Stress-induced formation of haploid plants through anther culture in cork oak (Quercus suber)

Induction of haploid embryos and regeneration of plantlets have been obtained, for the first time, in cork oak ( Quercus suber L.) by combining a starvation treatment in anther culture with a mild heat shock at 33°C for 5 days, followed by culture at 25°C in a simple agar medium without growth regulators. The same conditions had been shown previously to be optimal for embryogenic induction in isolated microspore cultures of several model species such as tobacco and wheat. These results support the notion that stress, particularly sucrose starvation, a heat shock or a combination of both treatments could be the major and general signal responsible for the inhibition of normal gametophytic development of the microspores and for the induction of the alternative embryogenic pathway. A similar approach may be used for the production of haploid and doubled haploids for plant breeding in other species that, like most forest trees, are still recalcitrant in anther culture.     

Phenological and growth patterns of the Mediterranean oak Quercus suber L.

The phenology of Quercus suber L., a dominant species of the montados in the Iberian Peninsula, was studied for 2 years in southwest Portugal. The seasonal progression of phenological events was analyzed in seven trees. Selected branches were examined monthly for shoot elongation, leaf number, branching, flowering, and fruiting. Radial stem growth and specific leaf weight were also studied. Active growth was observed from early spring to early summer. Reserves accumulated during winter and high photosynthetic activity in early spring apparently supported this strong development. The growth flush started with stem radial increment, which seemed to be impaired by spring rainfall. Male inflorescence production was the next phenological event. Old leaves were shed during new twig and leaf emergence. Shoot elongation and the number of new leaves produced were well correlated with the previous-year shoot's length, and were not clearly related to climatic factors. Radial growth resumed in autumn at a lower rate than in the previous spring, a possible consequence of a reserve depletion due to lower photosynthetic production in summer and investment on fruit maturation, which was complete by late autumn. Premature and excessive new leaf production were apparently subjected to self-pruning strategies related to the development of each tree's crown. Younger cork-oaks produced shorter and fewer shoots per module, and more sclerophyllous leaves than the older ones. A high intra-specific variability was observed in all the results.     

Synthetic seed production from encapsulated somatic embryos of cork oak (Quercus suber L.) and automated growth monitoring

Cork oak (Quercus suber) somatic embryos were coated with alginate for the production of synthetic seeds and their storability for commercialization was investigated. Also, the automatic monitoring of somatic embryo growth with a digital system of image capture was tested. A power regression model was fitted between size and fresh weight (Adjusted R-squared = 0.96). This method permitted growth assessment without contamination risk and opens the possibility of an automated control of culture growth for the future up scaling of plant production. Conversion rate of synthetic seeds was higher on medium supplemented with mineral nutrients than on medium without nutrients. Also, when the somatic embryos were coated without mineral nutrients added to the capsule, conversion rate was significantly lower. The addition of sucrose to the capsule had no significant effect on the conversion rate. No differences were recorded between 50 and 100 mM CaCl₂ for capsule complexation. Synthetic seeds were cold stored at 4°C for two months without significant loss of conversion capacity. The present study reports the first attempts to determine optimal storage time and conditions for conversion of encapsulated somatic embryos of cork oak.     

Micropropagation of juvenile and adult Quercus suber L.

This paper describes research on the application of tissue culture techniques to the micropropagation of cork oak (Quercus suber L.), a forest species of ecological and industrial importance in the Mediterranean area. Apical buds and nodal stem segments were employed as initial explants. Their origins were young seedlings, stump sprouts and sprouts formed on cuttings collected from old trees.The action of the mineral medium and growth regulators was studied in the multiplication stage. Media with low concentrations of ions, such as Sommer's or Heller's, are more suitable for growth and proliferation of explants than other media richer in salts. It was also observed that cytokinin (BA) must be present for the culture development. Adding low concentrations of auxin (NAA) to the medium improves the multiplication rate, especially in vegetative material of adult origin.The auxin type is the most important factor in the promotion of rhizogenesis. The method of application determines the quality of the root system. Treatment with low concentrations of IBA added to the rooting medium gives the best results.High sucrose concentration also improves rooting. Diluting the mineral rooting medium is slightly favourable, although there is no significant difference between it and the standard mineral concentration.Abbreviations D Durzan's - GD Gresshoff & Doy's - H Heller's - L Lepoivre's - MS Murashige & Skoog's - SH Schenk & Hildebrandt's - S Sommer's medium     

