Barley anther culture: assessment of carbohydrate effects on embryo yield, green plant production and differential plastid development in relation with albinism

Sugars and polyols were tested at different steps of anther culture in barley (Hordeum vulgare L.) to elucidate their influence on both the overall yield of androgenesis and the structure of plastids in relation to albinism. During the pretreatment period, the osmotic regulation in the medium was beneficial to microspore embryogenesis regardless of the type and concentration of the tested osmoticum. The use of mannitol (300 mOsm/kg), sorbitol (180 mOsm/kg), PEG (240 mOsm/kg) and sucrose (180 mOsm/kg) gave the best results in terms of green plant production, although the influence of each substance differed according to the studied parameter. Similarly, during anther culture the regulation of the osmotic pressure in the medium had various effects, according to the osmoticum used. The best results were obtained using mannitol (364 mOsm/kg), providing 139.7 green plants per 100 plated anthers. Plastids were examined by electron microscopy following both pretreatment and culture. In the presence of mannitol and PEG, plastids did not accumulate starch at any stage of the protocol but they started to differentiate into chloroplasts in the microspore-derived embryos. Using sorbitol and sucrose, plastids differentiated poorly but accumulated large amounts of starch, suggesting that these sugars are metabolized by micropores and microspore derived structures. However, the accumulation of starch was not correlated with the occurrence of albinism. These results indicated that, in barley, the osmotic regulation was favourable to switch the microspore gametophytic program toward a sporophytic program regardless of the nature of the osmoticum. In addition, during the pretreatment period, mannito was found to be the most suitable osmoticum for subsequent embryo development.     

DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation,differentiation, hormones and hypomethylating drugs

Summary The level of auxin - both natural and synthetic — in the medium has a strong effect on the level of 5-methyl-cytosine in the DNA of carrot cells in culture. This level may vary from approximately 15% to 70% of total cytosine without apparent effects on growth rate and cell morphology. No effect was seen with cytokinin. During somatic embryogenesis, in the absence of hormones, variations were seen in the level of methylation according to a characteristic pattern. If hypomethylation is induced with drugs such as azacytidine, ethionine or ethoxy-carbonyl-pyrimidine, embryogenesis is immediately blocked. A mutant was isolated which is resistant to the action of hypomethylating drugs. It shows variations in the methylation pattern and variations in indole-acetic acid metabolism. In addition its regeneration is often associated with the production of tumors.Deceased     

Cellular change and callose accumulation in zygotic embryos of Eleutherococcus senticosus caused by plasmolyzing pretreatment result in high frequency of single-cell-derived somatic embryogenesis

Summary. Eleutherococcus senticosus zygotic embryos were pretreated with 1.0 M mannitol or sucrose for 3–24 h. This pretreatment resulted in a high frequency of somatic-embryo formation on hormone-free medium. All the somatic embryos developed directly and independently from single epidermal cells on the surface of zygotic embryos after plasmolyzing pretreatment. Scanning electron microscopic observation revealed that the epidermal cells of hypocotyls rapidly became irregular and showed a random orientation before somatic-embryo development commenced. At the same time, the epidermal cells in the untreated control remained regular. Callose concentration determined by fluorometric analysis increased sharply in E. senticosus zygotic embryos after plasmolyzing pretreatment but remained low in the untreated control. Aniline blue fluorescent staining of callose showed that the plasmolyzing pretreatment of zygotic embryos resulted in heavy accumulation of callose between the plasma membrane and cell walls. On the basis of these results, we suggest that plasmolyzing pretreatment of zygotic embryos induces the accumulation of callose, and the interruption of cell-to-cell communication imposed by this might stimulate the reprogramming of epidermal cells into embryogenically competent cells and finally induce somatic-embryo development from single cells. Correspondence and reprints: Division of Forest Resources, College of Forest Sciences, Kangwon National University, Chunchon 200-701, Republic of Korea.     

