Development Of Hybrid Embryos Research Articles

Plant Hormone Pathway Is Involved in Regulating the Embryo Development Mechanism of the Hydrangea macrophylla Hybrid.

The research is aimed to elucidate the role of plant hormones in regulating the development of hybrid embryos in Hydrangea macrophylla. Fruits from the intraspecific cross of H. macrophylla 'Otaksa' × 'Coerulea' were selected at the globular, heart, and torpedo stages of embryo development. Transcriptome sequencing and differential gene expression analysis were conducted. The results showed that fruit growth followed a single "S-shaped growth curve, with globular, heart, and torpedo embryos appearing at 30, 40, and 50 d post-pollination, respectively, and the embryo maintaining the torpedo shape from 60 to 90 d. A total of 12,933 genes was quantified across the three developmental stages, with 3359, 3803, and 3106 DEGs in the S1_vs_S2, S1_vs_S3, and S2_vs_S3 comparisons, respectively. Among these, 133 genes related to plant hormone biosynthesis and metabolism were differentially expressed, regulating the synthesis and metabolism of eight types of plant hormones, including cytokinin, auxin, gibberellin, abscisic acid, and jasmonic acid. The pathways with the most differentially expressed genes were cytokinin, auxin, and gibberellin, suggesting these hormones may play crucial roles in embryo development. In the cytokinin pathway, CKX (Hma1.2p1_0579F.1_g182670.gene, Hma1.2p1_1194F.1_g265700.gene, and NewGene_12164) genes were highly expressed during the globular embryo stage, promoting rapid cell division in the embryo. In the auxin pathway, YUC (Hma1.2p1_0271F.1_g109005.gene and Hma1.2p1_0271F.1_g109020.gene) genes were progressively up-regulated during embryo growth; the early response factor AUX/IAA (Hma1.2p1_0760F.1_g214260.gene) was down-regulated, while the later transcriptional activator ARF (NewGene_21460, NewGene_21461, and Hma1.2p1_0209F.1_g089090.gene) was up-regulated, sustaining auxin synthesis and possibly preventing the embryo from transitioning to maturity. In the gibberellin pathway, GA3ox (Hma1.2p1_0129F.1_g060100.gene) expression peaked during the heart embryo stage and then declined, while the negative regulator GA2ox (Hma1.2p1_0020F.1_g013915.gene) showed the opposite trend; and the gibberellin signaling repressor DELLA (Hma1.2p1_1054F.1_g252590.gene) increased over time, potentially inhibiting embryo development and maintaining the torpedo shape until fruit maturity. These findings preliminarily uncover the factors affecting the development of hybrid H. macrophylla embryos, laying a foundation for further research into the regulatory mechanisms of H. macrophylla hybrid embryo development.

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders.

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

Chrysanthemum embryo development is negatively affected by a novel ERF transcription factor, CmERF12

Embryo abortion often occurs during distant hybridization events. Apetala 2/ethylene-responsive factor (AP2/ERF) proteins are key transcription factor (TF) regulators of plant development and stress resistance, but their roles in hybrid embryo development are poorly understood. In this study, we isolated a novel AP2/ERF TF, CmERF12, from chrysanthemum and show that it adversely affects embryo development during distant hybridization. Transcriptome and real-time quantitative PCR demonstrate that CmERF12 is expressed at significantly higher levels in aborted ovaries compared with normal ones. CmERF12 localizes to the cell nucleus and contains a conserved EAR motif that mediates its transcription repressor function in yeast and plant cells. We generated artificial microRNA (amiR) CmERF12 transgenic lines of Chrysanthemum morifolium var. 'Yuhualuoying' and conducted distant hybridization with the wild-type tetraploid, Chrysanthemum nankingense, and found that CmERF12-knock down significantly promoted embryo development and increased the seed-setting rates during hybridization. The expression of various genes related to embryo development was up-regulated in developing ovaries from the cross between female amiR-CmERF12 C. morifolium var. 'Yuhualuoying'× male C. nankingense. Furthermore, CmERF12 directly interacted with CmSUF4, which is known to affect flower development and embryogenesis, and significantly reduced its ability to activate its target gene CmEC1 (EGG CELL1). Our study provides a novel method to overcome barriers to distant hybridization in plants and reveals the mechanism by which CmERF12 negatively affects chrysanthemum embryo development.