Nutrient content and seasonal fluctuations in the leaf component of coark-oak (Quercus suber L.) litterfall

Nutrient content and seasonality of the leaf component in cork-oak litterfal were studied over a two year period in two cork-oak forest sites differing in biomass and edaphic condition in the north-eastern Iberian peninsula. Fallen senescent leaves compared to young leaves showed higher non-mobile nutrient concentrations and lower mobile nutrient concentrations, specially P, N, K, and Mg. At both sites, seasonal fluctuations affected both leaf production and leaf mineral content. The maximum leaf fall period correspond to the start of the vegetative growth and to the lowest N and P concentration in the falling leaves. The opposite was true for the winter, when litterfal was minimal and N and P content in falling leaves was at a maximum.The comparison between falling leaves and canopy leaves suggests that the analysis of fallen leaves can be a useful measure of N and P nutrition in cork-oak. We found site dependent differences in nutrient content and nutrient remobilization. In comparison with Q. ilex, although litter production was in the same range, nutrient retranslocation was greater for Q. suber.     

Water relations of crok-oak (Quercus suber L.) under natural conditions

Daily and annual courses of leaf transpiration, stomatal conductance and shoot water potential of four Quercus suber individuals were compared in a semi-natural stand in southwest Portugal, from spring 1989 to early summer 1990.The trees investigated showed annual patterns typical of evergreen sclerophyllous species but varied in their range of stomatal operation. This appeared to be related to differences in hydraulic conductivity in the root-to-leaf pathway.Maximum stomatal conductance and transpiration rates occurred from March to June.Water stress was found to be moderate and winter cold stress due to low air and soil temperatures appeared to have an influence on plant water balance through their effects on flow resistances.Abbreviations g_sw stomatal conductance - g_max maximum stomatal conductance - PAR photosynthetically active radiation - RH relative humidity of the air - T leaf transpiration - Ta air temperature - TL leaf temperature - T_max maximum leaf transpiration - W air-to-leaf vapor pressure difference - shoot water potential - PD predawn shoot water potential - MIN minimum shoot water potential     

Influx,efflux and net uptake of nitrate in Quercus suber seedlings

Nitrate influx, efflux and net nitrate uptake were measured for the slow-growing Quercus suber L. (cork-oak) to estimate the N-uptake efficiency of its seedlings when grown with free access to nitrate. We hypothesise that nitrate influx, an energetically costly process, is not very efficiently controlled so as to avoid losses through efflux, because Q. suber has relatively high respiratory costs for ion uptake. Q. suber seedlings were grown in a growth room in hydroponics with 1 mM NO₃ ^-. Seedlings were labelled with ¹⁵NO₃ ^- in nutrient solution for 5 min to measure influx and for 2 h for net uptake. Efflux was calculated as the difference between influx and net uptake. Measurements were made in the morning, afternoon and night. The site of nitrate reduction was estimated from the ratio of NO₃ ^- to amino acids in the xylem sap; the observed ratio indicated that nitrate reduction occurred predominantly in the roots. Nitrate influx was always much higher than net acquisition and both tended to be lower at night. High efflux occurred both during the day and at night, although the proportion of ¹⁵NO₃ ^- taken up that was loss through efflux was proportionally higher during the night. Efflux was a significant fraction of influx. We concluded that the acquisition system is energetically inefficient under the conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency embryogenesis in immature zygotic embryos of sunflower

