Search

Doubled haploid (DH) technology has become integral to maize breeding programs to expedite inbred line development and increase the efficiency of breeding operations. Unlike many other plant species that use in vitro methods, DH production in maize uses a relatively simple and efficient in vivo haploid induction method. However, it takes two complete crop cycles for DH line generation, one for haploid induction and the other one for chromosome doubling and seed production. Rescuing in vivo induced haploid embryos has the potential to reduce the time for DH line development and improve the efficiency of DH line production. However, the identification of a few haploid embryos (~10%) resulting from an induction cross from the rest of the diploid embryos is a challenge. In this study, we demonstrated that an anthocyanin marker, namely R1-nj, which is integrated into most haploid inducers, can aid in distinguishing haploid and diploid embryos. Further, we tested conditions that enhance R1-nj anthocyanin marker expression in embryos and found that light and sucrose enhance anthocyanin expression, while phosphorous deprivation in the media had no affect. Validating the use of the R1-nj marker for haploid and diploid embryo identification using a gold standard classification based on visual differences among haploids and diploids for characteristics such as seedling vigor, erectness of leaves, tassel fertility, etc., indicated that the R1-nj marker could lead to significantly high false positives, necessitating the use of additional markers for increased accuracy and reliability of haploid embryo identification.

Citrus rootstock breeding is important to improve their resistance to diseases, pests and adverse environmental conditions. The majority of citrus species have nuceller embryony. Although Clementine mandarin is monoembryonic, in some cases (intercrossing etc.) abortive seed formation can be seen frequently. In this study, Clementine mandarin × Carrizo citrange were crossed. In the study, interspecies hybridization (Citrus × Poncirus) was used. Embryo rescue technique was used to prevent the loss of hybrid individuals due to abortive seed formation. The seeds were gathered from the crossed plants 80, 100, and 120 days after pollination. The immature embryos were removed from the seeds and these embryos were transferred to control, 0.5, and 1.0 mg l-1 GA3 containing medium to germination. The highest germination rate with 100% was observed from 1.0 mg l-1 GA3 containing media harvested 120 days after pollination. Additionally, the highest rate of trifoliate seedlings was obtained from embryos gathered after 120 days of the pollination.

Wild species of the genus Cucumis, such as C. anguria, are resistant to various melon (C. melo) diseases such as Fusarium wilt race 1.2 and are anticipated as genetic resources of melon. However, in the interspecific crosses between C. anguria and melon, the abortion of the embryos was observed in the early growth stage and could not be regenerated even when cultured. Therefore, for embryo rescue, it is necessary to promote embryo growth during abortion occurrence. This study investigated the effects of fruit heating on embryo growth in interspecific hybrids of C. anguria and melon. In the heating device, the minimum night temperature around the fruit was maintained above 21.5°C. The ovule grain area of C. anguria × MR-1 with fruit heating was significantly enlarged compared to that without fruit-heating. It was also significantly enlarged compared with the ovule grain area of C. anguria self-pollination. Although, in C. anguria × MR-1 without fruit-heating, no embryo was observed until 10 days after pollination (DAP), and pre-embryos were observed at 13 DAP. Conversely, in C. anguria × MR-1 with fruit-heating, pre-embryos were even observed at 10 DAP. These results suggest that the possibility of embryo or ovule culture regeneration has increased due to embryo growth promotion.

This experiment was carried out at the High Rainfall Station of the International Institute of Tropical Agriculture (IITA) in Onne, Rivers State, Nigeria, to investigate if secondary triploid plantains established using in-vitro excised zygotic embryo propagules and tissue culture will differ significantly from those established by using ex-vitro propagules such as seeds obtained from open or hand pollinated parents. Seeds of secondary triploid plantain hybrids were obtained by crossing tetraploid female hybrids TMP4x 2796-5 and TMP4x 4698-1 by hand pollination with pollen from known diploid males TMP2x 1297-3 and TMP2x 1448-1. Another set of these female hybrids were exposed to open pollination. At maturity, seeds from ripened mature fruits from both methods of pollination had their zygotic embryos extracted and cultured in-vitro. Another set of seeds were grown ex-vitro in the soil. Both in-vitro and ex-vitro derived propagules obtained from open and hand pollinated parents were raised in the tissue culture laboratory and the nursery respectively. Three replications of the 12 treatments of eight-week-old secondary triploids were planted in the field at 3m x 2m spacing using an RCB (randomized complete block) design. Data collected were: number of days to 50% flowering (DTF), time to fruit filling (TFF), plant height and number of leaves at 50% flowering, bunch weight, number of hands/bunches, fingers/ bunch and total yield. Collected data were analysed using ANOVA in RCB design at (P = 0.05) while means were compared with Duncan’s Multiple Range Test at (P = 0.05). Inconsistent individual genotypically significant differences were found in some phenological and vegetative characters, yield constituents and total yield between ex-vitro and in-vitro derived secondary triploids indicating no definitive pattern in responses of the secondary triploids. However, only days to flowering (DTF) showed consistent significant notable differences out of all these traits. Generally, the ex-vitro derived secondary triploids flowered significantly later than in-vitro derived ones. The secondary triploids from open pollinated parental crosses flowered significantly earlier than those obtained from the hand pollinated parental crosses.

Immature lentisk (Pistacia lentiscus L.) seeds from plants grown in Liguria (Italy) were collected and surface sterilized with ethanol (70%) and then NaOCl (1%). The outer pericarp of the immature fruits was removed and shells were opened. Enhancement of Embryo germination was examined through the use of five sucrose concentrations (0, 15, 30, 45, and 60 g L-1) combined with five concentrations of GA3 (0, 0.1, 0.3, 0.5, and 0.7 mg L-1). After 14 days, the germination frequency was detected and after 16 days, seedling development was evaluated. The embryo development into plantlets increased up to 54.37% when 45 g L-1 of sucrose was supplied; at this concentration, all the parameters, such as plantlet weight, height, and root length were high, while at 60 g L-1 of sucrose, the plantlets showed many lateral root formations. The root shape was completely different among the treatments. The percentage of embryo germination, plantlet weight and height, root length, and main lateral root number were enhanced when GA3 was used in the concentration of 0.7 (mg L-1). No rooting at all was observed when GA3 was used in a concentration of 0.3 or 0.5 (mg L-1) or medium without GA3. This research increases the possibility to obtain a massive number of plants to be used as rootstock for pistachio cultivation in difficult soils.

Key message. The success of interspecific hybridization in hardy geranium (Geranium sp.) can be predicted by considering the genetic distance between parental genotypes and using the logistic regression model developed in this study. Hardy geranium is a popular ornamental plant known for its architecture, hardiness, prolonged flowering, and diverse colors in leaves and flowers. In ornamental breeding, the pursuit of novel trait combinations is never-ending. Even in Geranium, certain combinations of valuable traits have not yet been achieved. Interspecific hybridization can increase diversity; however, success remains low due to pre- and postzygotic barriers. Crossing success can be predicted by response criteria such as pollen tube growth (tube_length), seed development (seed_dev), and seed setting (seed_set). Within a collection of 42 Geranium genotypes and during two consecutive breeding seasons (years), we evaluated tube_length, seed_dev, and seed_set for 150, 1155, and 349 crosses, respectively. These crosses varied in four parental differences (variables): chromosome number (Chrom), DNA/chromosome (DNA), style length (Style), and genetic distance expressed as the Jaccard distance (cJaccard = 1 − Jaccard). Using logistic regression models has confirmed that most often, the success rate decreased with increasing parental distance. The most consistent association was seen in seed_dev in combination with cJaccard. The model was used to predict the number of crosses necessary to have 10 successful crossing products by taking into account the uncertainty in the model. These findings provide valuable guidance for future planning of interspecific breeding experiments in Geranium. By incorporating the genetic distance between parental genotypes, breeders can enhance the efficiency and success of hybridization efforts.

