Plant regeneration from isolated microspores of Triticum aestivum

Summary Wheat microspores were isolated, without prior anther culture, from a range of genotypes and cultured to regenerate self-fertile plants. Microspores were isolated using a microblender and competent microspores were enriched by gradient centrifugation. The use of maltose as the sole carbohydrate in the culture medium and co-culture of microspores with wheat or barley ovaries were critical for development of microspore-derived embryos. Results were also improved when spikes were pretreated by emersion of the basal ends of detached heads in water at 25°C for 2d. This procedure leads to highly reproducible production of plants.     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

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.     

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     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

Plant regeneration from long term suspension culture-derived protoplasts of hexaploid wheat (Triticum aestivum L.)

Highly regenerable callus cultures have been obtained from immature embryos of hexaploid wheat cv. Oderzo. Friable fast growing calli were induced at high frequency. Suspensions were initiated from the most friable callus lines: they became established in about two months. Suspensions consisted of cell aggregates of 30 to 1000 um in diameter. Upon plating on MS hormone-free medium, suspensions regenerated green plantlets, and their regenerative capability was maintained for at least 10 months. Protoplasts were isolated from 7–8 day old suspension cultures with a yield of 4–6×10⁶ protoplasts/g fresh weight cells. Protoplast culture was either in liquid medium or in a bead-type system with agarose beads. First divisions were detected at day 5. At day 14 visible colonies were detected and the plating efficiency was evaluated between 2 and 8% over the initial number of protoplasts plated. Protoplast-derived calli were cultured in the presence of 1 mg/l IAA and 0.5 mg/l zeatin and were used for reinitiating new suspension cultures. Upon plating onto MS hormone-free medium, with or without the addition of 0.1 mg/l GA3, calliclones were induced to differentiate. Regeneration of complete plantlets, with shoot and roots took about two months. Plantlets were grown in sterile conditions until 12–15 cm height, and were subsequently transplanted in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - PCV packed cell volume - MES morpholinoethanesulfonic acid     

High-frequency embryogenesis and plantlet regeneration from isolated microspores of indica rice

We report high-frequency embryogenesis and plantlet development from microspores isolated from anthers of two indica (IR-43, IR-54) and a japonica (T-309) rice cultivars, without prior nutrient preculture of anthers. Pretreatment stress of anthers with mannitol or a sugar-starvation medium, and use of maltose as the carbohydrate source in the microspore culture medium were found to be critical. Co-culture of microspores with rice ovaries was found beneficial but not essential. More than 60% of the microspores of the japonica variety Taipai-309 and 25–45% of the indica cultivars IR-54 and IR-43 showed induction of non-gametophytic development. Consequently, in the best treatments for IR-43 and T-309, more than 500 microspore-derived embryos could be obtained from a single dish (35 mm) containing about 80,000 microspores. Among the indica cultivars, the maximum response was obtained in the basal medium M-019. Plantlet regeneration occurred in about 9% (T-309), 7% (IR-43) and 2% (IR-54) of the transferred embryo-like structures. Received: 6 November 1996 / Revision received: 18 June 1997 / Accepted: 20 August 1997     

Plant regeneration through callus initiation from thin mature embryo fragments of wheat

A wheat regeneration system was developed using mature embryos. Embryos were removed from surface-sterilised mature caryopses (winter wheat Odeon cultivar and spring wheat Minaret cultivar) and ground to pieces through a sterile nylon mesh. The fragments were characterised by means of the image analysis technique. They were 500 M mean diameter and most of them were elongated. They were used as explants to initiate embryogenic calli on solid medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid. The morphogenic pathway of the initiated calli was followed for a 40-day culture period. Active cellular division occurred within 24 hours of cultivation. Several hundred calli were produced from 100 fragmented embryos within 3 days. A 90% callus induction rate was achieved and proembryos appeared by the 8th day of culture. The highest embryogenic calli induction rate of 47% was obtained when 2,4-dichlorophenoxyacetic acid was suppressed after a 3–4 week induction period. Two regeneration methods were finally compared. A total of 513 plantlets were produced. The optimal protocol produced 25–30 plants per 100 embryos. This regeneration method may be suitable for transformation applications.     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

Embryogenic cell suspensions of Triticum aestivum x Leymus angustus F1 hybrids: characterization and plant regeneration

Summary Embryogenic cell suspension cultures were established from Triticum aestivum X Leymus angustus F₁ hybrids, using compact nodular calli derived from inflorescence segments. Calli originating from leaf segments did not give rise to stable cell suspensions. Growth measurements of the cell suspensions revealed that they continued rapid growth up to 10 days after subculturing. Flow cytometric studies of the cell cycle over a 7 day culture period showed that the majority of cells were in G₁ phase while the rest were either in S or G₂. During the 7 days of culture, no significant differences in DNA distribution patterns were observed. The cells from suspension cultures produced somatic embryos when they were transferred to different solid media. The embryos germinated and gave rise to plantlets which were successfully rooted and transferred to soil.     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