In the present investigation, nutritional requirements for induction of a high frequency of well formed somatic embryos (SEs) from zygotic embryos (ZEs) of sunflower were assessed. Variables like genotype, embryo size (0.5–10 mm), sucrose concentration (30–240 g l⁻¹), carbohydrate source (sucrose, glucose, maltose), agar strength (0.2–1.0%), basal media (MS, Gamborg, Nitsch, White), photoperiod (light/dark) and temperature (20–36°C) were tested. All these variables except photoperiod had significant effect on the frequency of embryogenesis. Highest frequency of embryogenesis was facilitated by Gamborg basal salt media, 120–210 g l⁻¹ sucrose, 0.8–1.0% agar, smaller sized embryos (0.5–2 mm) and incubation temperature of 28–32°C. In addition to these, growth regulator combinations (2,4-D, 2,4-D+kinetin, BA+NAA) in varying concentrations were tried. Media supplemented with 2,4-D promoted direct embryogenesis, BA+NAA facilitated formation of single/multiple shoots while there was no response on 2,4-D+kinetin supplemented media. Zygotic embryos with well differentiated embryos were transferred to growth regulator free half strength MS medium for whole plantlet development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoinhibition in cork-oak leaves under stress: influence of the bark-stripping on the chlorophyll fluorescence emission inQuercus suber L.

Quercus suber is the primary source for industrial cork and becomes bark-stripped every 9–10 years. Recurring cork extraction is a major stress factor and the large water loss from the stripped trunk surface may affect the water balance and tree productivity. To evaluate the effect of bark-stripping, fluorescence emission and stomatal conductance of leaves were determined in groups of bark-stripped and control trees. Fv/Fm ratio was found to be significantly lower in bark-stripped trees indicating a reduced photosynthetic efficiency of PSII. Photosynthesis was not found to be stomata limited. The reduction in Fv/Fm resulted from a decline in maximum and variable fluorescence while the initial fluorescence of the dark-adapted state (Fo) remained constant. A general decline in photosynthetic efficiency of PSII was found in all trees during the summer, probably reflecting the prolonged environmental stresses during a hot and dry season. Additional stress caused by the bark-stripping seems to enhance the susceptibility to photoinhibition of the trees.     

Bark stripping in cork oak (Quercus suber L.): effect of an antitranspirant application on gas exchange and water relations of the stripped surface

Quercus suber L. is the primary source of industrial cork, which can be legally collected every 9 years. The main objective of this work was to test the efficiency of an application of an antitranspirant, at three different concentrations, after the bark stripping. For this purpose, several measurements of the gas exchange, water potential, total chlorophylls and the carotenoids contents were determined in cork oak trees, at two times in a day, morning and afternoon. The antitranspirant film was applied immediately after stripping. Transpiration rate showed a significant increase in the afternoon. The parameters, water potential, photosynthetic rates, stomatal conductance and the intrinsic water use efficiency, showed a significant decrease from morning to afternoon. The difference between pigments concentration was not significant throughout the day. Water potential and transpiration rate were high in the treatments with lower antitranspirant concentration. However, the treatment with a higher paraffin concentration showed larger photosynthesis rate. This result suggests that the loss of water observed for the stripping surface can be minimized by a larger concentration of the antitranspirant.     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

High frequency production of embryos inDatura innoxia from isolated pollen grains by combined cold treatment and serial culture of anthers in liquid medium

Summary This study concerns the development of pollen embryos as affected by various physical conditions of culture in media devoid of hormones. Freshly isolated pollen, from anthers ofDatura, failed to form embryos regardless of whether they were cultured on liquid or solid medium. In contrast, pollen isolated from anthers precultured on solid medium did form embryos and the response could be increased by prior cold treatment of anthers at 4 °C for 4 days. However, the best results were obtained when anthers were cultured from the very beginning in liquid medium and transferred serially to fresh medium. Under such conditions, the anthers dehisced, allowing spontaneous shedding of pollen grains. It was thus possible to have several fractions of shed pollen continuing their development into embryos. When serial culture was started with anthers from cold-treated buds not only were embryos formed in all the fractions of shed pollen but the frequency was also considerably higher than in any mode of culturing.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

