Isolated wheat microspore culture

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 × 10⁵ microspores per ml was necessary for continued microspore viability. One hundred and nine haploid or spontancously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 × 10⁵ microspores per ml is recommended for isolated microspore culture.Abbreviations LSmean least square mean - MES 2-N-morpholinoethane sulfonic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphtaleneacetic acid     

An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars

Fertile, green plants were regenerated from immature inflorescence explants from each of four Canadian wheat cultivars. The cultivars were representative of four classes of Canadian wheat. Explants from immature inflorescences of three size ranges were cultured on two types of media: MSI/MSR, which contains 1650 mg l^-1 NH₄NO₃and sucrose as a carbon source, and BII/BIR, which contains 250 mg l^-1 NH₄NO₃and maltose as a carbon source. Regeneration from all cultivars was significantly better on BII/BIR media than on MSI/MSR media. On BII/BIR media, `AC Karma', `Plenty', and `Fielder' gave the highest number of shoots per 10 explants, where the explants were derived from immature inflorescences 5.1 to 10.0 mm in length. 'Columbus' did not regenerate on MSI/MSR medium, and regenerated poorly on BII/BIR medium. Differences were found between cultivars with regard to the number of regenerant plants produced with the best treatments: `Plenty' produced 16.1 shoots per 10 explants, `AC Karma' 12.4, `Fielder' 6.4, and `Columbus' 2.2.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

An improved medium for in vitro grain development from detached wheat spikelets

Wheat spikelets detached from the spike at anthesis were cultured on solidified media and successfully produced mature grains. These grains resembled normal grains and contained well-developed, embryos. Lower concentrations of glutamine favored dry weight increase in developing grains. Such grains were indistinguishable from grains from greenhouse-grown plants in germination on moist blotting sheets. The technique of individual spikelet culture can be used to study physiology and development of wheat grains and kernels and to study host-pathogen interactions in wheat floret diseases such as Karnal bunt.     

Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture

Embryogenesis from isolated microspore cultures of wheat was improved by ovary co-culture when compared to a completely defined medium. This indicates that essential factor(s) in addition to PAA or its analogs may be supplied by the ovaries. Isolated microspores cocultured with 20 ovaries of wheat on the top of semi-solid MMS3 induction medium for 21–30 days gave the best response. Both the number and quality of the embryos was significantly increased. The maximum frequencies of dividing microspores and of embryogenesis were 94% and 2.4%, respectively. Up to 2583 embryos were formed per 100 anthers of cv Chris and between 18% and 43% of the larger embryos regenerated into green plants upon transfer. Genotype differences for both induction and embryogenesis phases were reduced using ovary co-culture. However, there was still a strong genotype influence on plant regeneration with cv Chris, with the F1 of Chris × Sinton displaying the highest frequencies. These results are important with respect to enhancing haploidy applications in wheat biotechnology and plant breeding.Abbreviations PAA Phenylacetic acid - MMS modified MS medium - MS Murashige and Skoog's medium 1962 - FHG Hunter's FHG medium 1988     

Small heat shock protein responses in leaf tissues of wheat cultivars with different heat susceptibility

The effect of heat stress on soluble proteins extracted from leaf tissues of bread (Triticum aestivum cv. Gönen-98, tolerant; cv. Cumhuriyet-75, susceptible; genome ABD) and durum (Triticum durum cv. Ege-88, tolerant; cv. Ankara-98, susceptible; genome AB) wheat cultivars differing in sensitivity to high temperature was examined by two-dimensional gel electrophoresis. At acclimation (37°C) and acclimation→high temperature (37°C→50°C) treatments compared to control (25°C), evaluation of gels revealed 31 proteins to be differentially expressed in first leaves as a result of heat stress in heat-susceptible and heat-tolerant cultivars of bread and durum wheats. All of the increased or decreased proteins in amount, newly synthesized and/or disappeared were in low-molecular-weight (LMW, 16.1–24.0 kDa) and generally acidic character (pI 4.8–6.9). The responses of the four cultivars were compared: Twenty-two of 31 proteins were detected as newly synthesized LMW heat shock proteins (LMW HSPs = small HSPs). The number of these sHSPs was different in cultivars which have the same genome. In addition, the number of the sHSPs in heat-tolerant cultivars was higher than in heat-susceptible cultivars. Some of the sHSPs were specific to cultivar. Most of the sHSPs synthesized at 37°C were also detected at 37°C→50°C treatment. It is suggested that sHSPs have special importance in two points: Firstly, sHSPs in cultivars showed abundance and diversity. Secondly, these proteins may play an important role in the acquiring of thermal tolerance.     