苜蓿抗乙硫氨酸愈伤组织的筛选和特性

用乙硫氨酸为筛选剂,通过幼苗和组织培养筛选得到乙硫氨酸抗性愈伤组织。该愈伤组织在含乙硫氨酸的培养基上表现出较高的半抑制剂量和相对生长量。作为天门冬氨酸途径的产物,甲硫氨酸、异亮氨酸和赖氨酸在所筛选的愈伤组织中分别增加到对照的两倍多,但苏氨酸保持正常水平,另外酪氨酸、半胱氨酸和亮氨酸也有所增加,而在所筛选的愈伤组织中缬氨酸浓度却下降。说明在所筛选愈伤组织中存在一个以上与氨基酸合成相关的酶发生改变。同工酶分析表明,该愈伤组织中出现对照中没有的分子量为44kD的超氧化物歧化酶和分子量为45kD的酯酶谱带。     

The influence of culture conditions on anther culture response of commercial varieties of Solanum tuberosum L.

The effect of media manipulatioss, temperature pretreatment, carbohydrate source, and seasonal variation on tetraploid potato anther cultures was investigated. The anther culture responses of three commercial Nordic potato varieties from Scandinavia and two from Germany were compared on different media manipulations. With most of the varieties, solid MS media gave better yields than other published media manipulations. Pretreatments at +6°C and at +30°C were studied on Pito and Danva varieties. The +6°C pretreatment and no pretreatment had the same effect on the anther culture response of cv. Pito, while with cv. Danva pretreatment at +6°C promoted embryogenesis. The +30°C pretreatment had no positive effect on anther culture response on either cultivar. The effect of maltose, melibiose and mannitol individually and in combination with sucrose were compared to normal sucrose medium in cv. Pito anther cultures. Anthers incubated on normal sucrose medium gave the highest embryoid and plant yields; the second highest plant yields were obtained on pure maltose medium. Strong seasonal variation was observed throughout the year in cv. Pito anther cultures. The percentage of anthers producing embryoids ranged from 15–20% during September and October to just 1–3% from February through May. The annual average embryoid production rate was 6.18%.     

Moderate desiccation dramatically improves shoot regeneration from maize (<Emphasis Type="Italic">Zea mays L.</Emphasis>) callus

Tissue and cell culture systems are vital to many areas of maize research and improvement. Efficient shoot regeneration remains a limiting factor for most elite lines. To enhance shoot regeneration, calluses derived from immature embryos of four genotypes were subjected to 6, 12, 24, 48, and 72 h of desiccation on sterile filter paper before shoot induction. We achieved up to 32% desiccation (measured as mass) and 48% imbibition (measured as mass gain in 48 h incubation) which was 3.4-fold higher than nondesiccated controls. This rapid desiccation/imbibition procedure enhanced shoot regeneration and development, but its efficiency was crucially dependent on the degree and duration of desiccation. All four genotypes calluses regenerated shoots best after 48 h of desiccation corresponding to a 23.7% average desiccation percentage, and regeneration frequencies reached 42–74%, which increased 1.5–2.1-fold compared with that of nondesiccated calluses. The effect of desiccation treatment on the shoot regeneration increase took place mainly during the early phase of induction. The weekly average shoot regeneration frequency of calluses with 48 h desiccation in the first wk reached 8.8% (the control was no regeneration); then it increased 3.0 and 2.3-fold in the second and third wk induction, respectively. We demonstrate that the moderate desiccation of calluses increases the yield of shoot regeneration and speeds up regeneration course in maize.     

Effects of Mannitol Pretreatment on Androgenesis of Barley (Hordeum vulgare L.)

The effects of mannitol pretreatment on androgenesis of barley were systematically studied in comparison with that of cold pretreatment and control. The results showed that mannitol pretreatment could significantly increase the frequency of pollen survival reaching 19.0% on the eighth day, while in cold pretreatment and control they were 8.4% and 6.6 %, respectively. Mannitol pretreatment could also improve the quality of pollen and inhibit starch production from microspore, which were quite advantageous to microspore division and development. The developing period was shortened 2--3 days as compared with cold pretreatment and control. The major developmental pathways of androgenesis after mannitol pretreatment were the equal division (B pathway). In addition, the majority of microspore nuclei were diploids. On the contrary, the major microspores pretreated with low temperature had fewer chromosomes than with mannitol pretreatment, the microspore nuclei were haploids.     

