Interspecific hybridization between Nicotiana repanda Willd. and N. tabacum L. through the pollen irradiation technique and the egg cell irradiation technique

Summary Two techniques were useful in overcoming hybrid inviability between N. repanda and N. tabacum. These techniques combine gamma-ray irradiation to pollen or to egg cells (in ovules) with in vitro culture of fertilized ovules. When in vitro culture of fertilized ovules from in situ hybridization of N. repanda x N. tabacum was combined without gamma-ray irradiation to pollen or to egg cells (in ovules), all of the resulting seedlings developed chlorosis and died. Furthermore, in the case of in situ hybridization of N. repanda x N. tabacum with gamma-ray irradiated N. tabacum pollen, no viable seeds were obtained. By using both techniques, combining gamma-ray irradiation to N. tabacum pollen or to egg cells in (N. repanda ovules) with in vitro culture of fertilized ovules, we were successful in obtaining flowering hybrid plants. Thus, it appears that it may be possible to overcome hybrid inviability to a certain extent using both the pollen irradiation technique and the egg cell irradiation technique, i.e., gamma-ray irradiation to pollen or to egg cells (in ovules) before pollination and in vitro culture of fertilized ovules.The research reported in this paper is in partial fulfillment of PhD requirements for the senior author     

In vitro induction of genetic variability in cotton (Gossypium spp.)

Summary Through the in vitro culture of excised embryos and ovules, interspecific hybrids have been obtained from cultivated and wild species of Gossypium. The hybrids matured upon transfer to the field. The anthers, ovules and embryos from both the diploid (2n=26) and tetraploid (2n=52) species underwent proliferation, and this response was genotypic. The diploid species invariably showed profuse callusing in comparison with the tetraploid. The callus showed various chromosome numbers, ranging from haploids to hexaploids, and from high polyploidy to aneuploidy. Hybrid callus culture may augment the genetic variability by providing a means for obtaining genetic exchange in interspecific hybrids. The implications of the in vitro induction of genetic variability for cotton improvement are discussed.     

In vitro hybridization in an incompatible cross Nicotiana glutinosa X Nicotiana megalosiphon

Summary Using conventional techniques interspecific hybridization between N. glutinosa (2n=24) and N. megalosiphon (2n=40) results in failure while the reciprocal is successful. Attempts were made here to obtain an F₁ hybrid of N. glutinosa X N. megalosiphon by embryo rescue. These reciprocal hybrids resembled each other in their phenotypic and chromosomal behaviour, i.e. there is broad spectrum of phenotypic variation coupled to chromosomal range from 2n-28 to 32. This may be due to the functional disharmony between chromosome sets of two unrelated species resulting in the elimination of chromosomes.     

In vitro attempts to overcome the cross-incompatibility between Vaccinium corymbosum L. and V. elliottii Chapm.

Summary Seed was readily obtained from V. corymbosum zygotes using embryo rescue techniques, even when embryos were cultured at proembryonic stages. Best in vitro seed development was obtained when ovules were cultured attached to placental tissues. Successful fruit and seed development in culture occurred only when the fruit was cut longitudinally or when the basal portion of the fruit was removed previous to plating. Addition of various vitamins, amino acids, and growth regulators to the nutrient medium did not increase seed production. Attempts to rescue hybrid embryos from V. croymbosum (tetraploid) x V. elliottii (diploid) crosses by in ovulo and in ovary culture gave a few presumably hybrid seed, but at a rate no greater than when normal crossing procedures are used.Florida Agricultural Experiment Station Journal Series No. 5748     

Wide hybridization in Brassica

Summary Crosses were made to obtain interspecific hybrids between B. fruticulosa (wild species , 2n = 16) × B. campestris (cultivar , 2n = 20). Although many pollen grains germinated and their tubes entered the style, only about 30% of the ovules received pollen tubes. Fertilized ovules aborted at various stages of development. A few hybrid seeds resulted from hand pollinations in the field, and they showed poor germination and seedling establishment. The in vitro culture of ovaries, ovules, and seeds increased the frequency of obtaining hybrid seeds and plants: the most effective method was ovary culture followed by ovule culture. The hybrid nature of the plants was confirmed through morphological, cytological, and electrophoretic studies. A meiotic analysis of F₁ hybrids (2n = 18) showed that they had 0–5 bivalents and were completely pollen sterile. Electrophoretic analysis of leaf esterases and acid phosphatases of F₁ hybrids revealed bands derived from each parent. Induced amphidiploids of F₁ hybrids contained mostly bivalents, and had about 50% fertile pollen.     

