Storage of pollen grains of Crotalaria retusa in oils

Summary Attempts were made to store pollen grains of Crotalaria retusa L. in a mineral oil (paraffin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15–30 days, while those stored in oils maintained some degree of germinability even after 60 days. Pollen samples stored in oils at –20° C did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

Treatment of maize pollen to reduce nuclease activity

Recently it has been reported that maize (Zea mays L.) pollen can be stored up to several hours in a hypertonic aqueous medium at 0°C without losing its germinability (Broglia and Brunori 1994). We found that both release and activity of pollen nucleases are diminished in a cold hypertonic aqueous medium. Nucleases can be washed off while preserving germination ability and thus preserving the possibility of passive uptake of exogeneous DNA into germinating pollen grains. Alternatively, active DNA transfer into non-germinating pollen grains in the storage medium itself may be facilitated, due to the very reduced nuclease activity in this medium.     

Invasion of sorghum seed by storage fungi at moisture contents of 13.5–15 % and condition of samples from commercial bins

In sorghum seed stored at 22–25° C for 535 days, invasion by storage fungi and loss of germinability increased greatly with small increases in moisture content between 13.5 and 15.5 %. Seeds dried for 18 hours at 70° C, then exposed to a relative humidity of 75 %, had a lower equilibrium moisture content, but were more heavily invaded by storage fungi and lost germinability fsater, than those that had been conditioned to 20 % moisture before storage at 75 % relative humidity. The 10 samples of Grade No. 2 sorghum examined averaged about 13.0 % moisture, germinated an average of 59 %, yieldedAlternaria from 75 % of the surface-disinfected kernels andAspergillus glaucus from 4 %; judged by these criteria, the lots from which the samples came were in good condition for continued storage.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Pollen viability and pollen vigor

Summary Investigations were carried out to correlate pollen viability, assessed on the basis of a fluorochromatic reaction (FCR) test, with pollen vigor, assessed on the basis of the time taken for in vitro germination in pollen grains subjected to high humidity (>95% RH) and temperature (38 °C) or storage stress of Nicotiana tabacum, Agave sp., Tradescantia virginiana, and Iris sp. Both high RH and temperature, as well as storage stresses, affected pollen vigor before affecting pollen viability. The results are discussed in the light of available data on the viability and vigor of stressed pollen and of aged seeds. The need for consideration of pollen vigor, particularly in stored pollen, the inadequacy of the methods presently used, and some of the methods suitable to assess pollen vigor are elaborated.     

Short-term storage of cotton pollen

Several methods of storing cotton pollen (Gossypium hirsutum L.) were evaluated. A successful pollen storage method that maintains fertility would enhance the crossing of breeding materials. Storing pollen at ultra-low temperatures in liquid nitrogen or at 5°C was not successful. No storage method maintained pollen fertility for more than 72 h. Cotton pollen did maintain adequate fertility up to 24 h at 10 and 15°C, at both low and high humidity when the pollen was stored in the detached flowers. Minimally acceptable pollen fertility was maintained in flowers stored at 15°C at 100% R.H. for 72 h. Use of these methods will allow for better utilization of parental plants when both parents do not flower on the same days.USDA-ARS, in cooperation with the New Mexico Agricultural Experimental Station, Journal Article No. 1161, Las Cruces, NM 88003, USA     

Cold storage ofPhytoseiulus persimilis (Phytoseiidae)

Phytoseiulus persimilis is commerelally mass-reared for use as a biological control agent for spider mites, primarilyTetranychus urticae, and cold storage is a potentially valuable aspect of mass-production. Cold storage ofP. persimilis in empty containers was found to be unsatisfactory, but provision of moisture during cold storage greatly increased survival. Provision of food further increased survival even though the mites were stored at temperatures below their threshold for development of 11°C. When food was provided, survival at 7.5°C was 97% after 4 weeks and 80% after 6 weeks. Subsequent longevity and fecundity of mites that survived 8 weeks at 7.5°C was comparable to mites taken directly from mass-rearing cultures. Survival of mites packaged in bran or vermiculite and held at 6°C for 10 days ranged from 75% to 85% and was not decreased by agitating the containers to simulate shipping. However, survival of mites held in bran or vermiculite at 5°C or 8°C for 4 weeks was poor, ranging from 0–19%, due to growth of mould in the media.Phytoseiulus persimilis can be successfully stored for 4 to 6 weeks in containers provisioned only with food and moisture; granular media used for distribution of the mites should be added just prior to shipping.     

