hybrid seedlings (15%) were successfully induced from diploid to tetraploid by a colchicine concentration of 2 g L. The tetraploid hybrids were able to produce flowers and set pods normally. [ABSTRACT FROM AUTHOR]

Roses are known to produce seeds with high concentrations of abscisic acid (ABA), both in the pericarp and in the testa tissues of the seed coat. No studies on roses have documented embryo morphological differentiation or the concentration of ABA in the embryo, which is known to inhibit premature germination. In this study, hip and seed growth of two hybrid roses were characterised from 3 to 60 days after pollination (DAP). An increase of about five times the hip mass at 3 DAP was necessary to obtain fully developed seeds. Fully developed embryos were found at 15 DAP and completely developed seeds at 30 DAP. The same pattern of hip mass increase was shown in both genotypes. In parallel, quantification of ABA in the developing embryos was carried out by ELISA. An exponential decay in ABA concentration was found in embryos of both genotypes, with basal levels (<0.5 pmol mg) registered at 30 DAP. These changes in ABA, during the embryo development, could be used to formulate time points for embryo rescue and understanding of the pollination to seedling stage. [ABSTRACT FROM AUTHOR]

Reproductive barriers often exist in 4 x-2 x crosses in Dianthus caryophyllus L. and seriously lead to low seed set and breeding efficiency. In this study, we inspected pre-fertilization and post-fertilization barriers in crosses between 'butterfly' (2 n = 4 x = 60) and the fertile breeding line 'NH10' (2 n = 2 x = 30) and 'NH14' (2 n = 2 x = 30) using fluorescence microscopy and paraffin section. The growth of the pollen tubes up to the ovary was observed in crosses of 'butterfly' × 'NH10' and 'butterfly' × 'NH14'. However, abnormalities of pollen tube including coiled and spiky and callose deposition in incompatible tubes has also been detected. In the cross 'butterfly' and 'NH10', the number of pollen tubes at the base of the style reached the highest value, 67.2 ± 10.8 at 48 h after pollination, but the number of ovules with micropylar penetration was low, 19 ± 4.5. Similarly, in the cross between 'butterfly' and 'NH14', pollen tubes at the base of the style at 48 h after pollination was 51 ± 6.2 and the ovules with penetrated micropyles per ovary at 48 h after pollination was 11 ± 2.4. It indicated the pollen tubes difficultly penetrated into the ovules in 4 x-2 x crosses. In addition, it was observed that non-fused of egg and sperm or non-fused polar nucleus resulted in endosperm without embryos, and embryos without endosperm. Embryos aborted at different developmental stages (prembryo to cotyledon embryo, main globular embryo), which might be related to a deficient endosperm development in reciprocal crosses. The results suggested that pre-fertilization and post-fertilization barriers were detected in these combinations. Due to the post-fertilization barriers in 4 x-2 x crosses, the embryo rescue might be contributed to obtain hybrid F1 plants. [ABSTRACT FROM AUTHOR]

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market, and mandarin triploid hybrids have this trait. Triploid citrus plants can be recovered by 4x × 2x hybridisations using non-apomictic genotypes as female parents. In this study, we characterise the type of seeds obtained in 4x × 2x hybridisations and the ploidy level of plants recovered from each type of seed. The majority of the plants recover were triploid (98.3 %), but a few diploid, tetraploid and pentaploid plants were also produced and their genetic origin was analysed by simple sequence repeat (SSR) markers. We also analysed the influence of parents and environmental conditions on the efficiency of recovery triploid hybrids. In this work, we present an effective methodology to recover triploid hybrids from 4x × 2x hybridisations based on in vitro embryo rescue and determination of ploidy level by flow cytometry that allow us to recover more than 4,400 triploid hybrids from more than 60 parental combinations. [ABSTRACT FROM AUTHOR]

Two methods of in vitro pollination of aspen were tested and compared, first with moist pollen by transferring swollen pollen grains from a wet agar surface to the stigma and, second, using dry pollen grains. In vitro pollination with dry pollen grains appeared to be more efficient and this method was applied in three subsequent years. Additionally, pollination was performed with selected pollen grains in order to obtain triploid plants. Diploid pollen was induced by heat-treatment of pollen mother cells, but the enrichment of unreduced pollen from natural pollen mixtures was also successful. The selection of diploid pollen, which is larger in diameter than haploid pollen, was performed by sieving using micro sieves. Five triploid plants from different poplar combinations were obtained from 1,227 zygotic embryos cultured after in vitro pollination of 2,676 ovaries with dry pollen over 3 years. Embryo rescue was used to facilitate the development of immature zygotic embryos. The results of this study demonstrate the possibility to effect pollination using selected single pollen grains with the help of in vitro techniques. [ABSTRACT FROM AUTHOR]

