Embryo Rescue Research Articles

Optimizing Recovery of Hybrid Embryos from Interspecific Citrus Crosses of Polyembryonic Rough Lemon (Citrus jambhiri Lush.)

The prevalence of rough lemon rootstock in India is presently threatened by the spread of Phytophthora root rot. Developing an alternative Phytophthora-resistant rootstock that also withstands other abiotic constraints is a priority for a sustainable citrus industry in India. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Citrus and Poncirus, a closely related genus. The production of hybrids accumulating the desired dominant traits, irrespective of the heterozygosity level of the parents, is an effective way to develop new candidate rootstocks. Rough lemon was hybridized with Swingle citrumelo, X-639 citrandarin, and GouTou Cheng. A reliable method was developed for greater recovery of hybrid seedlings from these crosses on the basis of morphology and simple sequence repeat (SSR) markers. The greatest proportion of hybrid seedlings resulted from embryo rescue at 95 days after pollination (DAP) (48.5% in rough lemon × X-639 citrandarin and 56.7% in rough lemon × Swingle citrumelo) on the basis of multifoliate leaves. The proportion of hybrid seedlings decreased with increase in embryo development stage after 95 DAP until the last stage of observation (125 DAP). Significantly higher proportion of multifoliate seedlings was recovered by in vitro culture compared with in vivo germination from the mature seeds (26.9% vs. 5.2%, respectively). Swingle and X-639 citrandarin are not homozygous for the multifoliate allele from Poncirus, and GouTou Cheng lacks the multifoliate allele; thus, unifoliate hybrids were selected from all crosses on the basis of SSR marker genotypes.

Morphological observations in durum wheat after embryo manipulations

In cereals, haploid or diploid embryo structures are suitable organisations in practice for amelioration or storage of genotypes or some other biotechnologies. It is possible to reveal of variation as different genotypes from heterozygote or landraces after making homozygote of recessive alleles by doubling of chromosomes using embryo rescue. In these genotypes, characters can be evaluated as qualitative or quantitatively, for instance; dark purple coleoptile could be observed in some doubled haploid (DH) genotypes. The amount of chlorophyll (a/b) and carotenoid have been evaluated in young leaves in DH-6 and DH-8 and some other durum wheat genotypes, while investigating their tolerances to salinity in laboratory conditions. Colour of grains (amount of carotenoid in harvested grains) in durum genotypes have also been evaluated with field experiment conditions in three different locations of Anatolia and also correlation (r) has been identified with other grain characters related with agronomical values. Here it is possible to determine the rate into correlation (path) of characters and direct and indirect effects of each other. Naturally, the relationships between amount of chlorophyl in young leaves and carotenoid in harvested grains and also harvest index (HI) of genotypes will be able to be interesting subjects to discuss.

Production of synthetic wheat lines to exploit the genetic diversity of emmer wheat and D genome containing Aegilops species in wheat breeding

Due to the accumulation of various useful traits over evolutionary time, emmer wheat (Triticum turgidum subsp. dicoccum and dicoccoides, 2n = 4x = 28; AABB), durum wheat (T. turgidum subsp. durum, 2n = 4x = 28; AABB), T. timopheevii (2n = 4x = 28; AAGG) and D genome containing Aegilops species offer excellent sources of novel variation for the improvement of bread wheat (T. aestivum L., AABBDD). Here, we made 192 different cross combinations between diverse genotypes of wheat and Aegilops species including emmer wheat × Ae. tauschii (2n = DD or DDDD), durum wheat × Ae. tauschii, T. timopheevii × Ae. tauschii, Ae. crassa × durum wheat, Ae. cylindrica × durum wheat and Ae. ventricosa × durum wheat in the field over three successive years. We successfully recovered 56 different synthetic hexaploid and octaploid F2 lines with AABBDD, AABBDDDD, AAGGDD, D1D1XcrXcrAABB, DcDcCcCcAABB and DvDvNvNvAABB genomes via in vitro rescue of F1 embryos and spontaneous production of F2 seeds on the Fl plants. Cytogenetic analysis of F2 lines showed that the produced synthetic wheat lines were generally promising stable amphiploids. Contribution of D genome bearing Aegilops and the less-investigated emmer wheat genotypes as parents in the crosses resulted in synthetic amphiploids which are a valuable resource for bread wheat breeding.

