Production of in vitro haploid plants from in situ induced haploid embryos in winter squash (<Emphasis Type="Italic">Cucurbita maxima</Emphasis> Duchesne ex Lam.) via irradiated pollen

The influence of pollen irradiation on the production of in vitro haploid plants from in situ induced haploid embryos was investigated in winter squash (Cucurbita maxima Duchesne ex Lam.). Pollen were irradiated at different gamma-ray doses (50, 100, 200 and 300 Gray) and durations (9, 11, 15, 21, and 28 July). Production of in vitro haploid plantlets was influenced by irradiation dose, irradiation duration, genotype, and embryo type and embryo stage. Embryos were only obtained from lower irradiation doses (50 Gray and 100 Gray) and earlier irradiation durations (9, 11, and 15 July). The greatest embryo number per fruit was procured from “G14” and “55SI06” genotypes at 50 Gray gamma-ray dose. Necrotic embryos were higher than normal embryos at delayed harvest times (5 and 6 weeks after the pollination). The convenient harvest time for embryo rescue was observed about 4 weeks (between 25 and 30 days) after pollination. All cotyledon and amorphous embryos had only diploid plants while late-torpedo, arrow-tip, and pro-cotyledon embryos produced 33.3, 50.0, and 66.7% haploid plant. The frequency of haploid plantlets was 0.11, 1.17, 10.96 and 0.28 per 100 seeds, 100 embryos, 100 plantlets and a fruit at 50 Gray gamma-ray dose, respectively.     

Enhancement of embryogenesis and plant regeneration from barley anther culture by low doses of gamma irradiation

Summary The effects of 0,5 and 10 Gy doses of gamma irradiation on the enhancement of embryogenesis and plant regeneration efficiency of three barley (Hordeum vulgare L.) genotypes, Igri, Arabi Abiad and AECS 76, were evaluated. Embryo yields at 5 and 10 Gy doses were significantly higher than those of the control (OGy). This effect was genotype-dependent. The most responsive genotype was Igri, with 592.8 embryos 32 anthers exposed to 10 Gy. However, despite a high embryo induction rate, the green plant regeneration rate was low. Arbi Abiad had a higher ability to generate green plants produced from, with 28. 13 plantlets obtained from 32 anthers at 10 Gy; irradiation had no significant effect on regeneration of Igri and AECS 76 genotypes. In general, the 10 Gy dose produced a much higher embryo yield than the 5 Gy dose. The root-tip chromosome number and the fertility of 298 regenerating green plants of cv. Igri revealed that 64% of the tested plants were spontaneously doubled haploids (DHs) and fertile.     

Gamma radiation effects on growth and yield attributes of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. with reference to enhanced production of psoralen

The present work describes radiation-induced effects on vegetative, reproductive traits and psoralen content in Psoralea corylifolia L. The effects of gamma radiation on Psoralea seeds were investigated by exposing seeds with doses of 2.5, 5, 10, 15 and 20 kGy at dose rate of 1.65 kGyh⁻¹ and studying the plant growth at three developmental stages: preflowering, flowering and post flowering (seed to seed) after irradiation. Irradiation with lower doses of gamma rays significantly improved vegetative traits while higher doses proved depressing for same parameters. Similar trend was followed in reproductive traits. Psoralen, showed highest concentration in seeds (7.56%) at 20 kGy and lowest in control roots (0.23%). Increment in psoralen was striking for higher gamma doses applied. These long-term changes in plant development may be attributed to alteration in plant genome induced by irradiation. The results show in depth development stimulation and enhancement of secondary metabolite in Psoralea corylifolia L. following low and high dose treatment respectively depicting the potential of gamma rays in plant biotechnology and metabolomics.     