The citrus fresh market demands the production of seedless citrus fruits, as seedy fruits are not accepted by consumers. The recovery of triploid plants has proven to be the most promising approach to achieve this goal, since triploids have very low fertility, are generally seedless and do not induce seeds in other cultivars by cross pollination. Triploid plants can be recovered by 2x × 4x sexual hybridization. In this work, we present an effective methodology to recover triploid plants from 2x × 4x hybridizations based on in vitro embryo rescue, ploidy level analysis by flow cytometry and genetic origin of triploid plants. The pollen viability of diploid and tetraploid citrus genotypes was analyzed by comparing the pollen germination rate in vitro . The pollen viability of tetraploid (doubled-diploid) genotypes is generally reduced but sufficient for successful pollination. Triploid embryos were identified in normal and undeveloped seeds that did not germinate under greenhouse conditions. The influence of parents and environmental conditions on obtaining triploid plants was analyzed and a strong interaction was noted between the parents and environmental conditions. The parental effect on the length of the juvenile phase was also demonstrated through observations of a large number of progeny over the last 15 years. The juvenile phase length of the triploid hybrids obtained with 'Fortune' mandarin as female parent and tetraploid 'Orlando' tangelo as male parent was shorter than the juvenile phase obtained with a clementine as female parent and tetraploids of 'Nova', 'W. Leaf' and 'Pineapple' male parents. Key message Effective methodology to recover citrus triploid plants from 2x × 4x sexual hybridizations and the parental effect on the length of the juvenile phase. [ABSTRACT FROM AUTHOR]

Anthers of snapmelon ( Cucumis melo L. var. momordica) were irradiated with varied doses of gamma rays (150, 200, 250, 300 and 350 Gray). Then pollen from irradiated anthers was used for pollinating female flowers. Results revealed that 250 Gray of gamma-irradiation was successful in inducing parthenogenesis and fruit development, whereas, low (150 and 200 Gray) or high (300 and 350 Gray) irradiation doses were not effective in inducing haploid embryos. Embryos at a range of developmental stages were dissected from fruits harvested after 21 days of pollination and cultured on E20A medium. Among these embryos cultured, only cotyledonary embryos germinated into plantlets. Chromosome counting, performed on the roots of regenerated plants, showed the haploid level ( n = 12). Ploidy analysis using flow cytometer, measurement of stomatal cells and counting of chloroplast in the guard cells also corroborated the haploid nature of regenerated plants. [ABSTRACT FROM AUTHOR]

Five intergeneric hybrids between the chrysanthemum cultivar 'Zhongshanjingui' (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While 'Zhongshanjingui' bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids' flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2 n = 45, of which GISH analysis was able to establish that 27 were inherited from 'Zhongshanjingui' and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content. [ABSTRACT FROM AUTHOR]

We report the first successful production of PRSV-P resistant backcross (BC) papaya plants following intergeneric hybridisation between C. papaya and a Vasconcellea species after 50 years of reports on unsuccessful attempts. This follows our previous reports of PRSV-P resistant F hybrids developed by intergeneric hybridisation between C. papaya and V. quercifolia. One PRSV-P resistant BC 1 (BC) plant was produced after 114,839 seeds were dissected from 940 fruits. The seeds yielded 1,011 embryos and 733 germinated in vitro from which 700 developed into plantlets that were screened in a glasshouse and in the field under high disease pressure and exposure to inoculation by viruliferous aphids. From the PRSV-P resistant backcross 1 (BC) male plant, 1465 plants [137 BC, 546 SbC (BC2 sib-crosses), 147 BC, 379 SbC and 256 BC] were grown from seed and inoculated with PRSV-P and virus resistant BC and BC plants were selected from these generations. Presence or absence of virus was confirmed by ELISA serological tests. BC plants generally developed mild symptoms of PRSV-P after periods ranging from 5 to 18 months in the field but many showed the ability to produce new growth free of symptoms. All control plants developed severe symptoms after 3 months in the field. Some BC and BC plants were free from viral infection after 18 months in the field. Subsequently they developed very mild symptoms on their leaves and a few ringspots on their fruit. They continued to grow vigorously and produce fruit for 3 years under high disease pressure provided by the infected controls and other susceptible plants. Good quality marketable fruit were produced on these plants. Application of these results should lead to restoration of the papaya industry in virus-infested regions of the Philippines and worldwide. [ABSTRACT FROM AUTHOR]