Coconut (Cocos nucifera L.) is widely recognized as one of nature’s most beneficial plants. Makapuno, a special type of coconut with a soft, jelly-like endosperm, is a high-value commercial coconut and an expensive delicacy with a high cost of planting material. The embryo rescue technique is a very useful tool to support mass propagation of makapuno coconut. Nevertheless, transplanting the seedlings is a challenge due to poor root development, which results in the inability of the plant to acclimatize. In this study, primary root excision was used in makapuno to observe the effects of primary root excision on lateral root development. The overall results showed that seedlings with roots excised had a significantly higher number of lateral roots, and shoot length also increased significantly. Using de novo transcriptome assembly and differential gene expression analysis, we identified 512 differentially expressed genes in the excised and intact root samples. ERF071, encoding an ethylene-responsive transcription factor, was identified as a highly expressed gene in excised roots compared to intact roots, and was considered a candidate gene associated with lateral root formation induced by root excision in makapuno coconut. This study provides insight into the mechanism and candidate genes involved in the development of lateral roots in coconut, which may be useful for the future breeding and mass propagation of makapuno coconut through tissue culture.

The production of the common bean (Phaseolus vulgaris L.), one of the most important sources of protein and minerals and one of the most consumed grain legumes globally, is highly affected by heat and drought constraints. In contrast, the tepary bean (Phaseolus acutifolius A. Gray), a common bean-related species, is adapted to hot and dry climates. Hybridization to introduce complex traits from the tepary bean into the common bean has been challenging, as embryo rescue is required. In this study, we report three novel interspecific lines that were obtained by crossing lines from prior common bean × tepary bean hybridization with Phaseolus parvifolius Freytag in order to increase the male gametic diversity to facilitate interspecific crosses. These interspecific lines enhanced the crossability of the common bean and tepary bean species while avoiding the embryo rescue process. Crossing these three interspecific lines with tepary beans resulted in 12-fold more hybrid plants than crossing traditional common beans with tepary beans. Whole-genome sequencing analysis of these three interspecific lines shows large introgressions of genomic regions corresponding to P. parvifolius on chromosomes that presumably contribute to reproductive barriers between both species. The development of these lines opens up the possibility of increasing the introgression of desirable tepary bean traits into the common bean to address constraints driven by climate change.

Seedlessness is one of the most desirable traits for table and raisin grapes. Stenospermocarpic cultivars are desirable because they have large berries with superior quality. Embryo rescue techniques have been widely used to get progeny seedling populations from crosses using seedless mother plants. Selection of the female parent, sampling time, and the growth medium are the most crucial to the success of this technique. This study investigated the effect of best sampling time and media composition on embryo rescue efficiency in a cold-hardy hybrid grape breeding program. We sampled ovules 5 to 9 weeks after flowering, and we tested four media compositions. The greatest percentages of embryo germination and normal seedlings were obtained when ovules were harvest at 8 weeks after flowering, indicating that it is suitable to harvest ovules at veraison, when the extraction of ovules is easier as a result of softer berry flesh. For the media composition experiment, all ovules were harvested at 8 weeks after flowering. Nitsch & Nitsch culture medium had very low germination, and the resulting seedlings performed the lowest compared with the other treatments. Lloyd & McCown Woody Plant Basal Medium (WPM) increased the number of embryos germinated significantly, and a number of normal seedlings and plantlets developed. Although there was no significant difference among the other three media containing WPM supplemented with different doses of plant regulators, the WPM Plus medium [with cytokinin (6-benzlaminopurine), indole-3-butyric acid, gibberellin, and casein hydrolysate] promoted the greatest percentage of established plants (46.98%). Therefore, the 8-weeks-after-flowering harvest time and the WPM Plus medium were selected for use in the embryo rescue protocol at the University of Minnesota grape breeding program.

[ES] Las variedades tradicionales son recursos genéticos que pueden actuar como fuentes de variación en los planes de mejora moderna, pero también pueden actuar como receptores de variación para conseguir una mejora de los recursos genéticos. Las razas locales aportan una serie de excelencias ya que son fruto de la domesticación y selección por los agricultores, están adaptadas a los climas donde se han desarrollado, son fuente inagotable de genes y su precio de venta es elevado. Por el contrario, suelen tener bajo rendimiento y una elevada susceptibilidad a nuevas enfermedades, sobre todo a virosis. Con el objetivo de conseguir una conservación o recuperación de variedades tradicionales, se debe actuar incorporando resistencias a virosis, para que su cultivo sea rentable y se mantenga en el tiempo. En este trabajo, se inicia un programa de mejora en el que se introgresan los genes de resistencia al virus del bronceado del tomate (TSWV) y al virus del moteado suave del pimiento (PMMoV) en 11 variedades tradicionalmente cultivadas en España, a partir de una línea de mejora resistente. En definitiva, la mejora de las variedades tradicionales toma sentido cuando estas finalmente consiguen ser más competitivas. Se consigue abordar la primera etapa del plan de mejora. Durante el ciclo de primavera ¿ verano del año 2021 se establecieron líneas híbridas entre las diferentes razas locales y la línea donadora de resistencias L275. Se consiguió mediante cruzamientos artificiales en los que las razas locales actuaron como parentales femeninos y la línea de mejora como parental masculino donador de polen. Aproximadamente 25-30 días después del cuajado, se aplicó con éxito la técnica de rescate de embriones inmaduros para conseguir acortar el ciclo generacional. Se consiguieron plantas de 9 de las 11 variedades tradicionales con esta metodología. La semilla de las 2 variedades restantes se recuperó cuando los frutos estuvieron completamente maduros. En el siguiente paso del programa de mejora, las plantas híbridas en madurez reproductiva se emplearon como parentales femeninos para la obtención del primer retrocruce; las variedades tradicionales actuaron en este caso como parentales masculinos. Durante esta etapa del programa las plantas se vieron afectadas por diferentes plagas e inclemencias meteorológicas, lo que alargó su duración. Se consiguieron frutos con semillas del primer retrocruce de 7 de las 9 plantas procedentes del rescate de embriones. La semilla del primer retrocruce está a la espera de ser evaluada por marcadores moleculares para que las plantas portadoras de las dos resistencias avancen hacia la siguiente etapa del plan de mejora: el segundo retrocruce. Los genotipos faltantes se iniciaron a partir de la semilla híbrida guardada para repetir el proceso de retrocruzamiento en el siguiente ciclo, aunque suponga un desfase con el resto de las razas locales., [EN] Landraces are genetic resources that can act as sources of variation in modern breeding programmes, but they can also act as recipients of variation to achieve an improvement ofgenetic resources. Local varieties provide a number of excellences since they are the result of domestication and selection by farmers, they are adapted to the climates where they have developed, they are an inexhaustible source of genes and their sale price is high. On the other hand, they tend to have low yields and a high susceptibility to new diseases, especially viruses. In order to achieve the conservation or the recovery of traditional varieties, action must be taken by incorporating resistance to viruses, so that their cultivation will be profitable and will be maintained over time. In this work, a breeding programme is initiated in which resistance genes to tomato spotted wilt virus (TSWV) and pepper mild mottle virus (PMMoV) are introduced into 11 varieties traditionally grown in Spain, from a resistant breeding line. In short, the improvement of landraces makes sense when they finally become more competitive. The first stage of the breeding plan is tackled. During the spring-summer cycle of 2021, hybrid lines were established between the different landraces and the resistance donor line L275. This was achieved by artificial crosses in which the traditional varieties acted as female parents and the breeding resistant line as the pollen-donor male parent. Approximately 25-30 days after fruit set, the immature embryo rescue technique was successfully applied to shorten the generation cycle. Plants of 9 of the 11 traditional varieties were obtained with this methodology. The seed of the remaining 2, was recovered when the fruits were fully ripe. In the next step of the breeding program, the hybrid plants at reproductive stage were used as female parents to obtain the first backcross; traditional varieties acted in this case as male parents. During this stage of the program, the plants were affected by different pests and inclement weather, which extended it duration. Fruits with seeds from the first backcross were obtained from 7 of the 9 plants coming from the embryo rescue. The seed from the first backcross is awaiting molecular marker evaluation so that the plants that carrying the two resistances can move on to the next stage of the breeding plan: the second backcross. The missing genotypes were started from the stored hybrid seed to repeat the backcrossing process in the next crop cycle, even if it means a lag with the rest of the landraces.