The transcription factor CmLEC1 positively regulates the seed-setting rate in hybridization breeding of chrysanthemum

Distant hybridization is widely used to develop crop cultivars, whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect. The LEAFY COTYLEDON1 (LEC1) gene has been extensively investigated as a central regulator of seed development, but it is far less studied in crop hybridization breeding. Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization. CmLEC1 encodes a nucleic protein and is specifically expressed in embryos. CmLEC1’s overexpression significantly promoted the seed-setting rate of the cross, while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum. The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development, namely, CmLEA (late embryogenesis abundant protein), CmOLE (oleosin), CmSSP (seed storage protein), and CmEM (embryonic protein), were upregulated in the OE (overexpressing) lines but downregulated in the amiR lines vs. wild-type lines. Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H, and this CmLEC1–CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA. Further, CmLEC1 was able to induce several other key genes related to embryo development. Taken together, our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants, which might involve activating CmLEA’s expression and interacting with CmC3H. This may be a new pathway in the LEC1 regulatory network to promote seed development, one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.

UTILIZACIÓN DE PLANTAS PUENTE EN EL MEJORAMIENTO DEL COMPLEJO Calibrachoa – Petunia

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.

Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system

Generation of functional spermatids from human spermatogonial stem cells (SSCs) in vitro is of utmost importance for uncovering mechanisms underlying human germ cell development and treating infertility. Here we report a three-dimensional-induced (3D-I) system by which human SSCs were efficiently differentiated into functional haploid spermatids. Human SSCs were isolated and identified phenotypically. Meiotic chromatin spreads and DNA content assays revealed that spermatocytes and haploid cells were effectively generated from human SSCs by 3D-I system. Haploid cells derived from human SSCs harbored normal chromosomes and excluded Y chromosome microdeletions. RNA sequencing and bisulfite sequencing analyses reflected similarities in global gene profiles and DNA methylation in human SSCs-derived spermatids and normal round spermatids. Significantly, haploid spermatids generated from human SSCs via 3D-I system were capable of fertilizing mouse oocytes, which subsequently enabled the development of hybrid embryos. This study thus provides invaluable human male gametes for treating male infertility.

Reproductive barriers to hybridizations between narrow-leaf and broad-leaf Anoectochilus roxburghii

ABSTRACTNarrow-leaf and broad-leaf Anoectochilus roxburghii were used as reciprocal parents to explore the reason for low seed setting rate. Pollen-pistil interaction was observed by fluorescence microscopy and hybrid embryo development by paraffin section technology. The results indicated that pollen tube growth could reach the embryo sac, and double fertilization could be completed. These findings showed that a pre-fertilization barrier was not the major factor in the cross of narrow-leaf and broad-leaf A. roxburghii. The endosperm development of the hybrid was abnormal and eventually promoted abortion of the embryos. A post-fertilization barrier appears to be the major factor for low seed setting rate in the cross of narrow-leaf and broad-leaf A. roxburghii. In addition, an efficient protocol of embryo rescue, the development of which is reported elsewhere, was used to compare the time needed for rescue between the reciprocal crosses.

Interspecific hybridization between Brassica napus and Brassica rapa ssp. chinensis genotypes through embryo rescue and their evaluation for crossability