Albinism is viewed as a major experimental bottleneck during wide hybridization in several species; the phenomenon is also widely reported in androgenesis and doubled haploid cultures. In this study, when chickpea ( Cicer arietinum) was crossed with distant wild relatives, C. judaicum and C. pinnatifidum, the only hybrid embryos that survived were those rescued using in vitro techniques. Fourteen to 21-day-old embryos were incubated in ML6 medium with 90 g l sucrose, 1 mg l zeatin and 0.25 mg l indole acetic acid. Germinating embryos were dissected from the ovule and transferred to fresh medium without growth regulators. Later, shoots were micropropagated on solid MS medium, supplemented with B5 vitamins, 30 g l sucrose, 0.1 mg l 6-benzyladenine and 0.01 mg l naphthaleneacetic acid. Although some post-hybridization barriers were overcome by rescue in vitro, regenerated hybrid plantlets failed to thrive in culture and few survived transfer to soil. Here we report findings from characterization of this valuable breeding material, as a step towards a better understanding of albinism in chickpea wide hybrids and other plant tissue cultures. Following proliferation, hybrids were phenotyped as green, pale green and albino. Genotype affected pod set, regeneration and albinism. Plastid ultrastructure studies using transmission electron microscopy revealed that thylakoid membranes were well-formed in green hybrids but not in albinos. Spectrophotometric analysis of chlorophyll a, chlorophyll b and total carotenoids revealed that chlorophyll content was three to fourfold lower in albino compared to green hybrids; moreover green hybrids had two to threefold lower total chlorophyll content than in vitro-grown plantlets of their parents. In contrast, carotenoids were higher in some albino phenotypes and lower in others; however overall differences in carotenoids were less marked between all genotypes compared to chlorophyll pigments. Genetic variability between different wide crosses provides an opportunity to select certain chickpea parents and wild species which give rise to more frequent green hybrid regenerants. In future, only these hybrids will be maintained and multiplied for transfer to the glasshouse in our program. [ABSTRACT FROM AUTHOR]

Plant ovules provide zygotes with a physicochemical environment that supports embryo differentiation, growth, and maturation. The exact nature of this embryogenesis-enabling environment is not well characterized, as evidenced by failed attempts to induce normal embryony from zygotes or proembryos (precotyledonary) on defined media. To identify factors required for cotton ( Gossypium hirsutum L.) zygotic embryony in vitro, we previously performed chemical and dissolved oxygen tension analyses of cotton ovule fluids and tissues at multiple stages of embryony in situ. Based on these analyses, we report herein the development of procedures that normalize embryo differentiation, growth, maturation, and germination in vitro, starting with proembryos. Our medium differed from Murashige and Skoog (MS) medium as follows (percentage of MS): N (30%, mostly from ten amino acids), P (815%), K (237%), Mg (85%), Ca (267%), S (506%), Fe (88%), and myoinositol (883%). Levels of other MS nutrients and vitamins, except sucrose, were kept at MS levels. Additionally, we included 100 mg L casein hydrolysate plus the following (mmol L): d-glucose (1.8), fructose (4.7), sucrose (62.0), arabinose (7.1), melibiose (3.5), malic acid (11.6), and citric acid (3.8). Mannitol was added to achieve a medium osmotic potential of −1.10 MPa, and an atmospheric O tension of 3.3 mol m at the surface of embryos was maintained during culture. When cultured on medium containing 8.0 μmol L indole-3-acetic acid, 80-90% of proembryos (as small as 100 cells) of cultivars HS-26 and B-27 increased four- to eightfold in surface area during the first 18 d in culture and germinated thereafter to produce viable plants. Increases in surface area of proembryos cultured on a modified MS medium previously used for somatic embryogenesis were from 0.2- to 0.6-fold. The described embryo culture medium should be useful for studying nutritional and molecular aspects of early embryony and possibly for plant zygote transformation protocols. [ABSTRACT FROM AUTHOR]