Meiotic study of Zea mays ssp. mays (2n = 40) x Tripsacum dactyloides (2 n = 72) hybrid and its progeny

Maize (2n = 40) x Tripsacum dactyloides (2n = 72) F1 hybrid plants (2n = 56) were obtained by embryo rescue and induction of somatic embryogenesis/organogenesis. Hybrid plants showed Tripsacum -like phenotypes, tolerance to stresses such as NaCl salinity and low temperatures. The more frequent meiotic configurations were 28II (24%), 24II + 2IV (19%) and 26II + 1IV (12%), with an average per cell of 0.55I + 25.18II + 1.19IV. Significant differences between plants were not observed. Pollen fertility ranged from 0% to 50%. After pollination with maize or Tripsacum , 20% of F1 plants have developed viable seeds, which originated the progeny. Thirty five percent of the progeny showed 2n = 56 chromosomes and F1 like-phenotypes, which suggests they have apomictic origin. The remaining plants were fertile and they showed maize-like phenotypes and different chromosome numbers (2n = 22, 24, 26, 28 and 30), because they kept the complete maize chromosome complement and some of the Tripsacum chromosomes. Meiotic cells showed pairing between chromosomes from both parental species, which suggests the possibility of genetic recombination between them.

Development of Interspecific Hybrids between a Cultivated Eggplant Resistant to Bacterial Wilt (Ralstonia solanacearum) and Eggplant Wild Relatives for the Development of Rootstocks.

Bacterial wilt caused by Ralstonia solanacerum is one of the most economically and destructive eggplant diseases in many tropical and subtropical areas of the world. The objectives of this study were to develop interspecific hybrids, as potential rootstocks, between the eggplant (Solanum melongena) bacterial wilt resistant line EG203 and four wild accessions (S. incanum UPV1, S. insanum UPV2, S. anguivi UPV3, and S. sisymbriifolium UPV4), and to evaluate interspecific hybrids along with parents for resistance to bacterial wilt strains Pss97 and Pss2016. EG203 was crossed successfully with wild accessions UPV2 and UPV3 and produced viable seeds that germinated when wild accessions were used as a maternal parent in the crosses. In addition, viable interspecific hybrids between EG203 and UPV1 were obtained in both directions of the hybridization, although embryo rescue had to be used. Hybridity was confirmed in the four developed interspecific hybrid combinations with three SSR markers. EG203 was resistant to both strains Pss97 and Pss2016, while UPV1 and UPV3 were, respectively, resistant and moderately resistant to Pss2016. The four interspecific hybrids with UPV2, UPV3, and UPV1 were susceptible to both bacterial wilt strains, indicating that the resistance of EG203, UPV1, and UPV3 behaves as recessive in interspecific crosses. However, given the vigor of interspecific hybrids between eggplant and the three cultivated wild species, these hybrids may be of interest as rootstocks. However, the development of interspecific hybrid rootstocks resistant to bacterial wilt will probably require the identification of new sources of dominant resistance to this pathogen in the eggplant wild relatives.

Production of Tetraploid and Triploid Hemp

To maximize yield, cannabidiol (CBD) hemp producers prefer female plants, and this is accomplished by using expensive feminized seed, vegetatively propagated female clones, or by removing male plants from dioecious seed lots. Hemp pollen drifts long distances on wind, and pollination of females reduces CBD content. Induction of triploidy is a common strategy used by plant breeders to produce sterile cultivars of agricultural crops. Triploid (3n) hemp, with three sets of chromosomes, was developed by crossing naturally diploid (2n) hemp with tetraploid (4n) hemp. Tetraploid plants used to create triploids were produced using pregerminated seeds and the mitotic spindle inhibitor colchicine. Seedlings from seeds of ‘Abacas’ × [(‘Otto2’ × ‘BaOx’) × (‘BaOx’ × ‘Colorado Cherry’)] treated with 0.05% colchicine or 0.02% colchicine for 12 hours and longer were significantly shorter than controls and ≤1 cm tall at 10 days after sowing. Surviving seedlings exhibited thickened cotyledons and hypocotyls, which indicated a potential change in ploidy. Tetraploid induction ranged from 26% to 64% for pregerminated seeds of five different hemp cultivars (Abacus × Wife, Cherry Wine, Mountain Mango, Wife, and Youngsim10) treated with 0.05% colchicine for 12 hours. Tetraploids had nearly twice the DNA content as diploids according to flow cytometric analysis. Tetraploid ‘Wife’ had larger stomates and reduced stomatal density compared with diploid ‘Wife’. Four triploid ‘Wife’ genotypes produced from crossing tetraploid ‘Wife’ with diploid ‘Wife’ were acclimated to greenhouse conditions after embryo rescue. DNA content and stomate size of triploid ‘Wife’ was intermediate between the parents. This is the first report of triploid plants of hemp. Future research will evaluate the sterility of triploid hemp.