Genetic inactivation of Leuciscus idus L. (ide) oocytes using UV irradiation

Oocytes of Leuciscus idus were genetically inactivated using ultraviolet (UV) irradiation. Eggs for the experiment were obtained from dark-coloured females, whereas milt was taken from yellow-coloured (recessive marker) males. The survival at the eleutheroembryo stage (free embryo) in all experimental groups fertilized with genetically inactivated spermatozoa was much lower than in control groups. All haploid embryos showed morphological abnormalities, such as a stunted body and a poorly formed retina, and the condition was referred to as the haploid syndrome. The androgenetic origin (haploid or diploid embryos) was checked using a recessive colour marker ('blond'). The optimal doses of UV irradiation were 3,456-4,608 Jm(-2) at which almost 100% haploid embryos were produced at a hatching rate of >15%. Lower UV-ray doses influenced abnormal embryo development. Ploidy level recognition showed a typical value of mean active nucleoli per cell in haploid and diploid (control fish and spontaneous androgenotes) specimens. Abnormal dark embryos were classified as aneuploids.     

A comparison of Hordeum bulbosum-mediated haploid production efficiency in barley using in vitro floret and tiller culture

Summary A high efficiency of Hordeum bulbosum-mediated haploid production in barley has been achieved using a floret culture technique in which florets pollinated with Hordeum bulbosum are cultured on modified N₆ media containing 0.5 mg/l kinetin and 1.2 mg/l2,4-D. Cultures were maintained at 25 °C with a 16 h photoperiod for 9 days before embryo rescue. In a comparison of haploid production efficiency using five F₁ hybrids from winter x winter and winter x spring barley crosses, 41.6 haploid plants/100 florets pollinated were produced using floret culture. Using detached tiller culture, 13.5 haploid plants/100 florets pollinated were produced. Higher efficiencies achieved with floret culture are attributed to the formation of larger, differentiated embryos. Such embryos lead to higher frequencies of plant regeneration. The F₁ from a winter x winter cross was inferior in haploid production compared to F₁s from winter x spring crosses. No genotype x technique interaction was observed.Oregon Agricultural Experiment Station Technical Paper No. 8653     

Non-ionic surfactants improved microspore embryogenesis and plant regeneration of recalcitrant purple flowering stalk (Brassica campestris ssp. chinensis var. purpurea Bailey)

As Brassicaceae species are mostly cross-pollinated, breeding homozygous parental lines by traditional approaches is time-consuming and costly. Alternatively, microspore culture has been widely applied to produce double haploid lines in a short time. This study aimed to establish a highly efficient microspore culture protocol for purple flowering stalk. Among the five genotypes studied, the highest and lowest embryo induction rates were observed in J18 and J17 (13.5 and 7.67 embryos per bud, respectively). Microspores of genotypes J17 and J18 were successfully induced to produce embryos in NLN-13 medium, but the frequency of microspore embryogenesis was low. Three non-ionic surfactants (Pluronic F-68, Triton X-100, Tween-20) were evaluated independently for their effect on microspore embryogenesis of purple flowering stalk. Microspores of the two genotypes were cultivated in NLN-13 medium supplemented with different concentrations (0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, and 1% (w/v)) of the three non-ionic surfactants to enhance microspore embryogenesis and plant regeneration. In both genotypes, supplementation with any of the three non-ionic surfactants at 0.0001% significantly increased the frequency of microspore embryogenesis; furthermore, at that concentration, Tween-20 significantly increased the number of plants regenerated from induced embryoids by 29.9% and 30% in J17 and J18, respectively. Moreover, the rate of double haploid formation among regenerated plants of the five genotypes was above 60%, which allowed the creation of 93 double haploid lines.

     

Stable haploid poplar callus lines from immature pollen culture

Embryogenesis and plant regeneration have been obtained from isolated immature pollen of two poplar hybrids ( Populus nigra L. × hybrid 'Aue1' and 'Aue2'). In total, 1487 calli or embryos, respectively, larger than 1 mm were generated in a 2-year study. By using a cytokinin containing induction medium, on average 19 calli per responsive immature catkin were formed. Additional supplementation with auxin in 2002 increased the frequency to 72 calli per catkin. Microsatellite marker analyses confirmed haploid origin in most regenerants studied. So far six out of eight obtained regenerative callus lines have maintained their haploid level up to 24 months of development. A number of haploid and doubled haploid plants of different lines are available and have been transferred to soil.     