Asymmetric hybridization between Nicotiana tabacum and N. repanda by donor recipient protoplast fusion: transfer of TMV resistance

Summary Genetically asymmetric hybrids were recovered by fusion of Nicotiana tabacum protoplasts with irradiated protoplasts of kanamycin-resistant, nopalineproducing plants of N. repanda. Hybrid calli were selected by culture on media containing kanamycin and were regenerated. These plants were morphologically similar to N. tabacum but produced nopaline, indicating they retained genes from N. repanda. Esterase isozyme profiles also indicated that the plants are somatic hybrids, but are more similar to N. tabacum than N. repanda. Chromosome counts showed most of the hybrids had 55–62 chromosomes, which is consistent with extensive, although incomplete elimination of N. repanda chromosomes. The hybrids were largely male sterile, but about half of them set seed when crossed with N. tabacum. Chromosome numbers of the progeny and the pattern of inheritance of kanamycin resistance indicated the continued elimination of N. repanda genetic material in these backcrosses. The N. repanda parent used in these fusions gave a hypersensitive response to TMV, whereas the N. tabacum parent was TMV sensitive. When inoculated with TMV, plants from two hybrid clones gave a hypersensitive response. Plants from the other clones became systemically infected with the virus.     

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Summary Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by -irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) — all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F₁ progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.Abbreviations Km ^r kanamycin resistant - Km ^s kamacysin sensitive - Nop ⁺ nopaline positive - Nop ^– nopaline negative     

Transmission of male cytoplasm during fertilization in Nicotiana tabacum

Serially sectioned embryo sacs of Nicotiana tabacum were examined during fertilization events using transmission electron microscopy. After pollen tube discharge, the outer membrane of the sperm pair is removed, the two sperm cells are deposited in the degenerate synergid and the sperm cells migrate to the chalazal edge of the synergid where gametic fusion occurs. During fertilization, the male cytoplasm, including heritable organelles, is transmitted into the female reproductive cells as shown by: (1) the cytoplasmic confluence of one sperm and the central cell during cellular fusion, (2) the occurrence of sperm mitochondria (distinguished by ultrastructural differences) in the zygote cytoplasm and adjacent to the sperm nucleus, (3) the presence of darkly stained aggregates which are found exclusively in mature sperm cells within the cytoplasm of both female cells soon after cell fusion, and (4) the absence of any large enucleated cytoplasmic bodies containing recognizable organelles outside the zygote or endosperm cells. The infrequent occurrence of plastids in the sperm and the transmission of sperm cytoplasm into the egg during double fertilization provide the cytological basis for occasional biparental plastid inheritance as reported previously in tobacco. Although sperm mitochondria are transmitted into the egg/zygote, their inheritance has not been detected genetically. In one abnormal embryo sac, a pair of sperm cells was released into the cytoplasm of the presumptive zygote. Although pollen tube discharge usually removes the inner pollen-tube plasma membrane containing the two sperm cells, this did not occur in this case. When sperm cells are deposited in a degenerating synergid or outside of a cell, this outer membrane is removed, as it apparently is for fertilization.     

Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L

Castor and Jatropha belong to the Euphorbiaceae family. This review highlights the role of biotechnological tools in the genetic improvement of castor and jatropha. Castor is monotypic and breeding programmes have mostly relied on the variability available in the primary gene pool. The major constraints limiting profitable cultivation are: vulnerability to insect pests and diseases, and the press cake is toxic which restrict its use as cattle feed. Conventional breeding techniques have limited scope in improvement of resistance to biotic stresses and in quality improvement owing to low genetic variability for these traits. Genetic diversity was assessed using protein based markers while use of molecular markers is at infancy. In vitro studies in castor have been successful in shoot proliferation from meristematic explants, but not callus-mediated regeneration. Genetic transformation experiments have been initiated for development of insect resistant and ricin-free transgenics with very low transformation frequency. In tropical and subtropical countries jatropha is viewed as a potential biofuel crop. The limitations in available germplasm include; lack of knowledge of the genetic base, poor yields, low genetic diversity and vulnerability to a wide array of insects and diseases. Great scope exists for genetic improvement through conventional methods, induced mutations, interspecific hybridization and genetic transformation. Reliable and highly efficient tissue culture protocols for direct and callus-mediated shoot regeneration and somatic embryogenesis are established for jatropha which indicates potential for widening the genetic base through biotechnological tools. Assessment of genetic diversity using molecular markers disclosed low interaccessional variability in local Jatropha curcas germplasm. The current status and future prospects of in vitro regeneration, genetic transformation and the role of molecular tools in the genetic enhancement of the two-oilseed crops are discussed.     