Positive effect of in vitro embryo rescue on breaking the dormancy of wild banana seeds compared to direct sowing in the greenhouse

Positive effect of in vitro embryo rescue on breaking the dormancy of wild banana seeds compared to direct sowing in the greenhouse

The process of embryo abortion of stenospermocarpic grape and it develops into plantlet in vitro using embryo rescue

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.

Challenges for Ex Situ Conservation of Wild Bananas: Seeds Collected in Papua New Guinea Have Variable Levels of Desiccation Tolerance.

Ex situ seed conservation of banana crop wild relatives (Musa spp. L.), is constrained by critical knowledge gaps in their storage and germination behaviour. Additionally, challenges in collecting seeds from wild populations impact the quality of seed collections. It is, therefore, crucial to evaluate the viability of seeds from such collecting missions in order to improve the value of future seed collections. We evaluate the seed viability of 37 accessions of seven Musa species, collected from wild populations in Papua New Guinea, during two collecting missions. Seeds from one mission had already been stored in conventional storage (dried for four months at 15% relative humidity, 20 °C and stored for two months at 15% relative humdity, −20 °C), so a post-storage test was carried out. Seeds from the second mission were assessed freshly extracted and following desiccation. We used embryo rescue techniques to overcome the barrier of germinating in vivo Musa seeds. Seeds from the first mission had low viability (19 ± 27% mean and standard deviation) after storage for two months at 15% relative humidity and −20 °C. Musa balbisiana Colla seeds had significantly higher post-storage germination than other species (p < 0.01). Desiccation reduced germination of the seeds from the second collecting mission, from 84 ± 22% (at 16.7 ± 2.4% moisture content) to 36 ± 30% (at 2.4 ± 0.8% moisture content). There was considerable variation between and (to a lesser extent) within accessions, a proportion of individual seeds of all but one species (Musa ingens N.W.Simmonds) survived desiccation and sub-zero temperature storage. We identified that seeds from the basal end of the infructescence were less likely to be viable after storage (p < 0.001); and made morphological observations that identify seeds and infructescences with higher viability in relation to their developmental maturity. We highlight the need for research into seed eco-physiology of crop wild relatives in order to improve future collecting missions.

Reconsidering the direct cross approach for gene transfer from Aegilops tauschii to hexaploid wheat using diversified D genome donors

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 development of woody and perennial Hibiscus species

Embryo development is the precursor of seeds, which are important to produce interspecific hybrid plants. The objective of this study was to investigate the embryo development of interspecific crosses between Hibiscus species and provide useful information, for the timely embryo rescue of potential F1 progenies. Hibiscus species were hand-pollinated and tagged on the day of anthesis. Starting two days after pollination (DAP), the pods were collected for observation under a stereomicroscope. Hibiscus species showed three stages of embryo development. Endosperm formation in the cross-pollinated ovules occurred from 2 to 7 DAP for H. sinosyriacus and 2–4 DAP for H. moscheutos in stage I. The ovules slowly increased in size. Embryo development was observed from 8 to 13 DAP in H. sinosyriacus and 5–8 DAP in H. moscheutos at stage II. The endosperm triggered the development of the embryo. Then, the endosperm supported the embryo histodifferentiation through globular, heart, and torpedo-shaped stages inside the growing ovule in stage III. After reaching the maximum ovule size, embryo growth until maturation occurred from 14 to 30 DAP in H. sinosyriacus, and 9–15 DAP in H. moscheutos at stage III. The embryo completely absorbed the endosperm, and the mature embryo fully occupied the ovule in stage III. As a result, ovules from cross-pollinated deciduous shrub-type H. sinosyriacus exhibited slower embryo development compared with the perennial H. moscheutos with fast embryo maturation. Embryo rescue may be possible starting at Stage II because the globular embryo can be observed under the microscope.

Development of Interspecific Hybrids (Abelmoschus Esculentus × A. Tetraphyllus) in Okra Using Embryo Rescue Approach

ABSTRACT: 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.