Effects of incubation temperature on microspore callus production and plant regeneration in wheat anther cultures

Anthers of wheat cultivars Orofen and Pitic 62 were incubated for 8 days at 15, 20, 25, 30, 35 and 40°C before transfer to 25°C. Compared with anthers cultured at 25°C constantly, anthers treated at 30°C produced 40% more microspore callus and green plants in both cultivars whereas those treated at 35°C produced 2–3 fold more green plants. Treatment at 40°C was deleterious. Possible modes of action of high temperature on callus production and albinism were discussed.     

Two pathways of plant regeneration in wheat anther culture

The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n=3x=21 were the most frequent.     

Cytogenetic structure of common wheat cultivars from or introduced into Spain

Summary Chromosome arrangements of twenty-eight cultivars of common wheat, Triticum aestivum L., from or introduced into Spain are compared with that of Chinese Spring taken as a pattern. All the cultivars analyzed differ from Chinese Spring by one or two reciprocal translocations. When 12 out of 28 cultivars were compared it was concluded that a minimum number of thirteen interchanges are present, involving at least ten different chromosomes of the complement. The interest of a reappraisal of the rôle of interchanges in the evolution of Gramineae is pointed out.     

In vitro pollen maturation and successful seed production in detached spikelet cultures in wheat (Triticum aestivum L.)

Summary Two spring wheat genotypes (cv Orofen and Chinese Spring) were compared for their in vitro pollen maturation capacity in detached spikelet cultures in a defined solid medium. Under these in vitro conditions Chinese Spring produced normal trinucleate pollen in 66.8% and Orofen in only 37.5%. In both cultivars the pollen maturation process from the middle uninucleate stage took approximately 3 days longer in vitro than in vivo. The pollen maturation time depended on the microspore developmental stage at the time that the culturing started. The viability, germination capacity, and fertilizing ability of the in vitro matured pollen also differed between the two genotypes. The seed set achieved in vitro (averagely 12.8%) offers promise for the practical application of this method for producing controlled or selected offspring.     

In vitro microspore selection in maize anther culture with oxidative-stress stimulators

Summary. In order to produce doubled-haploid maize plants tolerant of oxidative stress, in vitro microspore selection was carried out in anther culture with reactive oxygen species (ROS) progenitors such as paraquat, menadione, tert-butylhydroperoxide (t-BHP), and methionine combined with riboflavin. All the ROS progenitors reduced the anther induction, the formation of microspore-derived structures, and their regeneration potential. Abnormal cell divisions and progeny cell degradation could be observed during the development of microspores treated with ROS progenitors. Menadione and t-BHP influenced the microspore developmental pathway, as menadione induced the formation of embryoids, while t-BHP increased the proportion of calli in the microspore-derived structures. As the result of in vitro selection, 15, 10, 10, and 3 fertile doubled-haploid plants were obtained in cultures treated with paraquat, t-BHP, methionine combined with riboflavin, and menadione, respectively. Correspondence and reprints: Agricultural Research Institute, Hungarian Academy of Sciences, Brunszvik utca 2, 2462 Martonvásár, Hungary.     