Influence of anther pretreatment on the efficiency of androgenesis in barley

The change in the developmental pathway of microspores from gametophytic to sporophytic is induced by stress during pretreatment of spikes and anthers. In our experiments, anther culture of three barley cultivars was tested with regard to the effect of chilling at 4 degrees C for 28 days, starvation in 0.3 M mannitol solution for 4 days, and a combination of both methods. Chilling was shown to increase embryo/callus formation, while mannitol treatment favoured plant development, including development of green plants; simultaneous application of the two stress factors for 4 days proved to be ineffective. The tested cultivars exhibited a similar ability (calculated per 100 transferred embryos/calli) to develop plants without pretreatment; however, their responses to stress varied greatly. The collected data indicate that mannitol pretreatment, as compared to chilling, is more efficient in responsive cultivars.     

Callus induction in culture of Oenothera hookeri and Oenothera picensis anthers

In the present work we try to determine optimum conditions for callus induction in anther culture of Oenothera hookeri and O. picensis. The anther callus yield was increased when the anthers were cultured on modified MS medium supplied with 2 mg dm^-3 2,4-D and 2 mg dm^-3 NAA, in both species. In O. hookeri, best results were obtained when anthers were excised from 7.2 - 9 mm buds at the stage of vacuolated microspores, then pretreated at 4 °C for 2 d and grown under 16-h photoperiod. The response to anther culture of O. picensis was generally very poor compared with that of O. hookeri. The higher yield of calli was obtained when anthers were excised from 6.2 - 8 mm buds at the stage of vacuolated microspores and grown under continuous light. The cold pretreatment of buds decreased anther response in this species. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparative study of somatic embryogenesis in Secale vavilovii

Summary The ability of immature embryos, inflorescences and leaves of Secale vavilovii to form embryogenic callus was tested on Murashige and Skoog (1962) medium supplemented with different concentrations of 2,4-D. All cultured immature embryos formed calluses. The highest percentage of embryogenic callus production was from 1–2 mm embryos. Young leaves also formed calluses, mainly from the 10–15 mm basal segment, the percentages of embryogenic calluses being higher when cultures were maintained in darkness. Embryogenic calluses were obtained also from all the cultured immature inflorescences, in the three cases, rooted green plants were obtained and grown in soil. Comparison of the responses of the three explants used indicates that immature inflorescence is the most useful explant for obtaining regenerated plants in Secale vavilovii.     

The influence of osmotic pretreatment and inoculum age on the initiation and regenerability of switchgrass suspension cultures

Summary Experiments were conducted to study the influence of osmotic pretreatment and inoculum (callus) age on the initiation and induction of somatic embryogenesis from suspension cultures of ‘Alamo’ switchgrass (Panicum virgatum L.). Embryogenic 10-, 20-, or 30-d-old calluses (hereafter referred to as inocula), produced from in vitro-cultured inflorescences, were used as explants to initiate the suspensions. Inocula were cultured for 30 h on MS solid medium containing 0.1, 0.2 or 0.3 M each of sorbitol and mannitol as an osmotic pretreatment. They were then transferred to liquid MS medium with 5 μM 6-benzylaminopurine and 9 μM 2,4-dichlorophenoxyacetic acid and cultured for 28 d in 2-ml Multiwell™ plantes. Individual multiwell contents were transferred to 125-ml Erlenmeyer flasks containing 20 ml of the above liquid medium and cultured for an additional 2 wk. Cultures initiated from the 10-d-old inoculum produced a higher embryogenic response than those initiated from 20- or 30-d-old inocula. Cultures initiated with 30-d-old inocula produced nonembryogenic clusters and the number of embryogenic cells was low. More embryos and a higher regeneration frequency were produced by 0.3 M than by 0.1 or 0.2 M of each of the osmostica.     