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.

Wild bananas are believed to have genes for resistance to biotic and abiotic stress in nature, making them potential genetic resources for creating superior varieties. Wild banana seeds, such as Musa acuminata var. tomentosa are generally difficult to germinate in vivo, so that in vitro embryo culture technique is needed. This study aim ed to increase embryo germination and regeneration of wild banana M. acuminata var. tomentosa by soaking the seeds as hydropriming. The treatment comprised of soaking the seeds in sterile distilled water for four periods of time: 0 (control), 1, 4, and 7 days. A total of 45 embryos for each treatment were planted on petri dishes containing MS + 0.5 mg/L BA + 1 mg/L biotin + 1 mg/L proline. The results showed that hydropriming increased the rate of embryo germination and regeneration. Seeds soaked for 1, 4, and 7 days successfully resulted in embryo germination percentage s of 87%, 62%, and 62%, respectively, while the control unsoaked seeds germinated with a lower percentage of 42%. One-day soaking treatment was the most optimal treatment to increase the rate of germination and regeneration as well as obtained the best vigor as demonstrated by the highest average height of plantlets, number of leaves, and roots than other treatments. Thus, 1-day seed hydropriming is the best treatment for embryo rescue and regeneration of wild banana M. acuminata var. tomentosa

Acquisition and germination of seeds are the most desired targets for the improvement of vegetatively propagated crops. In the present study, we developed a potential embryo germination protocol for the Red Globe grape cultivar having a low seed germination rate. Three grape berries at different developmental stages, viz. 50, 60 and 70 days after flowering (DAF), were selected for in-vitro embryo germination. Three growth media, namely Emershad and Ramming (ER), Nitsch and Nitsch (NN) and Murashige and Skoog (MS), and plant growth regulators (benzyl amino purine (BA), 0.5, 0.7 and 0.9 mg/l; indole butyric acid (IBA), 1.0, 1.5 and 2.0 mg/l; and gibberellic acid (GA), 0.1, 0.3 and 0.9 mg/l) were screened individually in different combinations with three amino acids, namely cysteine, glutamine and proline (2.0 μmol/l each). The maximum embryos germination percentage recorded at 70 DAF was 63.33, 47.78 and 45.56% in ER, NN and MS media, respectively, supplemented with 0.9 mg/l BA, 2.0 mg/l IBA, 0.9 mg/l GA and 2.0 μmol glutamine. Glutamine was found to have the most significant impact, and it improved the rescued embryos germination. The present study provides a potential recipe for a medium that can facilitate efficient germination of grape embryos.

El género Alstroemeria comprende cerca de 90 especies restrictas a América del Sur, de las cuales, 10 se distribuyen en la Argentina. Su cultivo ha crecido en popularidad debido a la variedad de colores y calidad postcosecha de sus flores. El objetivo de este trabajo fue el estudio de la compatibilidad entre A. psittacina y 9 variedades de alstroemeria y el ajuste de la técnica de rescate de embriones. El estudio del crecimiento del tubo polínico permitió detectar distintas situaciones según las variedades. El número de embriones germinados y la obtención de plántulas normales varió respecto a la variedad y al tiempo al rescate. En el caso de los cruzamientos con “Belvedere”, la mayor cantidad de embriones germinados se produjo a los 14 días post polinización (DPP), para “Cartagena” a los 7 DPP y para “Costa Azul” no se detectaron diferencias significativas entre ambos tiempos. Se obtuvieron plántulas en los cruzamientos con “Belvedere”, “Cartagena”, “Costa Azul”, “Himalaya” y “Virginia”. La eficiencia reproductiva mayor fue en “Costa Azul” siendo 6,6 y 8,4 para los rescates a 7 y 14 DPP, respectivamente. Estos resultados permitirán el avance del plan de mejoramiento de Alstroemeria para la obtención de variedades argentinas. The Alstroemeria genus comprises about 90 species restricted to South America, 10 species are distributed in Argentina. Its cultivation has grown in popularity due to the variety of colors and post-harvest quality of its flowers. The objective of this work was the study of the compatibility between A. psittacina and 9 alstroemeria varieties and the adjustment of the embryo rescue technique. The study of the growth of the pollen tube allowed to detect different situations according to the varieties. The number of germinated embryos and the obtaining of normal seedlings varied with respect to the variety and time to rescue. In the case of crosses with “Belvedere”, the highest number of germinated embryos was produced at 14 days post pollination (DPP), for “Cartagena” at 7 DPP and for “Costa Azul”, no signifi cant differences were detected between both times. Seed-lings were obtained in the crosses with “Belvedere”, “Cartagena”, “Costa Azul”, “Himalaya” and “Virginia”. The highest reproductive effi ciency was in “Costa Azul” being 6,6 and 8,4 for rescues at 7 and 14 DPP, respectively.These results will allow the advancement of the Alstroemeria breeding program to obtain Argentine varieties. Instituto de Floricultura Fil: Pakoca, C. Cooperativa Argentina de Floricultores; Argentina Fil: Bugallo, Veronica Lucia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Floricultura; Argentina. Fil: Facciuto, Gabriela Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Floricultura; Argentina

Present study was conducted to achieve intergeneric hybridization in Juglandaceae for the first time. Due to embryo abortion possibility, the embryo rescue technique was conducted via callus induction from immature hybrid embryo culture. For this reason, embryos were sampled at 20, 30, 40 and 60 days after pollination and cultured on two enrichment media (WPM and DKW). The developmental stage of immature embryo, kind of medium and addition of growth regulators to the induction medium played the role in promoting better callus and plantlet formation. The best quality and quantity of callus induction were achieved in 60 days’ explant. Maximum induction was in WPM and there was no callus induction in PGRs free medium. Browning was the most obstacle to conquer in callus proliferation. Germination of remained mature hybrid embryo until fruit harvest time was induced on three media (MS, WPM, and DKW). There were no significant differences between medium and male parents effect on germination parameters. But, MS and WPM performed better for hybrid embryo germination. The first germination was 7 days in MS and WPM and about 10 days for DKW after culture. In all medium the roots were robust and more developed than the leaves. The microplant transferred to soil after hardening.