Effects of donor plant environment and light during incubation on anther cultures of some spring wheat (Triticum aestivum L.) cultivars

Four different growth environments (field, two phytotron greenhouses and one growth chamber) were compared, using two genotypes of spring wheat, one recalcitrant and one responsive. Field-grown plants gave inferior results. Large improvements could be made by improving the conditions, embryoid frequencies in the two genotypes reaching 77.1% and 183.9% per 100 anthers, respectively. High light intensity during the induction phase strongly suppressed induction in both genotypes, but stimulated regeneration of green plants in the recalcitrant genotype, which had the lowest regeneration ability. Weak, diffuse light did not inhibit induction while the positive effect on regeneration was maintained. Also, another recalcitrant genotype was grown in the field, together with two F₁-hybrids (recalcitrant x recalcitrant and recalcitrant x responsive). Evidence for a three-factor system was obtained.     

Response of durum wheat cultivars to immature embryo culture,callus induction and in vitro salt stress

Response of twenty eight cultivars of durum wheat (Triticum turgidum var. durum) to immature embryo culture, callus production and in vitro salt tolerance was evaluated. For assessment of cultivars to salt tolerance, growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and 2.1% w/v) added to the culture medium during two subsequent subcultures (4 weeks each). Comparison of cultivars for callus induction from immature embryo was based on callus induction frequency and fresh weight growth of callus (FWG). While, for salt tolerance, the relative fresh weight growth (RFWG) and necrosis percent of callus were used. There were significant differences among cultivars for potential of regeneration from immature embryo, and ‘Shahivandi’ a native durum wheat cultivar originating from western Iran was superior among the cultivars tested. The FWG distinguished cultivars more than callus induction frequency did for callus induction evaluation. Hence, a range of FWG from 1.23 to 14.65 g was observed in ‘Mexical-75’ and ‘Omrabi-5’ cultivars, respectively. Growing calli derived from cultivars ‘PI 40100’ and ‘Dipper-6’ showed superiority for tolerating salinity under in vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

A comparative evaluation of two methods of selecting locations used for testing spring wheat cultivars

Summary One of the considerations of regional cultivar evaluation programs is to optimize the number of locations used for testing. Although optimization of numbers of locations using cluster analysis has been previously attempted, no objective comparison of methods has yet been made. A new clustering method that uses the pairwise contribution of locations to the cultivar x location mean square as the distance measure (LB) was compared to another method that employs diallel correlations as the distance measure (CL). Data from six spring wheat (Triticum aestivum L. em Thell) cultivars grown at 13 locations for five years were used in the initial cluster analysis. Another set of data, from a separate year, consisting of yields of the original 6 cultivars and a set of 12 independent cultivars was then used to check the validity of the original groupings and to compare the two methods. When the 6 original cultivars were considered, the LB technique was found to be superior to the CL. When the 12 independent cultivars were used, neither method was considered to be superior. Because of the lack of flexibility on the part of the LB method, neither technique could be deemed as fully adequate.Contribution Number 842 of the Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada     

Factors affecting plant regeneration from immature inflorescence of two winter wheat cultivars

Inflorescence explants of two winter wheat cultivars, Triticum durum cv. Kızıltan-91 and T. aestivum cv. Bezostaja-01, were used to evaluate the effects of vernalization period of donor plants, callus age and medium composition on regeneration capacity. Donor plants were grown for 7 d and they were exposed to 4 °C for 1, 2, 3, 4, and 5 weeks. The maximum inflorescence formation was observed as 79 % at 4 weeks and 73 % at 5 weeks of vernalization period for Kızıltan-91 and Bezostaja-01, respectively. Among 6 different callus induction and regeneration mediums, I₁-R₁ and I₃-R₃ have to be the best responding mediums for Kızıltan-91 and Bezostaja-01, respectively. In Kızıltan-91, calli induced from donor plants, vernalized for 3 weeks, showed a significantly lower regeneration capacity than counterparts vernalized for 4 and 5 weeks. The highest regeneration capacity of 69 % was obtained from 6-week-old calli produced from 4 weeks vernalized Kızıltan-91 donor plants. In contrast to Kızıltan-91 cultures, the effects of vernalization period and callus age on regeneration capacity were not significant in Bezostaja-01 cultures. The maximum numbers of tillers were obtained from 6-and 15-week-old calli for Bezostaja-01 and Kızıltan-91, respectively. In contrast to vernalization period of donor plants, callus age had no effect on seed number.     