Embryogenesis and Regeneration of Green Plantlets from Wheat (Triticum aestivum) Leaf Base

A short-term regeneration system from leaf-base-derived callus of wheat (Triticum aestivum L.) was developed. Embryogenic callus formation and shoot regeneration were achieved from the first basal segments of 3–4-day-old seedlings. Callus formation frequency as well as plantlet regeneration frequency was dependent on the composition of basal medium and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). MS medium with 2,4-D 4.5–9.0 mol l^–1 was optimal for the culture of wheat leaf base. Effects of different combinations of plant growth regulators, which were added in either callus induction medium or shoot regeneration medium, were tested. Adding of BAP in callus induction medium shortened the time of shoot emergence but could not improve the producing of embryogenic calli and green plantlets. Optimal ratio of 2,4-D, BAP and NAA gave similar regeneration frequency to control. Existence of cytokinins in regeneration medium had no effect on increasing the regeneration frequency. The regenerants could grow to normal, fertile plants after they were transferred into soil.     

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.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

Pollen callus culture in Triticum aestivum

Summary Pollen shed between 4–8 d from anthers of Triticum aestivum cultured in liquid medium gave rise to calluses. Tillers were harvested at the mid-to late-unicellular pollen stages and chilled for 8 d at 4–5 °C before the anthers were dissected out. Pollen cultures gave about 6 times as many calluses on a per anther basis as anthers cultured on solid medium. With the most productive of 5 cultivars tested, pollen culture results in roughly one callus for each anther used, though the calluses formed by pollen culture were less productive for the regeneration of shoots than calluses derived from anthers cultured on solid medium. The ratio of green to albino shoots is roughly 1 1 for anther cultures but considerably less for pollen cultures.     

Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) somatic embryogenesis from isolated scutellum: Days post anthesis,days of spike storage,and sucrose concentration affect efficiency

Summary To improve the efficiency of somatic embryogenesis of isolated scutella from commercial wheat (Triticum aestivum L.) cultivars, two factorial experiments were conducted to examine effects of days post anthesis (DPA), days of spike storage (DSS) at 4°C, and sucrose concentrations (SC) on the percentage of scutella producing mature embryos and the number of mature embryos produced per responsive scutellum. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 DPA and stored at 4°C for 7, 10, 13, and 16d were placed on embryo induction medium [Murashige and Skoog plus 9.96 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 110 mg l⁻¹ casamino acids], incubated in darkness for 12–14 d and then under light for 2 wk. The interaction of DPA × DSS significantly affected the percentage of scutella producing mature embryos, while only DPA affected the number of mature embryos per responsive scutellum. In the second experiment, scutella isolated from spikes collected at 12 DPA and stored for 15, 16, 17, 18, and 19d were placed on embryo induction medium containing 2, 3, 4, and 5% sucrose. The interaction of DSS × SC significantly affected both the percentage of scutella producing mature embryos and the number of mature embryos per responsive scutellum. In general, DPA/DSS/SC combinations, 12/17/3, 12/18/3, and 12/19/2, yielded the numerically highest embryogenesis efficiencies.     

Plant regeneration from cultured immature embryos and inflorescences ofTriticum aestivum L. (wheat): Evidence for somatic embryogenesis

Summary Tissue cultures ofTriticum aestivum L. (wheat) initiated from young inflorescences and immature embryos possessed the potential for regeneration of whole plants. Both a friable and a compact type of callus were produced on Murashige and Skoog's medium with 2 mg/l 2,4-dichlorophenoxyacetic acid. The friable callus contained meristematic centers in which the peripheral cells ceased dividing, elongated, and could be easily separated. Roots were frequently formed in this type of callus. The compact, yellowish, and nodular callus arose from the epithelial and sub-epithelial cells of the embryo scutellum, and the rachis and glumes of the young inflorescence. Such callus had a smooth surface and characteristic chlorophyllous areas. Plants were regenerated only from the compact callus. The first sign of differentiation in the compact callus was the formation of a cleft or notch on the smooth surface, followed by the appearance of trichomes and the direct development of leafy structures which were not associated initially with any shoot meristems. Multiple shoots subsequently arose at the bases of the leafy structures, which are considered modifications of the scutellum, a definitive part of the cereal embryo. Accordingly, we suggest that while typical bipolar embryos are generally not formed, plant regeneration nevertheless takes place through embryogenesis and the precocious germination of the embryoids. Plants regenerated from immature embryo and inflorescence cultures were grown to maturity in soil, and were shown to have the normal chromosome number of 2n=6x=42.