Mungbean (Vigna radiata L. Wilczek) is an unique source of protein supplement with full of benefits as medicinal and climate friendly crop. Hence from past few decades, an increase has been observed in efforts of the improvement and expansion of cultivation of this pulse crop. Biotechnology in conjunction with conventional breeding techniques has a significant role in mungbean improvement programs. There is a necessity of major exploration in genetic resources, cytological, genetic, genomic, and tissue culture research to expand the vertical and horizontal base of this important crop to the level of other major legumes. Limited reports for development of transgenic Mungbean in comparison to other legume crops proves lack of an efficient plant genetic transformation protocol compatible with in vitro regeneration system. There is an utmost need to imply new technologies such as transgenic production, genome editing and conventional breeding along with embryo rescue techniques. Biotechnological approaches will speed up rather than replacement of conventional methods in mungbean breeding. The paper reviews in-depth studies on in vitro advancements of mungbean to understand the existing gaps for improvement in literature.

Undomesticated related species are the rich stock of genes providing resistance to various diseases, pests and unfavourable environmental conditions. The sexual incompatibilities are bottleneck in introgression of such genes from wild species to popular varieties. The present study was executed to standardize a protocol of embryo rescue so to obtain the hybrids of Abelmoschus esculentus × A. tetraphyllus IC141017. Crosses were made between four (Arka Anamika, Pusa Makhmali, Parbhani Kranti, Jammu Okra-05) cultivated varieties of okra (A. esculentus) and a wild species (A. tetraphyllus IC141017) in reciprocal manner. Out of the four popular cultivars used in crossing, fruit set was recorded in Arka Anamika, Pusa Makhmali, Parbhani Kranti only when A. tetraphyallus IC141017 was used as a male parent. Fruit showed distal cracking after 25 d of pollination. The seeds appeared healthy for up to 15 d after pollination and later shrivelled and became pale yellow. Cross combinations, viz., Parbhani Kranti × A. tetraphyllus IC141017, Arka Anamika × A. tetraphyllus IC141017 and Pusa Makhmali × A. tetraphyllus IC 141017 showed the highest shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. Greatest number of shoots per explant (0.92±0.12) as well as the highest frequency of shoot regeneration (86±0.12%) was reported in Parbhani Kranti × A. tetraphyllus IC141017. Arka Anamika × A. tetraphyllus IC 141017 (86±0.12%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.13%) resulted in high frequency of shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. It can be inferred that MS media containing on 0.5 mg l-1 NAA + 1.0 mg l-1 IBA showed positive results in regeneration of interspecific hybrids of okra.

Wild plant species related to crop plants form an important source of useful traits for fruit quality improvement and biotic and abiotic stress tolerance. Wide hybridization involving crop wild relatives and related taxa has gained momentum in the recent fruit crop improvement programs. It breaks the species barrier for gene transfer and makes it possible to transfer the genome of one species to another, which results in changes in genotypes and phenotypes of the progenies. The incompatibilities or barriers in wide hybridization occur as prezygotic and postzygotic barriers. Techniques of chromosome doubling, bridging species, protoplast fusion and embryo rescue are highly beneficial in recovering fertile progenies by overcoming various barriers in wide crossings. The present article reviews the techniques involved in distant crossing and their applications in fruit crop improvement.

In this essay, we respond to Dustin Crummett’s argument that one cannot consistently appeal to body count reasoning to justify being a single-issue pro-life voter if one is also committed to the usual response to the embryo rescue case. Specifically, we argue that a modified version of BCR we call BCR* is consistent with the usual response. We then move to address concerns about the relevance of BCR* to Crummett’s original thesis.

Nucellar embryony is the major obstacle in getting hybrid seedlings in interspecific crosses in citrus. Hence, the present study was conducted to standardize embryo age, culture media for maximizing germination, and subsequent identification of simple sequence repeat (SSR) markers to differentiate the hybrids. A factorial experiment was conducted with three embryo ages – 80–90, 110–120, and 130–140 days after pollination (DAP). The germination of the rescued immature embryos of acid lime × lemon crosses was initiated on three different growing media. The fruits harvested at 130–140 and 80–90 DAP had a higher number of functional seeds (90%). Older embryos (130–140 DAP) and middle-aged embryos (110–120 DAP) germinated in 100% on Murashige and Skoog (MS) medium supplemented with 500 mg·l−1 malt extract, 1.5 mg·l−1 gibberellic acid (GA3), and 0.02 mg·l−1 naphthalene acetic acid (NAA). MS medium supplemented with 500 mg·l−1 malt extract proved better for the germination of embryos taken at 80–90 DAP. Plantlet survival was the highest in younger embryos (80–90 DAP) cultured on MS basal medium (84.21%) and the lowest in older embryos cultured on MS medium supplemented with 500 mg·l−1 malt extract plus 1.5 mg·l−1 GA3 and 0.02 mg·l−1 NAA. The seedlings obtained from the culture 80–90 DAP had the highest root length (4.9 mm) and shoot length (5.3 mm) at 60 days after inoculation (DAI) on the above medium. SSR marker analysis revealed that CCSM-4 and CAC-33 markers expressed polymorphism between female and male parents, proving their ability to identify the hybrids of ‘Kagzi’ acid lime × ‘Konkan Seedless’ lemon.

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. 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.

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.

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.

Development of doubled haploid (DH) plants in wheat is an important technique for establishment of completely homozygous lines in the shortest time frame. In this study, we standardized the in vitro and in vivo variables for the production of wheat DH plants via crossing of three spring wheat genotypes with a wild grass (Imperata cylindrica). The experiment involves testing new treatments and protocol modifications for 2 years. Wheat florets were pollinated with I. cylindrica pollen after 2 days of emasculation, and next day pollinated spikes were injected with different auxin analogues: 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or 100 mg/l 3,6-dichloro-2-methoxybenzoic acid (dicamba) or 50 mg/l 2,4-D + 50 mg/l dicamba. A combination of 50 mg/l 2,4-D and 50 mg/l dicamba was superior to other treatments having maximum haploid embryos and haploid plants developed per emasculated florets. The responsiveness of embryo formation to the addition of silver nitrate (AgNO3) (the ethylene inhibitor) with the auxin treatments showed that the presence of silver nitrate in the medium may not be essential for wheat embryo induction. Time of embryo rescue had a significant effect on embryo and haploid plant production. The most successful time for embryo rescue was 24 days after pollination (DAP) resulting in maximum haploid plants developed (40% in wheat variety AKYK92 and 25% each in variety HD2888 and HS542). The immature embryos were cultured in different media, i.e. Murashige and Skoog (MS), half-strength MS, modified MS media, Gamborg’s B5 and modified B5 media. Maximum number of haploid plants were found in half-strength MS media in all accessions (60.0%) followed by modified MS media (40.00%). For colchicine concentration, during 2016–2017, maximum numbers of DHs with less plant mortality were obtained at 0.08 and 0.06% dose. Taking into account the first year results, refinement of the dose in the second year showed that colchicine doses of 0.08 and 0.075% were the best dose for maximizing DH induction in wheat; however, in this case the number of examined plants was very small. Therefore, we can treat the two doses as a guide for further research on larger samples.