The influence of various physical parameters on anther culture of broccoli (Brassica oleracea var. italica)

Embryo formation by cultured broccoli (Brassica oleracea L. var. italica) anthers was best in the pH range of 5.5 to 5.8. Manipulation of the initial medium pH showed, however, that embryos could be recovered throughout the entire pH range tested. Experiments designed to test the influence of anther density on embryo production exhibited an apparent population effect. Comparison of anthers cultured with and without filaments showed a significantly lower level of embryo formation with filaments attached. The importance of anther orientation with the adaxial surface up was also demonstrated. Detailed studies of the effect of temperature on anther response showed the importance of 35°C treatments. Other temperatures and a variety of temperature manipulations were either comparatively ineffective or inhibitory. The duration of 35°C exposure required for optimal response varied widely between 18 and 48 h. Wide variation in plant to plant response was observed despite attempts to optimize the manipulation of physical parameters. Individual plants were identified that reliably formed many thousands of embryos, whereas other plants failed to form embryos under all tested conditions.     

Somatic embryogenesis and plant regeneration in Quercus acutissima

Immature embryos of Quercus acutissima were collected weekly beginning 5 weeks post-fertilization and cultured on modified MS(Murashige and Skoog) medium containing 1,000 mg/l glutamine and 5 mM proline with different combinations of IBA(0.5–10.0 mg/l) and BA(0 or 1.0 mg/l) in light. The highest percentage of embryogenic cultures occurred on the medium containing 0.5 mg/l IBA or 1.0 mg/l BA and 0.5 mg/l IBA. Four weeks after initiation, the embryogenic cultures were transferred to MS medium without plant growth regulators and cultured for 4 weeks. The somatic embryos were then transferred to germination medium. The best germination results were achieved from WPM(Woody Plant Medium) containing 0.1 mg/l BA. Plantlets from somatic embryos were incubated on WPM supplemented with 0.2 mg/l BA for 4 weeks and plantlets with well developed shoots and roots were transplanted to perlite and peat moss(11, v/v) mixtures and placed in a culture room. After being hardened off for 8 weeks, they were transferred outdoors where they grew.Abbreviation BA N⁶-benzyladenine - IBA indole-3-butyric acid - GA₃ gibberellic acid - ABA abscisic acid - MS Murashige & Skoog Medium - WPM Woody Plant medium     

Fertility of Triticum aestivum plants derived from anther culture

The fertility of plants from wheat anther culture was studied. It was found that one half of the 36 plants with diploid root tips didn't set seeds at all, and that 41 of the 42 plants with haploid root tips were completely sterile. It was surprising that sterility was so widely distributed even among the plants with diploid root tips.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

The improvement in regenerated doubled haploids from anther culture of wheat by anther transfer

This study was conducted to determine the most suitable method of regeneration by comparing two approaches: transfer of anthers (with and without embryo-like structures) to regeneration conditions after a period of two to four weeks on induction medium (= anther-transfer treatment) and transfer of embryo-like structures to regeneration conditions after five to eight weeks on induction medium. The early transfer of anthers brought about a significant reduction in the number of embryos formed, but nevertheless significantly improved the frequency of plant regeneration. Combining an optimal date of anther transfer with the early addition of colchicine to the induction medium (100 mg l⁻¹ for 1 and 3 days) led to an increase in the number of doubled haploid regenerants. The results indicate that transferring the anthers after 28 days and adding 100 mg l⁻¹ colchicine to the induction medium on one day only caused a significant improvement in the ability of green plants to regenerate (7.0 compared to 0.50) as well as in chromosome doubling (success index: 4.0 compared to 0.33). This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.