In vitro efficiency of embryo rescue of intra- and interspecific hybrid crosses of sweet cherry and Chinese cherry cultivars

Hybrid cherry seedlings are very hard to obtain as the embryos easily abort during development and loose germination ability, especially some interspecific hybrids. An in vitro technique of embryo rescue has been developed for intra- and interspecific hybrids with the embryos by crossing 4 sweet cherry genotypes as maternal, and these have been planted in South China where the climate is not ideal for planting sweet cherry due to its hot and humid climate in summer and warm climate in winter. The success of embryo rescue approach is affected by several factors, in which the genotypes are one of the main factors for embryo germination and significant differences were found with the same genotypes that were used differently either as maternal or paternal in cross breeding. ‘Chaoyang-1’ (‘CY-1’) as mother performed the best, and ‘Zijing’ (‘ZJ-1’) and ‘CY-1’ were the best parents as fathers in cross breeding. The embryo development stage, breaking dormancy method and in vitro culture condition significantly affected the efficiency of seedlings production. When the development of their embryos reached to 0.5 to 0.7, then the young fruit was harvested and stored at 4 °C for 35 days were found to be the best initiating materials for culturing, and the embryo germination rates after removal of the embryo coat reached up to 69.84 ± 2.75 %, and their shoots were robustly and fastly grown. The medium when combined with different concentrations of plant growth regulators (PGRs) were tested, the optimal medium for immature embryo culturing was found to be 1/2 MS supplemented with 2 mg L−1 BA and 1.8 mg L−1 IAA, the multiplication of shoots culture media was MS +0.3 mg L−1 BA +0.4 mg L−1 IAA or MS +0.7 mg L−1 BA +0.1 mg L−1 IAA, the 1/2 MS medium supplemented with 0.3 mg L−1 IBA and 1.1 mg L−1 IAA showed the best for the rooting of shoots. Taken together, this system by integrating the genotypes, embryo development and in vitro culture condition with pre-treatments together significantly improved the rates of production of hybrid cherry seedlings. This is the first study to systematically explore all these factors on cherry embryo rescue in order to produce hybrid seedlings to breed new cultivars for the subtropical monsoon climatic areas like South China and provide new insights to keep the micropropagation of hybrid shoots with different PGRs.

Embryo rescue response and genetic analyses in interspecific crosses of oilseed Brassica species

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.

Effect of different in vitro and in vivo variables on the efficiency of doubled haploid production in Triticum aestivum L. using Imperata cylindrica-mediated chromosome elimination technique

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.

Inoculation of in vitro cultures with rhizosphere microorganisms improve plant development and acclimatization during immature embryo rescue in nectarine and pear breeding programs

In the present study, two fungi Cladosporium ramotenellum strain PGP02 and Phoma spp. strain PGP03 and the bacterium Pseudomonas oryzihabitans PGP01 were isolated from Pyrus and Prunus in vitro rescued embryos, whose plantlets showed a better growth than non-contaminated cultures. Upon identification, concentrated solutions of the three microorganisms were applied to pear (Pyrus communis L.) in vitro rooted plantlets, increasing in different ways biometric parameters such as plant fresh weight (FW), stem length and root length. Then, these microorganisms were tested in embryos derived from three directed crosses between early ripening nectarine varieties (Prunus persica cv. Nectarina). In a first cross, in vitro cultured embryos were inoculated with both fungi, C. ramotenellum PGP02 and Phoma spp. PGP03, at 2 × 107 esp mL−1, and the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1. In the following crosses, only the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1, was employed. The effects on number of germinated embryos, development of the subsequent plants, after 24 weeks of in vitro culture, as well as their ex vitro acclimatization performance were analysed. These microorganisms had no effect on the germination efficiency of nectarine embryos. However, the presence of the bacterium P. oryzihabitans PGP01 modified root system architecture in the three crosses, increasing root volume and thickness, which in consequence enhanced the acclimatization efficiency to soil in those crosses with poor acclimation efficiencies. These results enforce a breakthrough in the use of microorganisms along the in vitro embryo rescue used in early ripening peaches and nectarines breeding programmes, and the production of plants more resistant to the stressful conditions imposed by the acclimatization to soil.

Vibratome Sectioning and Clearing for Easing Studies of Cassava Embryo Formation.

This work describes the application of clearing on vibratome sections to study the embryo formation in cassava. This procedure provides high-resolution images and reduces significantly the number of sections that need to be analyzed per ovule. This methodology was instrumental for the development of the protocol for embryo rescue in cassava. It has been also applied to monitor the embryo formation response when optimizing seed setting from regular and broad crosses for cassava breeding. Broad crosses between cassava and castor bean (incompatible-euphorbiaceae species) were made aiming to induce doubled haploids through the elimination of the incompatible-male parent genome as done in cereals. Castor bean is widely available and provides continues supply of pollen. Our results suggest that this methodology is easy and effective to assess the response of hundreds of cassava ovules pollinated with castor bean pollen, allowing the identification of multicellular structures in the embryo sac without apparent formation of endosperm. The protocol is also useful when developing and optimizing a methodology to induce doubled haploids in cassava via gynogenesis or from ovules pollinated with irradiated cassava pollen.