Effect of pollination time,the hour of daytime,pollen storage temperature and duration on pollen viability,germinability, and fruit set of date palm (Phoenix dactylifera L.) cv "Deglet Nour"

Success artificial pollination with viable pollen is crucial process in the production chain of date palms. This study evaluated the impact of pollen storage temperature and duration, pollination time following spathe cracking, and the hour of daytime on pollen viability, germinability, fruit set and yield of 'Deglet Nour' date palm cultivar. In in vitro tests, fresh pollen showed the maximum viability (96.3%) and germination (85%) but it decreased thereafter upon the storage temperature (28, 4 and ?30 °C) and duration (3, 6, 9 and 12 months). In this respect, pollen stored at ?30 °C retained highest viability and germinability followed by those stored at 4 and then at 28 °C. In filed experiments, fruit set was 85, 75, 65, and 45% with pollination using fresh pollen, or pollen stored at ?30, 4 and 28 °C, respectively. Fruit set was 95%, 75%, and less than 50%, for pollination performed on the same day of spathe cracking, 6 and 12 days later, respectively. The highest fruit set percentage and yield/bunch were obtained with pollination performed between 12.0 pm and 15.0 pm in contrast to 8.0–11.0 am or 16.0–17.0.     

Effect of flowering stage and storage conditions on pollen quality of six male date palm genotypes

Availability of efficient male genotypes is critical for successful artificial pollination and regular bearing of female date palms. The effect of flowering stage and storage conditions on pollen quality of six male date palm genotypes encoded ‘ABD1′, ‘P4′, ‘P3′, ‘P8′, ‘P7′ and ‘P13′were evaluated. Pollen collected from spathes developed at the middle of flowering stage exhibited the best viability (90%) and germinability (85%) compared to other stages. Pollen viability was greater than 90%, except for ‘P8′ that exhibited 80%, while, germinability greatly varied among the genotypes. Pollen quality decreased during 4 months of storage upon genotype and temperature, with a minimum reduction at ?30 °C followed by 4 °C. Heat shock exposure (33 ± 2 °C) following storage revealed that pollen stored at ?30 °C or 4 °C should be used for pollination on the same day of take out to avoid dramatic quality loss. The ‘ABD1′, an early flowering genotype, proved highest pollen quality both at fresh stage and after storage. While, the ‘P3′, a late flowering genotype, retained its pollen quality during storage. However, the ‘P13′ genotype exhibited excellent pollen quality when fresh, but greatly loses germinability during storage.     

Effects of high humidity and temperature stress on pollen membrane integrity and pollen vigour in Nicotiana tabacum

Summary The prolonged exposure of pollen Nicotiana tabacum to high humidity at both room temperature and 38° C did not affect membrane integrity as revealed by the fluorochromatic reaction (FCR) test, but did affect pollen vigour. At room temperature germination was not affected, although tube growth was reduced; at 38° C, there was both a reduction in tube growth and delayed germination. When the pollen was subjected to 1 h hydration followed by 1 h desiccation (up to a maximum of four cycles) at room temperature, a reduction in the FCR, germination and tube length after each desiccation treatment was observed. Subsequent hydration fully restored the FCR, but only partially restored germination and tube growth. At 38° C, however, FCR, germination, and tube growth were drastically reduced. The implications of these results on the relationship between FCR and germinability, the responses of pollen exposed to humidity and temperature stress in the field, and on pollen storage are discussed.     