低温预处理对水曲柳体细胞胚胎发生的影响

用4℃低温预处理未成熟的水曲柳种子0-30d,取出种子内的合子胚为外植体诱导体胚发生,研究低温预处理影响体胚发生的结果表明：低温预处理过的外植体其体胚发生总数和子叶胚发生数均低于未作低温预处理的;随着预处理时间的延长,畸形胚发生数和发生比率与总的体胚发生数和发生率的变化趋势基本相同;处理20d的正常胚发生数和发生比率的绝对数虽然很低,但远高于不作低温预处理的。说明4℃低温预处理对水曲柳体胚发生没有促进作用,对畸形胚的发生也不能控制,总的来讲,适当的低温处理有一定的改善正常体胚发生的潜力。     

An improved method for dihaploid production in Nicotiana rustica through anther culture

Summary The efficiency of dihaploid production from anther culture in N. rustica has been improved by studying the effects of pretreatment temperature, pretreatment duration and initial anther stage on anther response, anther productivity and time to first plantlet production. Pretreatment was most effective on anthers at or around the stage of pollen mitosis. Pollen mitosis stage anthers pretreated at 9 °C for 15 days gave the best results. Both spontaneous and induced dihaploids were obtained. Small plantlets treated with 0.4% colchicine and 2% DMS solution for 5 h produced the maximum number of dihaploids (more than 50%). These considerable improvements in the efficiency of the techniques have made dihaploidy an attractive method for producing inbred lines in N. rustica. This will permit a large scale comparison of dihaploids with more conventional methods of inbreeding such as single seed descent and pedigree breeding.     

Effects of Different Pretreatment on Three Endogenous Hormones Contents and Peroxidase ctivity in Barley Anther

Endogenous hormones (ABA, IAA, IPA) contents and peroxidase activity during the mannitol and cold pretreatment periods were measured in two varietion ("Igri' and "Har- rington' ) barley (Hordeum vulgate L. ) anther. The results were as follows: Firstly, anther endogenous hormone contents were identical changes in both varieties during mannitol pretreatment period. The endogenous hormone contents of anthers were rapidly increased during the early period of pretreatment, reaching their maximum at the first 12 h or the 1st day. After then. the contents began to reduce until they reached a stable level. The result suggested that combined effect of some endogenous hormones, including ABA, was essential for changing the developmental pathway of pollen. Secondly, similar changes in peroxidase activity during the period of mannitol pretreatment were seen in both barley varieties. The activity of peroxidase was remarkably increased during the early period of pretreatment, being highest at the 3rd day. After that, it was reduced from the 3rd day to the 5th day, but increased again from the 5th day to the 7th day. Finally, there were two peaks of peroxidase activity during the cold pretreatment period, which were at the 2nd day and the 14th day for "Harrington' or the 5th day and the 21st day for "Igri'. The second peak was higher than the first one. A second increase in the peroxidase activity for both genotypes were observed from the 28th day to the 35th day during the cold pretreatment period as this phenomenon was in concert with that of mannitol pretreatment.     

Variability in maturation and germination from white spruce somatic embryos,as affected by age and use of solid or liquid culture

Summary The yield of morphologically normal Stage 3 somatic embryos of white spruce [Picea glauca (Moench) Voss], and subsequent germinability, was affected by culture age and use of solid and/or liquid culture growth conditions. Of the conditions that were compared, best results were obtained with cultures up to 3 yr old that had been continuously grown in liquid medium. Such material yielded up to 374 morphologically normal Stage 3 embryos per g f. wt. inoculum, when routinely pretreated using a 1 wk 2,4-dichlorophenoxyacetic acid-free period before maturation. By comparison the continual use of solid culture conditions resulted in lower yields (5/g f. wt. inoculum), and the use of solid medium in combination with liquid medium showed a greater affect of age on the production of normal Stage 3 embryos (348/g f. wt at 1.5 yr down to 19/g f. wt. at 3 yr) over the age range tested. In the absence of culture pretreatment, the oldest liquid cultures yielded only 44 normal Stage 3 embryos/g f. wt. inoculum, and the comparable solid to liquid cultures yielded 1.3/g f. wt. inoculum. The number of aberrant Stage 3 embryos in older cultures was reduced as a result of culture pretreatment; for example, in the oldest liquid cultures these represented 83% of the Stage 3 embryo population without pretreatment and 45% with pretreatment. Normal Stage 3 somatic embryo yield and germination characteristics (radicle and epicotyl development) were informative in distinguishing among the conditions studied. Germination characteristics were especially important when maturation responses were incapable of distinguishing among age classes. NRCC Contribution no. 38462.     