The genus Brassica contains several important crop species that are used for a variety of purposes. Brassica napus and Brassica rapa are the most important ones for their use as oilseed crops. In Brassica , interspecific hybridization is a potential and useful method for transferring valuable traits between species of commercial interest. For breeding Brassicas resistant to some diseases, i.e. clubroot, attempts of transferring resistant genes (CR) through interspecific hybridization have also been reported. The main step in the introgression process is the production of interspecific hybrids between the two species. However, the cross-incompatibility occurring in wide hybridization might hamper the possibility of obtaining hybrid progenies. For that reason, the crossability study is essential and may give an insight into the cross-compatibility relationship among the species, the direction of success of crossing, and the crossability barriers of some combinations, if any. In the present study, interspecific reciprocal crosses between B. napus and B. rapa ssp. chinensis genotypes were carried out in order to determine their crossability and to produce their F1 hybrids. Crossability was analyzed based on the pollen germination index (PGI) and the development of hybrid embryos in in vitro cultures. It was observed that the unilateral interspecific incompatibility occurred in crosses between B. napus × B. rapa ssp. chinensis. When B. napus cultivars were used as a maternal parent, pollen grains of B. rapa germinated well on the stigmas, while only a small number of pollen tubes could elongate near the ovules, resulting in a low development of hybrid embryos. Using the B. rapa ssp. chinensis as the pistillate parent in the crosses, it was found that the crossability was relatively higher than that in the case of the reciprocal cross. This resulted in a greater number of embryos obtained. Generally, it was concluded that the selection of parental components for hybridization is an important step for obtaining breeding success.

Reproductive barriers in interspecific hybridizations among Chimonanthus praecox (L.) Link, C. salicifolius S. Y. Hu, and C. nitens Oliver from pollen–pistil interaction and hybrid embryo development

To investigate interspecific cross-compatibility among Chimonanthus praecox (L.) Link, C. salicifolius S. Y. Hu, and C. nitens Oliver, pollen–pistil interaction by fluorescence microscopy and hybrid embryo development by whole mount clearing technique were observed. The results indicated that for all reciprocal crosses among the three species, pollen grains of the male parents could adhere to and germinate in the stigmas of female parents. For any cross combination, pollen tube growth could reach the embryo sac, and double fertilization could be completed. These findings indicated that no obvious barriers to interspecific hybridization existed at the stages of pollen germination and pollen tube elongation until double fertilization. Although certain proportion (29.6% at 9d and 36.3% at 15d after pollination) of abortions (e.g., non-fertilization and retarded or non-existent endosperm development) in normally pollinated embryos of C. praecox at different stages were also observed, a greatly increased proportion (66.1% at 8d and 76.7% at 15d after pollination in C. praecox×C. nitens, and 60.0% at 8d and 75.0% at 15d after pollination in C. praecox×C. salicifolius) of hybrid embryos did not complete double fertilization or completed double fertilization but underwent abortion due to the retardation of endosperm development. Comparing to normally pollinated embryos of C. praecox, the retardation of endosperm development in hybridizations occurred earlier. Moreover, the occurrence of abortions among different cross combinations was basically consistent. Thus, the retardation of the endosperm development of hybrid embryos after double fertilization (after the globular embryo stage) is an important factor leading to the failure of distant hybridization between Chimonanthus species.

ЦИТОЭМБРИОЛОГИЧЕСКИЕ АСПЕКТЫ НЕСОВМЕСТИМОСТИ У ФОРМ РОДА Cerasus Mill. ПРИ ОТДАЛЕННЫХ СКРЕЩИВАНИЯХ В СВЯЗИ С СЕЛЕКЦИЕЙ НА УСТОЙЧИВОСТЬ К КОККОМИКОЗУ

The harmful fungal disease caused by Blumeriella jaapii (Rehm) Arx (syn. Coccomyces hiemalis Higgins) results in an early exfoliation, an attenuation of trees, a reduction of productivity, an abatement of resistance to cold. The most virulent population of disease agent was revealed in Krasnodar region. In Northern Caucasian Zonal Scientific Research Institute of Orchard Culture and Viticulture the authors crossed a slightly infected varieties of cherries and crab cherries with immune samples of Eastern-Asiatic species — Cerasus lannesiana № 2, C. serrulata Hally Tolivetta, C. subhirtella Pendula. They carried out also a comparative cytoembryological analysis of different crossing combinations. The authors investigated the pollen fertility, the growth of pollen tubes in pistil tissues and embryogenesis. The most compatible combinations of crossing at the progamic stage of fertilization were Nord Star × C. subhirtella Pendula, Nord Star × 3-116 (seedling from free pollination C. serrulata Hally Tolivetta × Polyanka), Molodezhnaya × ( C. lannesiana № 2 × Frants Iosif). The abnormalities in development of hybrid embryos were appeared after 18-25 days from pollination on a stage of the heart. This period is recommended for isolation of embryos with subsequent growing in vitro in order to overcome the postgamic incompatibility.