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail. [ABSTRACT FROM AUTHOR]

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x × 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x × 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders’ rights. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop an efficient methodology to rescue embryos following interspecific crosses in the genus Hylocereus. Crosses between the diploids Hylocereus polyrhizus and H. undatus in both directions were performed. Fertilized ovules carrying embryos at very early pro-embryonic stages were excised from ovaries 5 days after pollination (DAP) and placed on half-strength basal MS medium containing 680 μM glutamine, 0.55 μM α-naphthaleneacetic acid (NAA), 0.45 μM thidiazuron (TDZ) and various concentrations of sucrose. After 30 days in culture, ovules were isolated from the surrounding tissue and transferred to the same fresh medium. Significant differences were found between the main effects (cross and sucrose concentration) in ovule response, i.e., increased ovule size and callus formation. The best responses were obtained in the cross: H. polyrhizus × H. undatus; and sucrose concentration of 0.09 M. In terms of embryo conversion, polyembryony and number of regenerated plants, the highest responses were observed on the culture medium supplemented with 0.17 M sucrose in both interspecific crosses. All tested plants were found to be diploid by flow cytometric analyses. Fluorescent amplified—fragment length polymorphism (fAFLP) confirmed the hybrid origin of the regenerated plants. This study reports on the success of a three-step embryo rescue procedure for Hylocereus species. The procedure developed here provides the means for producing plants from very-early embryo stage, thus expanding the prospects for vine-cactus breeding programs. [ABSTRACT FROM AUTHOR]

Aphids represent the most destructive of chrysanthemum pests to cultivation. Reliable variety sources of resistance and control methods are limited, so development of highly resistant breeding lines is desirable. An intergeneric hybrid between Dendranthema morifolium (chrysanthemum) variety ‘Zhongshanjingui’ and Artemisia vulgaris (mugwort) ‘Variegata’ was attempted. Most of the hybrid embryos aborted at an early developmental stage. Embryo rescue allowed the generation of hybrid plants, whose hybridity was confirmed by a combination of morphological, cytological and GISH analysis. The hybrids were vigorous, flowered normally, and their flower and leaf shape resembled those of the chrysanthemum more than those of the mugwort parent. The hybrids showed much higher resistance to chrysanthemum aphid ( Macrosiphoniella sanbourni) than maternal chrysanthemum by inoculation test. The leaves of the hybrid developed a higher density of trichomes and secretory glands compared to the maternal chrysanthemum. GC–MS analysis revealed that ~51% of the essential oil in the hybrid leaves were monoterpenoids and sesquiterpenoids, while the proportion in the chrysanthemum was ~37%, and in the mugwort was ~90%. It is inferred that higher aphid resistance in the hybrid mainly owed to the leaf micromorphology and bioactive essential oil content. [ABSTRACT FROM AUTHOR]

Interspecific hybridization was carried out between Lilium longiflorum and L. lophophorum var. linearifolium by using the cut style method of pollination, as a contrast, intraspecific hybridization between L. longiflorum ‘Gelria’ and L. longiflorum was also made, but no mature seeds and offspring were obtained from the two combinations under in vivo condition. Ovules excised from each carpel 5–35 days after pollination (DAP) were cultured on B5 or half-strength B5 medium containing sucrose at different concentrations in vitro. In L. longiflorum × L. lophophorum var. linearifolium, only 1.17% of ovules excised at 10 DAP developed into seedlings, and in L. longiflorum ‘Gelria’ × L. longiflorum, only 0.99% of ovules excised at 25 DAP developed into seedlings; none of the ovules excised at other different DAP in the two cross combinations produced any seedlings. The results showed that interspecific hybridization had a more serious post-fertilization barrier than the intraspecific hybridization, and that a lower concentration (3%) of sucrose led to better embryo development and higher percentage of seedlings in ovule cultures. All hybrid seedlings obtained were successfully transplanted to soil and grew normally. The progenies investigated were identified as true hybrids based on inter-simple sequence repeat (ISSR) analysis. [ABSTRACT FROM AUTHOR]