Effect of genotype,induction medium,carbohydrate source,and polyethylene glycol on embryogenesis in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) anther culture

This study conducted two experiments involving in vitro anther culture of Zea mays L. The first experiment tested 46 maize genotypes, including inbred lines, single and three-way cross hybrids, and line A188 as control, in three different induction basal media (IMSS, N6 and YPm) for their androgenic responses. The results showed that the embryos were established 2–3 weeks after the anthers of the few responsive genotypes were cultured. Most responsive genotypes produced embryos in at least one of the three basal media; therefore, genotype is more important than the type of medium for androgenesis in maize. The mean number of anthers that developed to embryo ranged from 19 embryos per Petri dish in YPm medium for the cross (DH5 × DH7) genotype to 0 for some maize genotypes. In the second experiment, this research reports for the first time the effect of carbohydrates and polyethylene glycol (PEG) as a non-metabolized osmoticum on the embryogenesis anther culture of maize. The genotype DH5 × DH7 was used for this experiment, and the media were varied by altering sucrose, maltose, and PEG concentrations. Results showed that the maximum embryogenesis (32 embryos per Petri dish) was obtained by YPm basal medium supplemented with 60 gl^?1 sucrose + 0.0125 M PEG and 30 gl^?1 sucrose + 30 gl^?1 maltose + 0.0125 M PEG. The lowest rate of embryogenesis was observed in YPm basal medium with 60 gl^?1 maltose and 0.0125 or 0.025 M PEG. Sucrose or a high concentration of maltose was found to be necessary for embryogenesis in anther culture of maize. Therefore, the addition of low levels of PEG and/or different sugars in the experimental design appeared to improve the protocol currently available in the world, especially for anther embryo yield and haploid plant regeneration in maize.     

Donor Plant Growth Factors Affecting Anther Culture of Maize and Sweetcorn (Zea mays)

Anthers from seven unselected commercial sweetcorn lines andten experimental maize lines were cultured on a liquid/solidbi-layer culture medium, containing 13 % maltose as the carbonsource. Mean anther efficiencies (number of embryos per 100anthers plated) of 0 to 27.6 % were recorded, with the maximumefficiency of 57.1 % from one plant. The anther efficiency wasfound to be dependent on genotype, microspore developmentalstage and the growth temperatures of donor plants. Immaturemicrospores were found to continue their development duringthe cold pretreatment of the spikes, and this in turn influencedthe level of response to culture. Direct regeneration of embryoidsto plants occurred most frequently when well formed uni- orbi-polar embryos were produced. The quality of embryo producedwas apparently inversely correlated with the number of embryosproduced. Zea mays, haploid culture, embryos, microspores     

Gamma irradiation of the female gametophyte in cabbage (Brassica oleracea L.)

Summary We examined the prospects for using the female gametophyte irradiation technique in cabbage to reduce the number of generations needed for cytoplasm transfer. Three different crosses were used with one nuclear and two cytoplasmic male-sterile lines as female parents. The doses applied ranged from 100 to 700 Gy. Differences between the genotypes were observed only in embryo and plant production with varying dose. Several plants derived from the various experiments exhibited one or two recessive paternal markers and an aneuploid number of chromosomes, which shows an irradiation-induced loss of maternal chromosomes. However, no androgenetic haploid plant was obtained. The aneuploid plants could sometimes be backcrossed, and in one case a shift in the segregation ratio towards the paternal (non-irradiated) phenotype was observed. We concluded that while the female gametophyte irradiation technique could reduce the number of generations necessary for cytoplasm transfer, the damage induced seems too restricted for this technique to be applied in cabbage breeding at present.     