In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen

The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4°C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at −20°C or −80°C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4°C for a couple of weeks and then at −80°C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).     

The response of anther culture to culture temperature in Triticum aestivum

Summary The response of anther culture to culture temperature was studied in detail using many varieties, F₁ hybrids and pollen-derived lines of wheat (Triticum aestivum) as materials. The suitable culture temperature for inducing pollen callus (or embryoids) in wheat anther culture ranged from 26 °C to 30 °C, varying with genotypes. But for the great majority of wheat genotypes the suitable culture temperatures lay between 28 °C and 30°C. The most significant genotypic variation in the response to culture temperature was observed in the comparison between the culture at 33 °C for eight days followed by culture at 25 °C (or 26 °C) and the continuous culture at 25 °C (or 26 °C). This genotypic variation in the response to culture temperature is a heritable character which may be controlled by multiple genes. The effect of culture at 30 °C for eight days followed by culture at 26 °C was similar to, or in some cases, better than that of continuous culture at 28 °C, and the effect of culture at 32 °C for eight days followed by culture at 28 °C was similar to that of continuous culture at 30 °C. In the range from 26 °C to 32 °C, the overwhelming majority of pollen calli emerged before the 40th day after anther inoculation, and the higher the culture temperature, the earlier and more concentrated the emerging period of the pollen callus. The pollen callus obtained at high temperatures above 28 °C should be transferred in time onto the regeneration medium at 25°–27°C to induce shoots.     

Androgenesis in isolated pollen cultures ofNicotiana tabacum: Dependence upon pollen development

Summary The influence of the stage of pollen development and of the growth conditions of donor plants on the performance of cultures of isolated pollen fromNicotiana tabacum, var. Badischer Burley has been studied. The method described includes cold treatment (4–5 °C for 3 days) and a pre-culture of the anthers for 7 days at 24 °C before the pollen is isolated. With this system reproducible results were obtained with pollen at the early binucleate stage collected from plants 11–13 weeks old. Another prerequisite for reproducibility is that the donor plants must have been grown for eight weeks in soil with an additional supply of mineral salts. Furthermore, the production of haploids by these pollen cultures was strongly influenced by the photoperiodic and temperature regime experienced by the donor plants; it was best (0.07%) with pollen from short-day plants (8 hours light per day at 18 °C) and rather weak (0.015%) with pollen from long-day plants (16 hours light per day at 24 °C). In contrast to other reports, haploid production from anther cultures was not influenced by the photoperiod or temperature.Cytological studies undertaken at the end of the pre-culture period showed that there were no differences in the percentage of potential embryos for the stages of the late uninucleate, 1. pollen mitosis and early binucleate pollen of long-day plants (1.5%). This value was considerably higher with pollen from short-day plants (7–9%), indicating that short-day conditions at 18 °C of the donor plants are favourable for the induction of androgenesis. However, only the potential embryos formed by the pollen at the initial binucleate stage were able to continue androgenetic development after isolation.     

Formation of Fusarium metabolites in barley grain

Summary The influence of moisture content and temperature during storage of grain on the formation of Fusarium metabolites was studied. Naturally and artificially contaminated barley grain samples were stored at 15%, 25%, and 30% moisture contents, and at temperatures of + 5°C + 25°C, and + 30°C. Time of storage varied between one week and six months and the occurrence of Fusarium species and metabolites was analysed. The only Fusarium metabolite detected was zearalenone. The extent of Fusarium contamination decreased during storage whilst the concentration of zearalenone increased. To avoid the danger of mycotoxicoses, grains must be dryed immediately after harvest and then stored at a low temperature.     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