Genetic maps are useful for detecting quantitative trait loci (QTL) associated with quantitative traits and for marker-assisted selection (MAS) in breeding. In this research, we used the wheat × maize method to develop a doubled haploid (DH) population derived from the synthetic hexaploid wheat (SHW) line TA4152-60 and the North Dakota hard red spring wheat line ND495. The population consisted of 213 lines, of which a subset of 120 lines was randomly selected and used to construct linkage maps of all 21 chromosomes and for QTL detection. The whole genome maps consisted of 632 markers including 410 SSRs, 218 TRAPs, 1 RFLP, and 3 phenotypic markers, and spanned 3,811.5 cM with an average density of one marker per 6.03 cM. Telomere sequence-based TRAPs allowed us to define the ends of seven linkage groups. Analysis revealed major QTLs associated with the traits of days to heading on chromosomes 5A and 5B, plant height on chromosomes 4D and 5A, and spike characteristics on chromosomes 3D, 4A, 4D, 5A and 5B. The DH population and genetic map will be a useful tool for the identification of disease resistance QTL and agronomically important loci, and will aid in the identification and development of markers for MAS. [ABSTRACT FROM AUTHOR]

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chromosome pairing was identified and most of the chromosomes were univalent, which resulted in complete male sterility in the hybrid. [ABSTRACT FROM AUTHOR]

The effect of the Embryo lethality mutant ( Eml) of rye was studied in crosses between hexaploid wheat and corresponding inbred rye line (L2). Histological analysis of hybrid embryos revealed morphological differences 16 days after pollination. Eml was found to arrest the formation of shoot meristem but had no influence on root meristem formation. The effect of Eml cannot be overcome by in vitro embryo rescue via direct regeneration on Kruse medium. The possibility of complementary interactions between wheat and rye genes and of changes in gene expression through increased variation in dosage-regulated gene expression during hybrid formation is discussed. [ABSTRACT FROM AUTHOR]

Interspecific hybrids from crosses between H. syriacus × H. paramutabilis and H. syriacus × H. sinosyriacus were obtained. In both cases unilateral incongruity was observed and reciprocal crosses yielded no fruits. In vitro embryo rescue, 11 weeks after pollination, increased the germination capacity of H. syriacus × H. sinosyriacus embryos, while this was not the case for H. syriacus × H. paramutabilis embryos. However, a lot of the generated H. syriacus × H. sinosyriacus seedlings were lost due to variegated and total albinism. In the progeny of H. syriacus ‘Oiseau Bleu’ × H. paramutabilis about 95% of the seedlings had an intermediate flower and leaf morphology compared to both parent plants. Leaves on the adult F1 hybrids showed a polymorphism. In total four different leaf types could be observed on the same plant. This leaf polymorphism also was seen in the progeny of H. syriacus ‘Melwhite’ × H. sinosyriacus ‘Lilac Queen’. In this progeny about 50% of the seedlings had an intermediate flower and leaf morphology compared with the parent plants. The hybrid nature of the seedlings of both progenies was also confirmed by AFLP analysis. Despite the low germination vigour of the pollen of the hybrids, a small F2 generation was obtained from H. syriacus ‘Oiseau Bleu’ × H. paramutabilis. [ABSTRACT FROM AUTHOR]

Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea ( C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F1 hybrids. The F1 hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F2, F3 and F4 progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F0 parental cross. As a route of introgression, diploid F1 hybrids were backcrossed with cultivated pigeonpea and BC1 progeny obtained by in vitro culture of aborting embryos. BC2 plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F1 and BC1 plants, it was possible to produce BC2 and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds. [ABSTRACT FROM AUTHOR]

Triploid hybrid embryos (AABB × SS = ABS) were produced by crossing two moderately salt-tolerant wheats, namely Triticum turgidum durum and Aegilops speltoides subsp. speltoides. The embryos were rescued by culturing on agar No. 1. Chromosome doubling was achieved by using 0.05% colchicine. The resulting hexaploid plants grew to maturity, and produced a considerable amount of seed (≈40,000). The synthetic hexaploid (C2) was tested for salinity tolerance, grown at 0, 100, 125, 150, 160, 170, 180 and 200 mM NaCl in a standard nutrient solution for 2 weeks. The criterion of salt tolerance was root growth in different NaCl concentrations. The amphidiploid material showed greater salinity tolerance than either parent, suggesting the presence of different genes for tolerance in the parents. [ABSTRACT FROM AUTHOR]