Utilizing Wild Cajanus platycarpus, a Tertiary Genepool Species for Enriching Variability in the Primary Genepool for Pigeonpea Improvement.

The use of crop wild relatives in the breeding program has been well recognized to diversify the genetic base along with introgression of useful traits. Cajanus platycarpus (Benth.) Maesen, an annual wild relative belonging to the tertiary genepool of pigeonpea, possesses many useful traits such as early maturity, high protein content, photoperiod insensitivity, and pod borer tolerance for the genetic improvement of cultivated pigeonpea. Using this cross incompatible wild Cajanus species, an advanced backcross population was developed following the embryo rescue technique. In the present study, a pre-breeding population consisting of 136 introgression lines (ILs) along with five popular varieties (used as checks) was evaluated for important agronomic traits during 2016 and 2017 rainy seasons and for grain nutrient content during 2016, 2017, and 2018 rainy seasons. Large genetic variation was observed for agronomic traits such as days to 50% flowering, number of pods per plant, pod weight per plant, grain yield per plant, and grain nutrients [protein content, grain iron (Fe), zinc (Zn), calcium (Ca), and magnesium (Mg)] in the pre-breeding population. Significant genotype × environment interaction was also observed for agronomic traits as well as grain nutrients indicating the sensitivity of these traits to the environments. No significant correlations were observed between grain yield and grain nutrients except grain Zn content which was negatively correlated with grain yield. Overall, 28 promising high-yielding ILs with high grain nutrient content were identified. These ILs, in particular, ICPP # 171012, 171004, 171102, 171087, 171006, and 171050 flowered significantly earlier than the popular mega variety, ICPL 87119 (Asha) and thus hold potential in developing new short-duration cultivars. The comprehensive multi-site assessment of these high-yielding, nutrient-rich accessions would be useful in identifying region-specific promising lines for direct release as cultivars. Moreover, these ILs are expected to replace the popular existing cultivars or for use as new and diverse sources of variations in hybridization programs for pigeonpea improvement.

In Support of Winge's Theory of "Hybridization Followed by Chromosome Doubling".

Polyploidy—or chromosome doubling—plays a significant role in plant speciation and evolution. Much of the existing evidence indicates that fusion of unreduced (or 2n) gametes is the major pathway responsible for polyploid formation. In the early 1900s, a theory was put forward that the mechanism of “hybridization followed by chromosome doubling” would enable the survival and development of the hybrid zygote by providing each chromosome with a homolog with which to pair. However, to date there is only scant empirical evidence supporting this theory. In our previous study, interspecific-interploid crosses between the tetraploid Hylocereus megalanthus, as the female parent, and the diploid H. undatus, as the male parent, yielded only allopentaploids, allohexaploids, and 5x-and 6x-aneuploids instead of the expected allotriploids. No viable hybrids were obtained from the reciprocal cross. Since H. undatus underwent normal meiosis with regular pairing in the pollen mother cells and only reduced pollen grains were observed, the allohexaploids obtained supported the concept of “chromosome doubling.” In this work, we report ploidy level, fruit morphology, and pollen viability and diameter in a group of putative hybrids obtained from an embryo rescue procedure following controlled H. megalanthus × H. undatus crosses, with the aim to elucidate, for the first time, the timing and developmental stage of the chromosome doubling. As in our previous report, no triploids were obtained, but tetraploids, pentaploids, hexaploids, and 5x- and 6x-aneuploids were found in the regenerated plants. The tetraploids exhibited the morphological features of the maternal parent and could not be considered true hybrids. Based on our previous studies, we can assume that the pentaploids were a result of a fertilization event between one unreduced (2n) female gamete from the tetraploid H. megalanthus and a normal (n) haploid male gamete from H. undatus. All the allohexaploids obtained from the embryo rescue technique where those that regenerated from fertilized ovules 10 days after pollination (at the pro-embryo stage), showing that the chromosome doubling event occurred at a very early development stage, i.e., at the zygote stage or shortly after zygote formation. These allohexaploids thus constitute empirical evidence of “hybridization followed by chromosome doubling.”

Speed breeding short-day crops by LED-controlled light schemes

Key messageA 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.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.