In Vitro Development of Hybrid Embryos After In Vitro Fertilization and Intracytoplasmic Sperm Injection of Bovine Oocytes with Bos gaurus hubbacki Sperm.

In Vitro Development of Hybrid Embryos After In Vitro Fertilization and Intracytoplasmic Sperm Injection of Bovine Oocytes with Bos gaurus hubbacki Sperm.

Reproductive barriers in the intergeneric hybridization between Chrysanthemum grandiflorum (Ramat.) Kitam. and Ajania przewalskii Poljak. (Asteraceae)

In order to reveal the causes of low seed set in the intergeneric cross between Chrysanthemum grandiflorum (Ramat.) Kitam. ‘Zhongshanjingui’ (female parent) and Ajania przewalskii Poljak. (male parent), we systematically investigated megasporogenesis and megagametogenesis of female parents, pollen germinability of male parents, pollen–pistil interaction and hybrid embryo development using paraffin section and light, fluorescence and scanning electron microscopy. The results indicate that development of megagametophyte of female parent belongs to Polygonum type and 84.6% ovules developed to normal 8-nucleate embryo sac at the stage of pollination; the pollen germinability in vitro of male parent was 39.3%; a large number of pollen grains germinated normally and no callose deposited on the female stigmas within 24 h after pollination. Furthermore, about 75% ovules were fertilized, whereas only 17.2% ovules developed to normal maturing embryos at 15 days after pollination due to abortion at various developmental stages. Taken together, these results suggest that sterile ovules of female parent, pollen viability and pollen–pistil interaction have no significant influence on seed set in the cross. A high percentage of embryo abortion or degeneration, i.e. post-fertilization barrier, may be the main factor for low seed yield.

Interspecific hybridization in the genus Leucadendron through embryo rescue

Abstract Interspecific hybridization of Leucadendron was attempted by using species from different sections and subsections to assess the crossability between these species. An embryo rescue protocol was used to raise interspecific hybrids. For the crosses that produced embryos, the most crucial factor responsible for successful culture was the time at which the embryos were incubated. A medium containing MS basal nutrients with sucrose (2%) and phytogel (3 g L − 1 ) was found to be suitable for initiating growth and development of hybrid embryos of L. orientale × L. rubrum , L. tinctum × L. rubrum and L. rubrum × L. salignum .

Buffalo and cattle hybrid embryo development is decreased by caffeine treatment during in vitro fertilization

Water buffalo are renowned for difficulties in the implementation of assisted reproductive technologies, with both males and females being problematic. In this study, we used cattle oocytes to assess the effect of treatments with heparin and caffeine on buffalo spermatozoa and subsequent fertilization and embryo development in vitro. There was no significant difference between buffalo and bovine spermatozoa in the events associated with fertilization. Fertilization of cattle oocytes with buffalo spermatozoa resulted in 7.8% of oocytes developing into hybrid embryos. A difference in the developmental capability of hybrid embryos compared with the cattle control was observed. This has not been previously reported. The subsequent transfer of a limited number of hybrid embryos did not produce a viable pregnancy. However, control treatments in this experiment also failed to achieve pregnancy, so objective data is not available to provide conclusions about the developmental competence of the buffalo and cattle hybrid embryos. Optimal spermatozoa capacitation treatments achieved 61% fertilization and 21% zygote cleavage into two cell embryos. There was no significant difference in fertilization or development due to heparin or spermatozoa concentrations. However, treatment of buffalo and cattle spermatozoa with caffeine significantly decreased embryo cleavage but also tended to decrease embryo development to the blastocyst stage. These studies suggest that problems with reproduction in buffalo may reside with biological mechanisms associated with the oocyte that are often complicated by poor male reproductive performance. Selection for bull fertility would prevent some of these complications.

Heterologous in vitro fertilization and sperm capacitation in an endangered African antelope, the scimitar-horned oryx (Oryx dammah).