The effect of lecirelin (GnRH) dosage on the reproductive performance of nulliparous and lactating rabbit does

The present study attempted to evaluate the effect of different doses of lecirelin intramuscularly administered at the moment of insemination on parameters such as the conception rate, the total number of rabbits per litter, the number of stillborns per litter and the abortion rate in nulliparous and lactating crossbred rabbit does. The experiment was performed with 295 crossbred female rabbits Hyplus strain PS 19 that were housed in a rabbit farm. Six days before insemination, the light-dark cycles were as follows: 16-h light (70 lx and more):8-h dark, and all does were subcutaneously injected with 25 IU of eCG 48 h before insemination. The doses of lecirelin were as follows: 0.05, 0.1, 0.2, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 microg/doe, and were tested in both nulliparous and lactating female does; the doses were applied at the moment of insemination. The conception rate in nulliparous does ranged significantly from 10.0% (0.05 microg) to 89.5% (1.5 microg). The conception rate in lactating does was the lowest at a dose of 0.05 microg (10.0%), as compared with the doses starting from 0.3 microg, and was confirmed statistically significant. The lowest number of rabbits per litter was found at a lecirelin dose of 0.1 microg (6.64). This finding was statistically significant, as compared with doses equal to or exceeding 0.3 microg. No statistical differences were found for doses ranging from 0.3 to 4.0 microg in nulliparous does (9.06 versus 10.29, respectively). The lowest number of rabbits per litter in lactating does was found at a dose of 0.05 microg (8.28) while the highest number of rabbits was detected at a dose of 4.0 microg (10.73), without statistically significant differences among individual doses. Generally, the number of rabbits per litter in lactating rabbit does was higher than that in nulliparous does (P<0.05). One negative finding is that abortions occurred in lactating does at the intramuscular application of a dose equal to or exceeding 1.5 microg, and in nulliparous does at a dose equal to or exceeding 2.0 microg.     

Quantitative studies of embryogenesis in normal and 5-methyltryptophan-resistant cell lines of wild carrot

The frequency of embryo formation was determined in normal and 5-methyltryptophan-resistant (5-MT^r) cell lines of wild carrot (Daucus carota L.) grown in the presence or absence of 2-isopentenyladenine (2-ip) and 2,4-dichlorophenoxyacetic acid (2,4-D). 2-ip stimulated the intitation of embryo formation and also accelerated embryo development. 2.4-D inhibited embryo differentiation at several stages: at 0.1 mg/l, it stopped regeneration at the earliest stage, resulting in callus growth instead of embryo formation; at 0.04 mg/l 2,4-D, some globular embryos were produced, but they did not develop into more advanced embryos. Variant cell lines with higher levels of auxin (indole-3-acetic acid, IAA) were used to study the effect of an elevated endogenous concentration of auxin on embryogenesis. IAA at these concentrations suppressed regeneration in the same manner as the exogenous auxin, 2,4-D, did. This result confirms the hypothesis that high levels of IAA are responsible for the suppression of regeneration in the 5-MT^r cell lines.     

Influence of CCC, putrescine and gellam gum concentration on gynogenic embryo induction in Allium cepa

The induction of haploid plants by in vitro gynogenesis is a promising practice in onion breeding. In order to increase the frequency of embryo regeneration and haploid plant production in Valcatorce INTA, Cobriza INTA and Navidena INTA cultivars, putrescine and CCC were used, either as a component of the culture media or by spraying or injecting them to the umbels. Additionally, two concentration of gellam gum were tested. A higher number of gynogenic embryos was achieved by using 7 g dm⁻³ gellam gum, and this number was not affected by the addition of putrescine to the media. CCC sprayed at the umbels significantly increased the gynogenic embryo rate, which was more than three times higher than the control. Cobriza INTA showed the highest induced embryo rate (4.76 %).     