Key message A simple and rapid speed breeding system was developed for short-day crops that enables up to five generations per year using LED lighting systems that allow very specific adjustments regarding light intensity and quality. Abstract Plant breeding is a key element for future agricultural production that needs to cope with a growing human population and climate change. However, the process of developing suitable cultivars is time-consuming, not least because of the long generation times of crops. Recently, speed breeding has been introduced for long-day crops, but a similar protocol for short-day crops is lacking to date. In this study, we present a speed breeding protocol based on light-emitting diodes (LEDs) that allow to modify light quality, and exemplarily demonstrate its effectiveness for the short-day crops soybean (Glycine max), rice (Oryza sativa) and amaranth (Amaranthus spp.). Adjusting the photoperiod to 10 h and using a blue-light enriched, far-red-deprived light spectrum facilitated the growth of short and sturdy soybean plants that flowered ~ 23 days after sowing and matured within 77 days, thus allowing up to five generations per year. In rice and amaranth, flowering was achieved ~ 60 and ~ 35 days after sowing, respectively. Interestingly, the use of far-red light advanced flowering by 10 and 20 days in some amaranth and rice genotypes, respectively, but had no impact on flowering in soybeans, highlighting the importance of light quality for speed breeding protocols. Taken together, our short-day crops’ speed breeding protocol enables several generations per year using crop-specific LED-based lighting regimes, without the need of tissue culture tools such as embryo rescue. Moreover, this approach can be readily applied to a multi-storey 96-cell tray-based system to integrate speed breeding with genomics, toward a higher improvement rate in breeding.

The current paper presents the estimation efficiency results of in vitro fabric culture methods used to develop the initial material for triticale breeding in the Povolzhie. A culture of immature embryos (embryo rescue) was used to obtain wheatrye amphigaploids when cross breeding durum wheat with rye. The hybrid kernels formation varied from 10.4 to 47.7%. When polluting 2223 flowers of durum wheat there were formed 768 kernels, 604 of which had embryos with different degrees of differentiation. The largest frequency of hybrid kernels formation was found in the hybrids where the mother forms were the durum wheat varieties “Amazonka” (47.7%), “Diona” (37.5%), and “Aksinit” (37.6%). Fertile amphidiploids (primary hexaploid triticale) were obtained after doubling the number of chromosomes. In vitro immature embryo cultivation was also used to obtain triticale hybrids with soft wheat in those cases when it served as the maternal parent in cross breeding. The efficiency of haploid production in the triticale anthers depended on the genotype. The frequency of embryogenic anthers formation varied from 6.7 to 17.4%, plant regeneration from the new forms was 11.4–31.4%. The 68 obtained of the 338 plants were green and 270 were albinos. In vitro microclonal propagation using segments of young heads as explants allowed preserving and propagating sterile plants for further work to restore their fertility.

Consumer demand for seedless grape with high quality and large berry has been increasing. Breeding of hypotetraploid grape was suggested as one of promising methods to satisfy it, but low frequency of hypotetraploid occurrence and low seed germination by abortive embryo were indicated as the major problem to hamper the development of hypotetraploid grape. Hence, this study was carried out to evaluate the basic efficiency of in ovulo embryo culture after the cross between hypotetraploid (‘Hanareum’) and tetraploid (‘Honey Black’ and ‘Kyoho’) grape cultivars on the establishment of hypotetraploid grapes. Embryos and plantlets were hardly obtained in ovules cultured at six after the cross pollination (WAP), but ovules inoculated at 10 WAP produced more embryos as well as plantlets regardless of cross combination. Furthermore, we found that embryo formation was not affected by the basal media in ovules cultured at six WAP, but utilization of specific medium can be more beneficial for embryo formation when ovules were cultured at 10 WAP. A total of 17 plants were obtained from ovules cultured at 10 WAP, and above 50% of plants were identified as hypotetraploid grapes. These results indicate that in vitro embryo rescue after cross pollination between hypotetraploid and tetraploid grape can enhance the efficiency for the breeding of hypotetraploid grapes.

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several genetic and methodological issues. This work aimed to regenerate viable plants from immature ovules of stenospermocarpic table grape hybrids by applying a three-step in vitro culture protocol consisting of embryo development, embryo germination-rooting, and plantlet formation. The influence of parental genotypes (six “seedless × seedless” crosses), ovule sampling time (30, 40, 50 days after pollination (DAP)), and extent of embryo germination induction (4, 6, 8 weeks) was assessed on ovule fertilization, embryo development and germination, rooting, and plantlet formation to establish the best rescue time for each combination hybrid. Our optimized protocol included immature ovule isolation for 40 DAP and embryo germination induction for 8 weeks. As for genotypes, the most efficient embryo germination was recovered from hybrids of Thompson, Superior, and Regal cultivars, whereas the highest percentage of viable plants was derived from 50-DAP ovules of Luisa × Thompson progeny. Such an optimized protocol could be useful to maximize the efficiency of future breeding programs for grape seedlessness.

In vitro plant tissue culture and biotechnology used to assist and support the development of plant breeding when classical methods of propagation must be accelerated or it was necessary to overcome barriers inaccessible by classical approaches. In asparagus, to improve multiple breeding tasks, a high number of in vitro methods have been used, such as plant regeneration methods through organogenesis, embryogenesis, manipulation of ploidy, protoplast isolation, genetic manipulation (protoplast fusion, genetic transformation), embryo rescue and germplasm preservation (in vitro, in vitro slow growth, cryopreservation). Plant tissue culture methods can overcome multiple problems in asparagus breeding such as, barriers of self and cross-incompatibility between asparagus species through embryo rescue of interspecific hybrids and protoplast fusion or genetic transformation, introgression of new genes, clonal propagation of elite genotypes of asparagus, mass screening, and the generation of haploid and polyploid genotypes, among others, becoming the tool of choice for asparagus breeding programs. Some of these in vitro methods are still under development.

A genetically determined resistance to the Asparagus virus 1 was identified within the Mediterranean wild relative Asparagus amarus. Field experiments under climatic conditions in Germany showed for A. amarus a relative low yield potential (~20%) compared with various cultivars of A. officinalis. Analyses of volatile organic compounds revealed a number of significant differences. Furthermore, the wild relative is hexaploid (2n=6x=60), expresses a strong bitter taste and is frost tolerant up to -15°C. A backcross programme was started for introgression the AV-1 resistance from the wild relative into the genome of garden asparagus. Interspecific hybrids were obtained by reciprocal crosses between A. amarus and tetraploid A. officinalis via embryo rescue approach. Four F1 progenies were cloned in vitro and transplanted in the field. Two progenies showed resistance to AV-1, while two progenies were susceptible.

Powdery mildew (PM), caused by Erysiphe cruciferarum, is an epidemic of oil rapeseed (Brassica napus L.) growing worldwide, but PM resistant germplasm is rare in this species. We screened 102 accessions of B. napus and other cruciferous species and found an Ethiopian mustard (Brassica carinata) cultivar 'White flower' immune to PM in both the field and greenhouse. Outcrossing in the female parent 'White flower' was promoted by using a chemical gametocide tribenuron-methyl, to obtain hybrid seeds of distant hybridization with an elite B. napus cultivar 'Zhongshuang11'. Three true F1 hybrids with B. carinata cytoplasm were obtained without using embryo rescue, which showed complete male sterility and light yellow petals. The hybrid plants and the progenies derived from backcrossing were validated using morphological traits, seed quality, and molecular markers. Five lines in the BC1F3 generation, named 'W7-1', 'W7-4', 'W7-6', 'W8-1', and 'W8-3', and one BC2F2 line 'W3PS-1', whose young leaf was yellow green, were identified to be resistant or moderately resistant to PM. The seed quality and some morphological traits of these lines resembled the parent 'Zhongshuang11', indicating that the resistance gene(s) has been preliminarily introduced into B. napus.