Scimitar-horned oryx sperm function was studied using protocols developed for domestic cattle. Objectives were to assess sperm 1) viability and motility in vitro over time, 2) capacitation in heparin- or calcium-supplemented medium, and 3) function in an in vitro fertilization system using heterologous (domestic cow) oocytes. Seminal aliquots were washed, and sperm were resuspended in 1) Talp with 5% fetal calf serum (TALP), 2) TALP + 10 microM heparin, 3) TALP + 20 microM heparin, and 4) TALP + 10 mM CaCl. At 0, 3, and 6 h, aliquots were evaluated for sperm motility, viability (using Hoechst 33258), and ability to acrosome-react when exposed to lysophosphatidylcholine (LC). Sperm function was assessed by evaluating fertilization and embryo development after coculture of in vitro-matured domestic cow oocytes with oryx sperm. Overall mean percentages of motile and viable sperm remained high at 6 h (> 60% and > 70%, respectively). Fewer (p < 0.05) sperm incubated in TALP + 10 microM heparin for 6 h contained intact acrosomes after exposure to LC, but there were no differences between LC and control samples after incubation in TALP without heparin. LC-treated sperm in TALP + 10 mM CaCl contained fewer (p < 0.05) intact acrosomes at 3 and 6 h (52.6% and 31.2%, respectively) than paired controls (83.6% and 70.0%, respectively). Oryx sperm from all males were capable of fertilizing cow oocytes (range 17 of 26 [65.4%] to 25 of 26 [96.2%]). Of the 55 2-cell embryos produced, 34 (61.8%) developed to > or = 8 cells. Of the 24 uncleaved oocytes, 7 (29.2%) were polyspermic. These data demonstrate that processed sperm from the endangered scimitar-horned oryx remain vigorous in vitro for at least 6 h. Capacitation can be induced using cattle sperm-processing techniques, with sperm appearing most responsive to elevated CaCl concentrations. Most interesting was the successful production and development of hybrid embryos after coincubation of oryx sperm with cow oocytes, suggesting that the two bovid species have similar fertilization mechanisms.

Production of New Intergeneric Hybrids between Raphanus sativus and Brassica Wild Species.

Intergeneric reciprocal crosses between Raphanus sativus and three Brassica species, such as B. fruticulosa (ssp. cossoneana strain 201 and ssp. mauritanica strain 401), B. maurorum and B. oxyrrhina, were carried out in order to analyze their crossability and to produce their Fl hybrids. Crossability was analyzed based on the pollen germination index (P. G. I.) and the development of hybrid embryos which was evaluated in vivo by conventional crossing and in vitro by ovary culture. When R. sativus was used as the pistillate parent, pollen grains of the Brassica. species germinated well on the stigmas while only a small number of pollen tubes could elongate near the ovules in a few R. sativus pistils, resulting in a low development of hybrid embryos. Using the Brassica species as the pistillate parent in the intergeneric crosses, the crossability was relatively higher than that of the reciprocal cross. When R. sativus was used as the pistillate parent, one F1 hybrid was produced by embryo culture in the cross of R. sativus × B. fruticulosa-401. When the Brassica species were used as the pistillate parent, 200 F1 hybrids were obtained through ovary culture followed by embryo culture from all cross combinations and 12 F1 hybrids were obtained in the cross of B. oxyrrhina × R. sativus by conventional crossing without embryo rescue. Fl hybrids were intermediate between their parents in several characteristics except for flower color and root thickening. In the cross of the Brassica species × R. sativus, 12 amphihaploid Fl hybrids formed trivalents and a number of bivalents at metaphase I(MI)and their pollen fertility ranged from O% to 70.2%. Induced 6 amphidiploid Fl hybrids showed relatively stable chromosome associations at MI and a higher pollen fertility than that of the corresponding amphihaploid. It is suggested that these new Fl hybrids could become valuable materials in the breeding of the tribe Brassiceae.