ORGANOGENESIS FROM HELIANTHUS ANNUUS INBREDS AND HYBRIDS FROM THE COTYLEDONS OF ZYGOTIC EMBRYOS

Dormant mature and immature zygotic embryos of sunflower (Helianthus annuus L.) inbreds and hybrids were dissected and cultured on modified Murashige and Skoog's medium supplemented with 0.1 mg/l benzyl adenine (BA), 0.5 mg/l naphthalene acetic acid (NAA), adenine sulfate, and casamino acids. For certain inbreds and hybrids, adventitious shoot formation occurred from callused cotyledonary tissue, particularly along the cut edges. The developmental stage of the zygotic embryo was critical. Eighty percent of immature H. annuus ‘Mammoth Russian’ embryos produced adventitious shoots from cotyledons, while mature embryos did not. The organogenic potential of mature cotyledons diminished as soon as one day after germination was initiated. Different forms of regeneration were observed in two inbred lines, and when these were crossed, both forms of regeneration were observed at a lower frequency from the immature embryos of the hybrid. Rooted shoots were successfully grown to maturity in a greenhouse.     

Gamma radiation resistance of Bacillus anthracis spores

The aim of the presented study was determined the effectiveness of action the gamma radiation on water suspension B. anthracis spores. The irradiation was performed using a Cobalt 60 (Co 60) source, by using single and fractionary irradiation doses. In the investigations was used B. anthracis stain "Sterne" 34F2. The obtained results show, that gamma radiation effectively inactivates B. anthracis spores. On the efficiency of sterilization process influence the irradiation's method and the number of spores in 1 ml suspension. In the suspension 1.5 x 10(9) spore in 1 ml, sporicidal doses gamma radiation amount to 25.0 kGy (single dose) or 41.5 kGy (fractionary dose). The volume suspension about definite inoculum of spores, subjected working the gamma rays has not influence on sporicidal effectiveness of radiation sterilization.     

Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)

Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2 mg·L⁻¹) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1 mg· L⁻¹ sodium nitrophenol and 0.01 ~ 0.2 mg· L⁻¹ of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1 mg· L⁻¹ sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01 mg·L⁻¹ significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.

     

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.     

DNA fragmentation by gamma radiation and electron beams using atomic force microscopy

Double-stranded pBS plasmid DNA was irradiated with gamma rays at doses ranging from 1 to 12 kGy and electron beams from 1 to 10 kGy. Fragment-size distributions were determined by direct visualization, using atomic force microscopy with nanometer-resolution operating in non-tapping mode, combined with an improved methodology. The fragment distributions from irradiation with gamma rays revealed discrete-like patterns at all doses, suggesting that these patterns are modulated by the base pair composition of the plasmid. Irradiation with electron beams, at very high dose rates, generated continuous distributions of highly shattered DNA fragments, similar to results at much lower dose rates found in the literature. Altogether, these results indicate that AFM could supplement traditional methods for high-resolution measurements of radiation damage to DNA, while providing new and relevant information.     

Production of double haploids in oat (Avena sativa L.) by pollination with maize (Zea mays L.)

The aim of the study was to optimize the method of oat haploid production by pollination with maize. Seventeen oat genotypes were used in the experiment. Various factors influencing the growth and development of ovaries and embryo production were investigated: genotype, time of pollination, growth regulators and time of their application. Emasculated before anthesis, oat florets were pollinated with maize pollen after 0, 1 or 2 days. Next, one of two auxins analogues (2,4-D or dicamba) were applied to oat pistils. These auxins had no significant influence on the number of enlarged ovaries and embryos. The time of application of these growth regulators had a significant influence on embryo production. Haploid embryos were obtained from all used genotypes, although the frequency of enlarged ovaries and obtained embryos did not differ markedly between the genotypes. On average, 85% of ovaries were enlarged and 11.7% of them produced haploid embryos. Depending on the regeneration medium, 24–41% of embryos were germinated, of which 12% had developed into green plants. A strong significant difference in the number of germinating embryos and haploid plants was observed between the kind of regenerating medium used. There were no albino plants and all the obtained plants were haploid.     

Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts

Summary With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.