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. 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.

Cereal breeding programs are interested in increasing the number of generations per year to reduce the time needed to develop new cultivars. A common method to accomplish this is to extend the photoperiod to speed up plant growth. For oat, this method is problematic because the species responds to changes in light and temperature. Current methods of fast generation cycling in oat require embryo rescue, which is labor intensive and has a low success rate. Recently a method was developed using increased photoperiod and foliar mineral supplement to reduce generation time for wheat and barley. We evaluated this newly published method in oat and found that anthesis occurred 15 ± 3 days faster, however there was a 3-fold reduction in seed count and a 2-fold reduction in inflorescence weight. In addition, we measured endogenous ascorbate to evaluate the physiological status of the plants under fast generation cycling conditions. For oat, fast generation cycling would be effective to more rapidly advance populations using single seed descent, but not as useful when seed yield is important.

Turuncgil islahinda; tur ve cesitler arasinda uyusmazlik, uzun genclik kisirligi, gametik kisirlik ve polyembriyoni gibi nedenlere bagli olarak bircok engel bulunmaktadir. Poliembriyonik turuncgillerin tohumlarindaki zigotik embriyo gelisim sirasinda nuseller embriyolar tarafindan dejenere edilmektedir. Buna gore, zigotik embriyolarin cimlenmesini ve gelisimini saglamasi nedeniyle embriyo kulturu islah calismalarinda yararli bir aractir. Bu calismada, yeni anac bireylerinin elde edilmesi amaciyla Yerli mandarin x Carrizo sitranji melezlenmistir. Embriyo kurtarma icin uygun embriyo gelisim asamasinin belirlenmesi amaciyla olgunlasmamis embriyolar tozlanmadan 80, 100 ve 120 gun sonra alinmistir. Daha sonra, alinan embriyolar 0, 0.5 ve 1 mg l -1 GA 3 iceren Murashige ve Tucker (MT) besi ortaminda cimlendirilmistir. Sonuclar; en yuksek cimlenme oranlarinin tozlanmadan 120 gun sonra alinan embriyolarda 1 mg l -1 GA 3 iceren ortamda %100 ile ve 0.5 mg l -1 GA 3 iceren ortamda %95 oldugunu gostermistir. Tozlanmadan 80 gun sonra alinan embriyolarin 0 mg l -1 GA 3 (kontrol) ve 0.5 mg l -1 GA 3 iceren ortamlarda cimlenme olmamistir. Genel olarak, ortama GA 3 ilavesi kontrole gore cimlenme oranini artirmaktadir. En fazla uc yaprakli bitki orani %33.33 ile tozlanmadan 100 ve 120 gun sonra alinan ve sirasiyla 0.5 ve 1 mg l -1 GA 3 iceren ortamlardan elde edilmistir. Sonuclar, turuncgillerde embriyo kurtarma icin en uygun zamanin tozlanmadan sonraki 120 gun oldugunu ve ortamda GA 3 bulunmasinin cimlenmeyi artirdigini gostermistir.

Cross-incompatibility between peanut cultigen and species outside section Arachis, impedes the transfer of desirable traits such as high resistance, good quality and high productivity from incompatible species in cultivar development. To recover hybrids from 4 incompatible crosses involving 3 wild species, viz., A. pusilla Benth., A. appresipilla Krapov. & W.C. Greg. and A. paraguariensis Chodat & Hassl., an in situ embryo rescue technique was exploited, resulting in hybrid seeds from all the 4 crosses. The key of this technique is to apply growth substances to pollinated flower bases, and the next step is to wait and harvest seeds directly, whose hybridity is to be tested by miniature inverted-repeat transposable element markers. The technique is advantageous over its in vitro counterpart in earlier intervention, easier operation, and shorter duration required for obtaining hybrids. These superiorities warrant its wide use in the genus Arachis as well as in other plant taxa where post-zygotic hybridization barriers also exist.

Seedlessness in commercial table-grape cultivars is caused by stenospermocarpy, which results in abortion of the embryo before complete development. The technique of embryo rescue and culture in vitro, before abortion, allows crosses between seedless cultivars as parents, with the main advantage of increasing the frequency of seedless individuals in the progeny. The present work aimed to evaluate the efficiency of table-grape breeding by controlled hybridizations and embryo rescue in the development of new table-grape cultivars for tropical semiarid conditions in the northeast of Brazil. The crosses were carried out in experimental fields of Embrapa Semiarido in Petrolina, PE, and Juazeiro, BA, in the Sao Francisco River Valley, between 2011 and 2017. During this period, 689 inflorescences were pollinated, resulting in 55.9% fruitfulness, of which 11,685 seed traces were obtained and inoculated in vitro. It was possible to rescue 16.9% of immature embryos, obtaining 56.1% success in seedling germination. The responses observed depended strongly on the genotypes used in the crosses. Genotypes ‘CG 351’ and ‘Marroo Seedless’ used as female parents showed the best results for fruitfulness and the formation and germination of embryos, which means that the number of seedlings obtained by the embryo rescue technique is correlated with the genotype used as parent. Also noteworthy is the progress in results obtained in 2015 and 2016 compared with the period 2011-2014, when ‘Marroo Seedless’ was used as a female parent in crosses using pollen of ‘A1105’, ‘A Dona’, ‘BRS Linda’, ‘BRS Isis’, ‘BRS Clara’, ‘BRS Vitoria’, ‘CG 351’, ‘Feal’ and ‘Jupiter’, obtaining high rates of fruitfulness, embryo rescue and embryo germination in all combinations.

The widely divergent species 4xTrifolium ambiguum and 2xT.occidentale are inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids (T. repens × T. ambiguum × T. occidentale) have the potential to achieve introgression of stress resistant traits from both wild species into white clover. Trifolium ambiguum and T. occidentale are geographically, adaptionally and phenotypically contrasting species in the white clover section (Trifoliastrum) of the genus. T. ambiguum occurs as a high-altitude polyploid series (2x, 4x, 6x) in W Asia and NE Europe. T. occidentale is a diploid coastal species, occurring at sea level in W Europe. This study investigated hybridisation between 4xT. ambiguum and 2xT. occidentale and considered the significance of the hybrids for introgression breeding of white clover. Partially fertile F1 hybrids between 4xT. ambiguum and 2x and 4xT. occidentale were generated by embryo rescue. Hybrid plant morphology and fertility varied widely and hybrids generally expressed traits from both species. Advanced generation (F2–F5) 4x hybrids were highly fertile and constitute a new synthetic allotetraploid species. FISH analyses of 4x hybrids showed multivalent chromosome configurations with homoeologous associations between T. ambiguum and T. occidentale chromosomes. Crosses of the hybrids with T. repens produced fertile tri-species progeny. These very divergent species remain inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids have the potential to achieve introgression of stress resistance traits from both wild species into white clover.