Effect of temperature and moisture content on Egyptian peanut seed-borne fungi

In peanut seeds adjusted to various levels of moisture content (8.5 %, 13,5 %, 17.5 % and 21 % on a dry-weight basis) and stored for six months at 5°, 15°, 28° and 45 °C, seed-borne fungi were monthly identified and counted using the dilution plate method, and the germinability of seeds was tested. The total count of seed-borne fungi (recovered at 28 °C) increased regularly and the germinability declined with the rise moisture content and with the lengthening of the storage period. At 5°, 15° and 28 °C,A. fumigatus was almost the most dominant fungal species followed by several fungi such asAspergillus flavus, A. niger, A. terreus, Penicillium funiculosum, P. notatum, Pyrenochaeta decipians andScopulariopsis brevicaulis. The degree of dominance of each fungal species depended upon the conditions of storage and the length of storage period.A. flavus gained its highest count in seeds adjusted to 13.5 % stored at 15 °C for 1 month.Penicillium predominated at 17.5 % and 21 %, and at 13.5 % and 17.5 % when the seeds were stored for long periods at 5° and 15 °C respectively.The total count of thermophilic fungi (recovered at 45 °C) significantly increased with the rise of moisture content and with the lengthening of the storage period between 1 and 2 months and regularly declined after 4 months.A. fumigatus was so extremely dominant that it was the main component of the total fungal flora. Several fungi truly thermophilic were isolated also. They were,Mucor pusillus, Humicola grisea var.thermoidae, H. insolens, H. lanuginosa (Thermomyces lanuginosus) andSporotrichum thermophile.     

Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 mol m^–2 s^–1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 mol m^–2 s^–1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 mol m^-2 s^-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 mol m^-2 s^-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 mol m^-2 s^-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - NOA 2-naphthoxyacetic acid     

Effect of pollen-style interaction on the pollen tube growth of Gossypium hirsutum

Summary All possible crosses among 5 strains of Gossypium hirsutum were made, and the pollen tubes were grown in vivo for 4 h before being fixed, stained and measured. Temperatures ranging from 18.5 to 40.0 °C were tested for pollen germination and pollen tube growth. The optimal temperature for pollen tube growth was 30.0 °C. Relative humidity levels of 0 to 100% were used as a pre-pollination treatment of the pollen. Significant differences among the mean pollen tube length of the strains occurred due to pollenXstyle interactions. The strains also differed in the number of styles which did not support pollen tube growth. These differences were also due to pollenXstyle interactions. Pollen and style strains could be ranked according to their relative contribution to pollen tube length.College of Agricultural Sciences Publication Number T-4-189     

Longevity and temperature response of pollen as affected by elevated growth temperature and carbon dioxide in peanut and grain sorghum

It is important to understand the effects of environmental conditions during plant growth on longevity and temperature response of pollen. Objectives of this study were to determine the influence of growth temperature and/or carbon dioxide (CO₂) concentration on pollen longevity and temperature response of peanut and grain sorghum pollen. Plants were grown at daytime maximum/nighttime minimum temperatures of 32/22, 36/26, 40/30 and 44/34 °C at ambient (350 μmol mol⁻¹) and at elevated (700 μmol mol⁻¹) CO₂ from emergence to maturity. At flowering, pollen longevity was estimated by measuring in vitro pollen germination at different time intervals after anther dehiscence. Temperature response of pollen was measured by germinating pollen on artificial growth medium at temperatures ranging from 12 to 48 °C in incubators at 4 °C intervals. Elevated growth temperature decreased pollen germination percentage in both crop species. Sorghum pollen had shorter longevity than peanut pollen. There was no influence of CO₂ on pollen longevity. Pollen longevity of sorghum at 36/26 °C was about 2 h shorter than at 32/22 °C. There was no effect of growth temperature or CO₂ on cardinal temperatures (T_min, T_opt, and T_max) of pollen in both crop species. The T_min, T_opt, and T_max identified at different growth temperatures and CO₂ levels were similar at 14.9, 30.1, and 45.6 °C, respectively for peanut pollen. The corresponding values for sorghum pollen were 17.2, 29.4, and 41.7 °C. In conclusion, pollen longevity and pollen germination percentage was decreased by growth at elevated temperature, and pollen developed at elevated temperature and/or elevated CO₂ did not have greater temperature tolerance.