Plant Recovery of Selfs and Interspecific Hybrids of Arachis by In Vitro Culture of Peg Tips

Embryo abortion is a barrier to interspecific hybridization between cultivated and wild species of Arachis L., and in vitro culture of embryos prior to abortion is potentially useful for overcoming this problem. In Arachis, the peg tips encase the embryos as they move from above to below ground. The objectives of this research were to enhance the in vitro techniques to rescue selfed embryos of Arachis species and to apply this technique to obtain hybrid plants. Arachis hypogaea L. and four diploid wild species—A. glandulifera Stalker, A. duranensis Krapov. and W.C. Gregory, A. batizocoi Krapov. and W.C. Gregory, and A. valida Krapov. and W.C. Gregory—which have different degrees of compatibility with A. hypogaea were used. Ten‐day‐old peg tips were cultured on the combined MS and B5 media with 1‐naphthaleneacetic acid (NAA), gibherellic acid (GA3), and and 6‐benzylaminopurine (BAP) for 90 d. In vitro‐developed ovules, embryos, or seeds were subcultured to regenerate plants. The results indicated that A. hypogaea had higher frequencies of pod and seed production than the wild species. Arachis glandulifera produced more pods and seeds than the other three wild species, and no significant difference in pod and seed production was found among A. duranensis, A. valida, and A. batizocoi. Selfed plants were recovered from all live species. In vitro development of hybrid embryos to the cotyledonary stage was observed in all eight interspecific crosses among A. hypogaea and the four Arachis dipioids. Mature hybrid plants were recovered from seven of eight combinations. This is in contrast to plants recovered from five of eight hybrid combinations (including reciprocals) when pods were left on the plant. Arachis glandulifera × A. hypogaea hybrids which previously had been attempted, but not obtained, were recovered through peg tip culture followed by somatic embryogenesis. This study demonstrated that Arachis hybrid proembryos can be rescued through peg tip culture.

Gene dispersal from transgenic crops

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.

Production of Intergeneric Hybrids between Raphanus and Sinapis and the Cytogenetics of Their Progenies.

Reciprocal crosses were made to analyze the intergeneric crossability and to obtain intergeneric hybrids between Raphauus sativus and four Sinapis species (S. alba. S. arvensis. S. pubescens and S. turgida ). Pollen germination in the pistil and development of hybrid embryos depended on the direction of each cross. The crossability was relatively higher when Sinapis species were used as pistillate parents, whereas S. pubescens was compatible with R. sativus only when used as pollen parent. By embryo rescue technique and/or conventional method, hybrid plants were produced in all intergeneric crosses between R. sativus and four species of Sinapis except for the cross of S. pubesceus x R. sativus due to strong prefertilization barriers. When Sinapis species were used as pistillate parents,

The fate of genes escaped from transgenic plants

One possible consequence of releasing transgenic plants into the environment is introgression—the transfer of gene constructs from transgenic crop populations to, and establishment in, recipient wild species, especially weedy species related to the transgenic crop. This escape process can be broken down into two components: the production of a fertile inter‐specific hybrid from a cross between a transgenic crop and related wild species, and the subsequent spread of the transgenic gene construct through a population of wild relatives. The first of these components is a relatively well understood process which depends on the occurrence of a sequence of ‘biological accidents’ to break through reproductive barriers between species. The key determinants of this include pollen dispersal synchrony and gametic compatibility, and hybrid embryo development and fertility. The second component (spread of transgenic gene construct through a recipient population) depends on the overall fitness of the hybrid and its offspring. Hybrids between crop and wild species can be expected to have lower fitness than pure wild genotypes, as they carry alleles coding for traits selected for under domestication but potentially disadvantageous in the wild. An example of such a trait is the solid rachis commonly found in cereal crops, whereas their wild relatives have fragile rachides which presumably aid seed dispersal. The probability of a transgenic gene construct being able to increase its frequency in the recipient population, and the rate of that increase, is dependent on the net fitness of the hybrid and its progeny from backcrosses with wild‐type individuals. That net fitness in turn depends upon the fitness advantage conferred by the transgenic gene construct, the number of ‘disadvantageous domesticated alleles’ carried by the hybrid, and the linkage relationships between these two factors. We are using simulation studies to address these issues.