High frequencies of homoeologous and even non-homologous chromosome recombination in Brassica hybrids can transfer useful traits between genomes, but also destabilise synthetic allopolyploids. We produced triploid hybrids (2n = 3x = ABC) from the cross B. napus (rapeseed, 2n = 4x = AACC) × B. nigra (black mustard, 2n = 2x = BB) by embryo rescue and allohexaploid hybrids (2n = 6x = AABBCC = 54) by chromosome doubling of the triploids. These hybrids demonstrated resistance to blackleg disease (causal agent: Leptosphaeria maculans) inherited from their B. nigra parent. In order to assess the possibility of transfer of this resistance between the B genome and the A and C subgenomes of B. napus, as well as to assess the genomic stability of allohexaploids from the cross B. napus × B. nigra, frequencies of non-homologous chromosome pairing in these hybrids were assessed using classical cytogenetics and genomic in-situ hybridization. Meiosis was highly irregular, and non-homologous chromosome pairing between the B genome and the A/C genomes was common in both triploid hybrids (observed in 38% of pollen mother cells) and allohexaploid hybrids (observed in 15% of pollen mother cells). Our results suggest that introgression of blackleg resistance from the B genome into the A or C genomes should be possible, but that allohexaploids from this genome combination are likely unstable.

Interspecific hybridization was carried out between sterile analogues of cultivated sunflower lines with normal cytoplasm and wild annual Helianthus bolanderi accession E-009 from collection of DAI-General Toshevo. Hybrid plants were produced using classical breeding methods and the biotechnological method embryo rescue. The degree of crossability and the inheritance of some morphological traits were determined. The obtained F1 progenies were characterized from morphological and phytopathological point of view. Hybrid forms distinguished with resistance to stem canker, phoma and downy mildew were obtained. The hybrid plants, carriers of Rf genes for CMS Pet 1, could be used in sunflower breeding programs for developing restorer lines.

Production and use of double haploids is important for attainment of systematic genetic gains, and indeed several plant breeding programmes have benefited from it. Gynogenesis, the in vitro culture of unfertilized ovules and/or embryos has specifically been exploited in several economically important crops but not cassava. In this study, we examined possibilities of generating doubled haploids (DH) in cassava through gynogenesis, by bagging female flowers of selected varieties to prevent pollination. A total of 2,466 flowers across 32 elite cassava varieties were bagged for a period of one-to-three days. Early embryo rescue and ovule culture were done at 7 to 42 days after anthesis. Consequently, 517 fruits (21%) were harvested and dissected to obtain 97 seeds from which 47 unique embryos and 18 callus lines were obtained in vitro. Only six of the rescued embryos (12.8%) regenerated into plantlets. Upon undertaking ploidy analysis and single nucleotide polymorphism (SNP) genotyping, it was established that all samples analyzed (regenerated plants and calli) were diploid. SNP genotyping revealed increased homozygosity (up to 85.7%), but no doubled haploids were noticed. The knowledge generated is a significant contribution towards understanding cassava flowering biology and thus a foundation to on-going efforts towards developing protocols for generation of cassava DH. Key words: Anthesis, embryo rescue, gynogenesis, homozygosity, ploidy.

Seedlessness is a primary breeding objective for citrus fresh fruit market, and triploids have been proven to have great value to produce seedless fruits. In this study, aiming to produce triploid plants for developing some seedless cultivars, four 2x × 4x interploid crosses were conducted using three elite but seedy cultivars as seed parents and one newly flowered doubled diploid and two allotetraploid somatic hybrids as pollen parents. As a result, a total of 1454 developed and 3409 undeveloped seeds from 341 fruits were obtained. Using an optimized embryo rescue approach, 669 developed and 1301 undeveloped seeds germinated in vitro, with an average germination rate as 52.5% for the crosses using ‘Nadorcott’ tangor and ‘Bendizao’ tangerine (polyembryonic) as seed parents and 31.0% for the crosses using ‘Orah’ mandarin (monoembryonic) as seed parent. Then by shoot and root induction, totally 1354 plantlets were regenerated, among which 401 and 54 plants were proved to be triploids and tetraploids by flow cytometry (FCM) analysis and chromosome counting. Hybrid nature of the selected triploid progenies, as well as two different origins (doubled diploid and hybrid origins) for the selected tetraploid progenies was further confirmed by single nucleotide polymorphism (SNP) markers.

The embryo rescue case (ERC) is a thought experiment that is used to argue against the view that embryos have a right to life (i.e., are persons). I will argue that cognitive science undermines the intuition elicited by the ERC. I will show that whether or not embryos have a right to life, our mental tools will make it very difficult to believe that they do have the said right. This suggests that the intuition elicited by the ERC is not truth-indicative. The upshot of this is that we have an undercutting defeater for our intuition that embryos do not have a right to life. Thus, the ERC is a bad argument against embryonic personhood.

Interspecific hybridization is an excellent way to create germplasm resources and is considered an important driver of genome evolution. We selected one Chinese cabbage variety and one red cabbage variety for hybridization. Heterologous haploid offspring were obtained by embryo rescue, and heterologous diploids were obtained by colchicine-induced chromosomal doubling. The field traits and simple sequence repeat markers of 98 F2 plants were investigated. The result showed that the parental traits were separated in the hybrids, with traits tending to shift from those of Chinese cabbage to those of red cabbage, and trait values showed normal distributions. Simple sequence repeat patterns varied, with the number of missing bands being significantly greater than that of novel bands in hybrids. Thus, the phenotypes of the early formed allopolyploids were unstable and accompanied by dramatic changes in the genome. This study not only increased the genetic resources available for B. napus but also laid a theoretical foundation for exploring trait segregation in early formed allopolyploids.

The international ornamentals industry is benefiting from perennial plants developed in New Zealand (NZ) such as begonia Bonfire™, limonium Sinzii™, and the gentian Showtime™ series. The 'Bonfire' series of begonia, with its distinctively new flower shape, was released in the year 2000. While flower and foliage colour were important; architectural form, vigour and floral productivity were key selection criteria. Understanding the underlying physiology of plant architecture (physiologically informed breeding) was key in the introduction of subsequent new cultivars to the series. These screening criteria allowed us to evaluate annually ten times more begonia seedlings per season. Planting of limonium Sinzii continues to expand exponentially: in 2018/19 plantings almost doubled compared with those of the previous year. The limonium Sinzii range resulted from successful hybridisation between Limonium sinuatum and L. perezii. Embryo culture, chromosome doubling, and backcrossing were some of the in vitro and in vivo tools and strategies applied to achieve the current range. The sterile gentian 'Little Pinkie' was another plant developed using wide crosses and embryo rescue. As with other gentians in the Showtime series, a physiologically informed breeding strategy was used. In the case of 'Little Pinkie', an understanding of how crown buds arise was used to generate at least double the number of shoots on propagules. This has underpinned the success of this crop for us, and our industry partners. The preceding examples illustrate how, for each crop species, our breeding programmes have diversified germplasm, through exploiting a diverse skill base within the team. Our conclusion is that our most successful examples of breeding programmes have been those in which plant breeders, plant physiologists, in vitro specialists, horticulturalists, and marketers are all actively engaged.