Factors affecting somatic embryogenesis in anther cultures of Chinese pink <Emphasis Type="Italic">(Dianthus chinensis</Emphasis> L<Emphasis Type="Italic">.)</Emphasis>

In this study, we aimed to maximize the rates of somatic embryogenesis achievable in anther cultures of Chinese pink (Dianthus chinensis L.) (2n = 2x = 30). The genotype of the donor plant was found to be a major factor in determining the success rate. Conditions imposed during anther culture (notably medium composition and light conditions) and pretreatments (namely, cold, heat, and mannitol incubations) were also found to influence somatic embryo induction. For example, the highest levels of embryogenic callus induction were achieved when the donor buds had been cold pretreated and the subsequent anther culture was maintained in darkness. Furthermore, there appeared to be an interaction of genotype with culture conditions. Thus, in cultures of the cultivar (cv.) ‘Carpet’, the highest rates of embryogenesis were obtained when the anthers had received a 5-d heat-shock, but such a thermal treatment did not generally produce a significant effect. Likewise, a 3-d mannitol pretreatment was optimal only for the cross-hybrid line ‘HC’. Assessment of the ploidy of the plants regenerated from the anther cultures revealed both diploid and tetraploid plants. Histological and cytological observations showed that all of these (both from n-pollen and 2n-pollen lines) derived from anther wall cells. Spontaneous chromosome doubling was inferred to have occurred during the embryogenic callus culture period.     

ORIGIN AND DEVELOPMENT OF POLLEN EMBRYOIDS AND POLLEN CALLUSES IN CULTURED ANTHER SEGMENTS OF HYOSCYAMUS NIGER (HENBANE)

The dedifferentiation of pollen grains of Hyoscyamus niger (henbane) into embryoids and calluses was examined by culturing identical segments of the same anther in a mineral salt-sucrose basal medium and in the basal medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid, respectively. Addition of auxin enhanced anther efficiency but did not affect the number of embryogenic pollen grains of an anther segment transformed into calluses. In anther segments cultured in the basal medium, the organogenetic part of the pollen embryoid was formed by the division of the generative cell alone, or by the division of both generative and vegetative cells. More or less similar pathways were followed by pollen grains of anther segments cultured in a medium containing auxin to form calluses. Culture of anther segments in a medium containing a high concentration of auxin (50.0 mg/l) led to a significant reduction in the yield of calluses which were formed almost entirely by the division of both generative and vegetative cells. The bearing of these observations on the role of auxin in determining the pathway of differentiation of embryogenic pollen grains in cultured anther segments is considered. The appearance of embryogenic pollen grains in close proximity to the tapetum as seen in longitudinal sections of cultured anther segments has suggested a role for a gradient of tapetal factors in embryogenic induction.     

低温预处理影响籼稻花药培养效率的研究

低温预处理使籼稻花药在培养初期退化花粉百分率低于对照;多核花粉百分率明显高于对照。经过低温预处理的花药来见新的可溶性蛋白谱带出现;淀粉酶同工酶谱带少和弱于对照,过氧化物酶同工酶中碱性区活性初期弱而后期强于对照。低温处理使药壁组织衰退进程有所延缓,但未能制止衰退,也未出现如粳稻中所见的药壁细胞充实发育的现象。低温预处理只能稍微延缓籼稻药壁组织的衰退进程,有限度地促进籼稻花药培养效率。     

The effect of starch and ineubation temperature in anther culture of potato

Three Andean tetraploid potato genotypes (2n=48) and 7 anther-derived dihaploids (2n=24) originating from two of the tetraploids were used in anther culture. Relative number of embryos/vial was significantly higher when the anther culture media was gelatinized with 3% potato starch than when Gelrite or wheat starch (3%) were used as gelatinizing agents. The degree of anther culture response varied between tetraploids but also within a group of related dihaploids. Additionally, the embryo production of individual genotypes, tetraploids as well as dihaploids, was dependent on the incubation temperature (10, 15, 20, 25, 30°C) of the anther culture. The incubation temperature of the anther culture was also important for the regeneration rate. Direct regeneration was mostly stimulated when the anther culture was incubated at 20°C.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid     

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.