Cytological analysis on the distribution and origin of the alien chromosome pair conferring blue aleurone color in several European common wheat (Triticum aestivum L.) strains

Summary Meiotic chromosome pairing and Giemsa C-banding analyses in crosses of several European blue-grained wheat strains with Chinese Spring double ditelosomic and other aneuploid lines showed that Triticum aestivum Blaukorn strains Berlin, Probstdorf, Tschermak, and Weihenstephan are chromosome substitutions, in which the complete wheat chromosome 4A pair is replaced, whereas the strains Brünn and Moskau are 4B substitutions. The alien chromosome pair in all of these strains is an A genome chromosome (4A) from diploid Triticum monococcum or T. boeoticum not present in common tetraploid and hexaploid cultivated wheats. The Blaukorn strain Weihenstephan W 70a86 possesses, in addition to a rye chromosome pair 5R compensating for the loss of part of chromosome 5D, a 4A/5DL translocation replacing chromosome pair 4B of wheat.     

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.     

Leaf senescence and grain filling affected by post-anthesis high temperatures in two different wheat cultivars

High temperature is a major factor affecting grain yield and plant senescence in wheat growing regions of central and east China. In this study, two different wheat cultivars, Yangmai 9 with low-grain protein concentration and Xuzhou 26 with high-grain protein concentration, were exposed to different temperature regimes in growth chambers during grain filling. Four day/night temperature regimes of 34°C/22°C, 32°C/24°C, 26°C/14°C, and 24°C/16°C were established to obtain two daily temperatures of 28 and 20°C, and two diurnal day/night temperature differences of 12 and 8°C. Concentration of a lipid peroxidation product malondialdehyde (MDA), activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT), chlorophyll concentration (SPAD) in flag leaves and kernel weight were determined. Results show that activities of SOD and CAT in leaves increased markedly on 14 days after anthesis (DAA) for the high-temperature treatment (34°C/22°C) and then declined. As a result, MDA concentration in leaves increased significantly under high temperature (34°C/22°C and 32°C/24°C). Compared with optimum temperature treatment, high temperature reduced the concentration of soluble protein and SPAD values in flag leaves. Grain-filling rate increased slightly initially, but decreased significantly during late grain filling under high temperature. As a result, final grain weight was reduced markedly under high temperature. Decreases in the activities of SOD and CAT and increases in MDA concentration in leaves were more pronounced with a 12°C of day/night temperature difference when under high temperatures. Kernel weight was higher under 12°C of day/night temperature difference under optimum temperatures (24°C/16°C and 26°C/14°C). The responses to high-temperature regimes appeared to differ between the two wheat cultivars with different grain protein concentrations. It is concluded that a larger diurnal temperature difference hastened the senescence of flag leaves under high-temperature conditions, but retarded senescence under optimum temperature treatments of 26°C/14°C and 24°C/16°C.     

Carbon monoxide sensitivity of cytochrome oxidase in wheat leaves in relation to dwarfing genes (Rht) in wheat cultivars

Summary Leaves of young seedlings of a number of tall cultivars of wheat, lacking the dwarfing Rht genes, readily responded to a brief 2 min exposure to CO, as assessed by in vivo aerobic assay of nitrate reductase. This test depends on the inhibition of cytochrome c oxidase by CO, which in turn renders cytosolic NADH available for the reduction of nitrate to nitrite in vivo. Semi-dwarf cultivars of wheat (Rht present) did not respond to CO in this way. Since CO forms a complex only with reduced cytochrome a₃, the results indicate differences in the redox state of cytochrome a₃, during in situ respiration of leaves from tall and semi-dwarf plants which are likely to be under genetic control.     

Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment,isolation methods,and hormones

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.