Key message Statistical analysis of the chromosomal composition in a population of 210 primary plants regenerated from two intergeneric wheat–barley cross combinations revealed the random nature of uniparental elimination for barley chromosomes. Abstract Uniparental chromosome elimination is a common process in interspecific and intergeneric cereal hybrids. To characterize the frequency of paternal chromosomes, a population of 218 independent green plants was generated from two wheat (♀) × barley (♂) cross combinations via embryo rescue. The chromosomal composition of 210 primary plants was analyzed with chromosome-specific DNA markers representing all seven barley chromosomes. The analysis revealed an equal proportion of haploid and full hybrids (20.5% and 19.5%, respectively), while the rest of the population contained hypoploids (partial hybrids) with no preference for any possible numbers (one to six) of barley chromosome additions. Contrary to the previous reports, there was no statistical bias or preferential elimination for any individual barley chromosome (1H–7H) in this population. The reasons for the apparent contradiction and the implications of the above findings for cereal breeding are discussed. Electronic supplementary material The online version of this article (10.1007/s00299-019-02405-1) contains supplementary material, which is available to authorized users.

In order to cultivate new high-quality lily cultivars with good ornamental and edible traits, hybridization between Lilium brownii var. viridulum and L. davidii var. unicolor was conducted with cut style pollination and embryo rescue. 39 progenies were obtained. FISH with 5S rDNA and 45S rDNA as probes were used to analyze the parents and four progenies. The results showed that all of them are diploid (2n=2x=24); L. brownii has 10 loci of 5S rDNA on chromosomes (CH)-1, -2, -4, -8 and -11 and has 6 loci of 45S rDNA on CH-3, -9 and -10; L. davidii has 6 loci of 5S rDNA distributed on chromosomes CH-8, CH-10, and CH-11 and has 8 loci of 45S rDNA on chromosome CH-1, CH-5, CH-7 and CH-10; all four progenies have 8 loci of 5S rDNA and 7 loci of 45S rDNA, unambiguously, it was concluded that they are true hybrids coming from L. brownii and L. davidii.

The unreduced (2n) gametes have long been used in triploid breeding of citrus. In lemon, the previously reported mechanisms for 2n megagametophyte formation were controversial, whereas that for 2n pollen production is still unknown. Herein, the frequency of and mechanism underlying 2n megagametophyte and 2n pollen formation in ‘Eureka’ lemon were investigated based on cytological observation and genotyping of the triploid hybrids between ‘Eureka’ lemon and ‘Early gold’ sweet orange. As a result, 4.79% of the viable pollens of ‘Eureka’ lemon were identified as the 2n pollen with a larger diameter (70.16 ± 3.92 µm). The 2n pollen might be resulted from the formation of parallel spindles at meiosis stage II. Among the 204 plantlets regenerated from embryo rescue following the sexual cross, 12 were triploids as identified by flow cytometry. According to the analysis of heterozygosity transmission using 13 pericentromeric single nucleotide polymorphism (SNP) markers and 20 randomly distributed simple sequence repeat (SSR) markers, 11 triploids were identified to be originated from the fertilization of 2n megagametophytes of ‘Eureka’ lemon, with a frequency of 5.39%. Among them, nine 2n megagametophytes were supposed to be arisen from the second division restitution (SDR), whereas the other two were from post-meiotic genome doubling (PMD). These results to understand the mechanism underlying 2n gamete formation in lemon are valuable for its efficient polyploid breeding.

La técnica de hibridación a través de plantas puentes, utiliza cruzamientos indirectos o intermedios como instrumento para cruzar parentales con diferente nivel de ploidía. El objetivo de este trabajo fue obtener híbridos que puedan ser utilizados como puentes en cruzamientos con especies ornamentales del género Petunia y/o Calibrachoa. Lo anterior permite sortear las barreras de incompatibilidad que surgen cuando la hibridación es directa entre especies. Para la obtención de híbridos se realizaron cruzamientos dirigidos entre: P. integrifolia, P. inflata, P. interior y P. axillaris subespecie axillaris; con C. linoides, C. ovalifolia, C. thymifolia, C. caesia, C. x hybrida var. Kabloom blue y C. x hybrida var. Kabloom yellow. El primer paso fue la detección de combinaciones putativas, para lo cual se comparó la tasa de fructificación definida como la formación de frutos cada 10 cruzamientos intergenéricos, encontrando resultados variables para cada combinación. Se obtuvo una planta híbrida entre P. inflata x C. x hybrida 'Kabloom yellow', en que los estudios morfológicos, de viabilidad del polen y meióticos confirman su naturaleza. Con el híbrido puente obtenido se realizaron cruzamientos recíprocos con todas de las especies de Petunia y Calibrachoa involucradas, obteniendo 31 plantas híbridas que comparten características morfológicas de ambos géneros en las combinaciones con P. inflata y P. integrifolia. La identificación de cruzamientos intergénericos con alto porcentaje de fructificación abre una ventana para futuros estudios sobre el desarrollo de los embriones híbridos. Esto permitirán conocer el momento oportuno para aplicar técnicas de rescate embrionario con el objeto de aumentar el número de plantas híbridas puentes. The hybridization technique through bridging plants uses indirect or intermediate crosses as an instrument to cross parents with different levels of ploidy. The objective of this work is to obtain hybrids that can be used as bridges in crosses with ornamental species of the genus Petunia and/or Calibrachoa. This can overcome the incompatibility barriers that arise when hybridization is direct between species. To obtain hybrids, directed crosses were carried out between: P. integrifolia, P. inflata, P. interior and P. axillaris subspecies axillaris; with C. linoides, C. ovalifolia, C. thymifolia, C. caesia, C. x hybrida var. Kabloom blue and C. x hybrida var. Kabloom yellow. The first step was the detection of putative combinations. In order to do so, the fruiting rate, which was defined as the formation of fruits every 10 intergeneric crosses, was compared, and variable results were found for each combination. A hybrid plant was obtained between P. inflata x C. x hybrida 'kabloom yellow', where morphological, pollen viability and meiotic studies confirm its nature. With the bridge hybrid obtained, reciprocal crosses were carried out with all of the Petunia and Calibrachoa species involved, obtaining 31 hybrid plants sharing morphological characteristics of both genera in combinations with P. inflata and P. integrifolia. The identification of intergenetic crosses with a high fruiting rate opens doors for future studies on the development of hybrid embryos. This would allow determining the optimum time to apply embryo rescue techniques in order to increase the number of hybrid bridging plants. Instituto de Floricultura Fil: Milicia, Víctor José. Universidad Nacional de Lomas de Zamora. Facultad de Ciencias Agrarias; Argentina Fil: Rodríguez, María Fabiana. Universidad Nacional de Lomas de Zamora. Facultad de Ciencias Agrarias; Argentina Fil: Hagiwara, Juan Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Floricultura; Argentina Fil: Soto, Maria Silvina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Floricultura; Argentina

Somatic embryogenesis is the best demonstration of totipotency in higher plants in which somatic cell produce whole plant like zygotic embryo. It is also demonstrated that immature, weak, hybrid or sometimes inviable embryos can be saved through in vitro culture to prevents its degradation. It may help to cross the reproductive barriers when interspecific hybrids developed. Brasssica is an economically valuable oil yielding and vegetable crop and India is the largest producer of oil seed rape in the world. Various factors affect the embryo rescue in Brassica like growth stage of the embryos, types and composition of the rescue medium etc. The embryo regeneration potential can improve through the modification of culture conditions in both zygotic as well as somatic embryo. Except the embryo culture other parts like ovule, ovary culture can also be done to developed interspecific hybrids. This chapter is focused on the embryo rescue techniques in the genus Brassica and summarizes possible ways of improving the technique used.