A comparative study of genotypic and environmental response to androgenesis in Nicotiana rustica

Summary A total of six genotypes of Nicotiana rustica comprising the two F₁'s (V₂ × V₁₂ and V₁ × V₅) and their parents were evaluated for their efficiency in haploid production. Excised immature flower buds with pollen at late uninucleate to early binucleate stage were pretreated for 21 days at 5 ° or 7 °C, or for 15 days at 9 °C before culturing on Nitsch's medium+ 0.1 mg/l NAA. The effects of genotype, pretreatment and their interaction were tested on anther response, anther productivity and days to first plantlet formation. Highly significant genotype X pretreatment interaction and differences between genotypes were observed for all three characters. Significant differences between pretreatments were observed for anther productivity only. The performance of V₁₂ both in respect of anther productivity and response was highest whereas that of V₅ was the lowest. Analysis of variance showed that a simple additive genetic model was not adequate to explain the above variation due to significant additive genetic and dominance interactions with the pretreatment.     

Durum wheat haploid production using maize wide-crossing

While anther culture or pollinations with Hordeum bulbosum have provided suitable methods for haploid production in bread wheat, they have been largely unsuccessful in durum wheat. Pollinations with maize were used in an attempt to produce haploid seedlings and, from these, fertile doubled haploids of durum wheats. Moreover, the effect of various concentrations and combinations of a synthetic auxin, 2, 4-dichlorophenoxyacetic acid (2,4-D), kinetin, and an ethylene inhibitor, silver nitrate (AgNO₃), on embryo recovery were also investigated. Haploid seedlings were recovered from Triticum turgidum ssp. turgidum cv Rampton Rivet pollinated with maize following in-vivo treatment of ovaries with 2,4-D for 2 weeks and subsequent embryo culture. The recovery of haploid seedlings from T. turgidum ssp. durum cv. Wakona pollinated with maize necessitated the addition of AgNO₃, to the 2,4-D treatment. Overall, haploid seedlings were produced in 1.7% and 3.3% of pollinated florets for Rampton Rivet and Wakona respectively. The success of the present work represents a significant breakthrough for haploid production in durum wheats. Wide hybridization with maize followed by in-vivo treatment of ovaries with 2,4-D alone, or in combination with AgNO₃, may provide a widely-applicable method of haploid production in tetraploid wheats.     

Genotype, ploidy and physiological state in relation to isoperoxidases in Nicotiana

Peroxidase and isoperoxidase patterns of tobacco have been comparatively analyzed in relation to parental origin and ploidy level (including hypohaploidy status) at different ages and physiological states. The interspecific hybrid N. sylvestris × N. tomentosiformis shows an additive pattern of parental peroxidases, which also resembles that of N. tabacum cv. Wisconsin and cv. Samsun. Enzyme activity (on dry weight and protein bases, decreases from the diploid to the hypohaploid forms of Wisconsin and Samsun with respective quantitative differences in the zymograms, when the plants are compared at the same chronological and vegetative stages. Abnormally vegetatively ageing hypohaploids recover all peroxidasic isoenzymes and reach an enzyme activity as high as those of haploids and diploids. Flowering hypohaploids, however, generally behave like haploids and diploids in changing their balance between acidic and basic peroxidases.     

The use of IAA to overcome interspecific hybrid inviability in reciprocal crosses between Nicotiana tabacum L. and N. repanda Willd.

Summary Flowering hybrid plants were obtained from reciprocal crosses between N. tabacum L. (2n=48) and N. repanda Willd. (2n = 48), in which cross incompatibility and hybrid inviability were manifested. Stylar pollination and ovule culture were used to overcome the cross incompatibility of stylar barriers and ovular death. It was shown that application of 2 mg/l indole-3-acetic acid (IAA) during the growth period, from the fiveto six-leaf stage to the flowering stage, is a useful and easy method to overcome hybrid inviability.     

Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridization

Interspecific crosses were made by hand-pollination of Allium cepa with pollen of 19 species belonging to nine sections of two subgenera of the genus Allium. In all cases viable plantlets were obtained from ovary culture. The efficiency depended on the relationship of the pollen donor to A. cepa. The hybrid character of the regenerants was checked by morphological comparisons with the parents and/or by one or more cytological methods such as flow cytometric DNA measurement, karyotype analysis, and genomic in situ hybridization (GISH). Hybrids were confirmed for 18 new species combinations. The viable hybrid of the most distant cross resulted from crossing A. cepa with A. sphaerocephalon. The relevance of the verification methods and the potential use of the hybrids for breeding purposes are demonstrated.     

The molecular path to in vitro shoot regeneration

Plant regeneration through de novo shoot organogenesis in tissue culture is a critical step in most plant transformation and micropropagation procedures. Establishing an efficient regeneration protocol is an empirical process and requires optimization of multiple factors that influence the regeneration capacity. Here, we review the molecular process of shoot induction in a two-step regeneration protocol and focus on the role of auxins and cytokinins. First, during incubation on an auxin-rich callus induction medium (CIM), organogenic callus is produced that exhibits characteristics of a root meristem. Subsequent incubation on a cytokinin-rich shoot induction medium (SIM) induces root to shoot conversion. Through a detailed analysis of the different aspects of shoot regeneration, we try to reveal hinge points and novel candidate genes that may be targeted to increase shoot regeneration capacity in order to improve transformation protocols.     

Bioelectric activity is required for regional specificity of sensory ganglion projections to spinal cord explants cultured in vitro

Summary Chronic blockade of spontaneous nerve impulses by means of tetrodotoxin leads to abnormally diffuse afferent projections into spinal cord cross-sections cultured for two to six weeks in vitro. In addition, even untreated explants which show a low level of spontaneous cord discharges failed to develop the normal degree of dorsal pathway selectivity. It is therefore concluded that centrally generated neuronal activity may play an important role in eliminating exuberant connections which, during early development, are transiently present in this part of the nervous system.     

Fluorescent in situ hybridization on tissue microarrays: challenges and solutions

Tissue microarray (TMA) technology has provided a high throughput means of evaluating potential biomarkers and therapeutic targets in archival pathological specimens. TMAs facilitate the rapid assessment of molecular alterations in hundreds of different tumours on a single slide. Sections from TMAs can be used for any in situ tissue analysis, including fluorescent in situ hybridization (FISH). FISH is a molecular technique that detects numerical and structural abnormalities in both metaphase chromosomes and interphase nuclei. FISH is commonly used as a prognostic and diagnostic tool for the detection of translocations and for the assessment of gene deletion and amplification in tumours. Performing FISH on TMAs enables researchers to determine the clinical significance of specific genetic alterations in hundreds of highly characterized tumours. The use of FISH on archival paraffin embedded tissues is technically demanding and becomes even more challenging when applied to paraffin embedded TMAs. The problems encountered with FISH on TMAs, including probe preparation, hybridization, and potential applications of FISH, will be addressed in this review.     

In vitro fertilization and seed development inTrifolium

Summary Pistils ofTrifolium repens L. andT. ambiguum Bieb. were cultured on an agar-based modified Murashige-Skoog medium. Pistils with and without accessory floral parts were removed from flowers of selected clones ofT. repens, hand-pollinated under aseptic conditions, and planted on the medium. Pistils cultured without accessory floral parts showed no evidence of fertilization after 2 weeks. However, 52% of thoseT. repens pistils cultured with calyx lobes and pedicels contained ovules with maturing embryos 12 days after in vitro cross-pollination. Pistils fromT. ambiguum intraspecific cross-pollinations could not be cultured successfully under the same conditions; however, addition of various combinations of auxin, cytokinin, and gibberellic acid enhanced embryo growth. Fertilization and partial embryo development occurred in interspecific crosses betweenT. ambiguum andT. repens orT. hybridum only whenT. ambiguum was used as the pistillate parent. These results indicate that embryological development under in vitro conditions closely parallels in situ development although growth regulator requirements may vary among species. This work is Technical Contribution 1785 from the South Carolina Agricultural Experiment Station and was supported by SCAES-USDA Cooperative State Research Agreement No. 616-15-65.