Embryo yield in wheat anther culture is influenced by the choice of sugar in the culture medium

Summary The effect of employing different sugars in wheat anther culture has been investigated using four Spring wheat cultivars. The most responsive cultivar, Orofen, gave a three to four-fold increase in embryo yield when maltose was used in place of sucrose, with 50 embryos being produced for every 100 anthers cultured. Measurement of sugar concentrations in the culture media indicated that sucrose was more rapidly hydrolysed than maltose. However, neither the osmotic potential of the medium nor the concentration of glucose appeared to be critical factors in determining embryo yield.     

Donor Plant Growth Factors Affecting Anther Culture of Maize and Sweetcorn (Zea mays)

Anthers from seven unselected commercial sweetcorn lines andten experimental maize lines were cultured on a liquid/solidbi-layer culture medium, containing 13 % maltose as the carbonsource. Mean anther efficiencies (number of embryos per 100anthers plated) of 0 to 27.6 % were recorded, with the maximumefficiency of 57.1 % from one plant. The anther efficiency wasfound to be dependent on genotype, microspore developmentalstage and the growth temperatures of donor plants. Immaturemicrospores were found to continue their development duringthe cold pretreatment of the spikes, and this in turn influencedthe level of response to culture. Direct regeneration of embryoidsto plants occurred most frequently when well formed uni- orbi-polar embryos were produced. The quality of embryo producedwas apparently inversely correlated with the number of embryosproduced. Zea mays, haploid culture, embryos, microspores     

Effect of maltose on the response of potato anthers in culture

Anthers of the Solanum tuberosum genotype H3703 were cultured on medium containing equimolar concentrations of sucrose or maltose. It was found that significantly more pollen embryos became plants after culture on maltose and hence the yield of plants per 100 anthers cultured increased significantly. Mechanisms by which carbohydrate source may influence response to anther culture are discussed.     

Improved embryoid induction and green shoot regeneration from wheat anthers cultured in medium with maltose

Summary Anthers from spring wheat (Triticum aestivum L.) genotypes, including six F₁ hybrids, were cultured in a modified liquid N6 medium containing either sucrose or maltose. In every case, use of maltose resulted in greater microspore callus induction and green shoot regeneration than culture in sucrose-containing medium. Induction in maltose medium also allowed green shoots to be recovered from crosses that showed only a poor response in other media and from two genotypes that did not respond to modified N6 medium with sucrose. Replacement of sucrose with maltose generally resulted in microspores having a more embryogenic mode of development in which distinct embryoids often formed. The most responsive genotype produced over 200 green shoots/100 anthers when cultured in medium with maltose.NRCC publication no. 31494     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

矮牵牛花药培养及植株再生研究

采用花粉发育双核期的矮牵牛花药,研究不同浓度植物生长调节剂配比及不同浓度蔗糖和麦芽糖对花药诱导率的影响。结果表明,采用6-BA和IBA组合诱导效果较好;NH+6-BA 1.5mg/L+IBA 1.5mg/L花药诱导率较高;麦芽糖诱导效果明显比蔗糖好。     

Anther culture with different genotypes of opium poppy (Papaver somniferum L.): effect of cold treatment

This study concerns anther culture and the production of microspore-derived calluses and plants of the opium poppy (Papaver somniferum L.). It was confirmed that growth regulators were necessary for microspore callus production. Cold treatment (7 d at 7°C) of the buds prior to culture lead to a twofold increase in the frequency of responsive anthers and in the number of calluses per 100 anthers plated. Callus was produced from cultured anthers of several genotypes, covering a wide genetic background. Step by step removal of growth regulators from the culture medium promoted organogenesis and plant regeneration. Most regenerated plants were diploid. The overall process of microspore embryogenesis closely resembled that described in previous reports on somatic callus production and plant regeneration from poppy hypocotyls in vitro.     

Osmotic potential of media affecting green plant percentage in wheat anther culture

The percentage of green plants in anther culture is known to be controlled by the genetics of anther donor materials. The objective of this study was to determine whether components in the culture media also would have a significant influence on the percentage of green plants from wheat anther culture. Anthers of a spring wheat cultivar, Pavon 76, were cultured on potato 4 (P4) induction media with various modifications. Addition of 200 g/l ficoll to the liquid P4 medium significantly increased the percentage of green plants even though the final yield of green plants per 100 anthers was lower than the liquid medium. A higher concentration of maltose (135 g/l) produced significantly higher percentage of green plants than the medium containing 90 g/l maltose or sucrose. These results demonstrate culture medium effects on albinism, indicating that the percentage of green plants in wheat anther culture can be increased by optimizing medium osmotic potential.     

Haploid plants regenerated via anther culture in wild barley (Hordeum spontaneum C. Kock)

Summary Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l^–1 maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.Abbreviations IAA indolacetic acid - BAP 6-benzylaminopurine     

Microspore embryogenesis and fertile plantlet regeneration in a salt susceptible × salt tolerant rice hybrid

Shed microspore embryogenesis and fertile plantlet regeneration were observed in a salt susceptible × salt tolerant indica rice F₁ hybrid involving IR 24 and CRM 30. The in vitro culture response and regeneration of green plantlets in the hybrid were superior to those of the parents. Direct embryogenesis and plantlet regeneration with multiple tillers were observed in shed microspore embryos. In intact anther culture, plantlet development from microspore involved a callus phase. The number of multiple tillers developed through secondary embryogenesis was almost equal in both the cases. However, the results indicate that regeneration of green plantlets was higher in case of shed microspore culture in liquid medium containing the synthetic polymer Ficoll 400 than from intact anthers cultured on a semi-solid system. Shed microspore culture produced a number of double haploids, which may result in far reaching consequences in genetic improvement of rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

在甜椒花药培养中马来酰胼对小孢子发育的抑制作用

选用甜椒的小孢子单核期花药,用100、300、500、2000ppm的马来酰肼溶液浸泡处理,并设对照,进行无激素MS固体培养基培养。分别取样用各种组化方法对花药内部多糖、蛋白质、核酸及ATP酶进行组化反应和形态学上的观察。与对照组相比,处理组花药外部形态和内部结构出现许多变异。小孢子内多糖,蛋白质含量减少;绒毡层无明显变化;两组中,核酸的含量均无明显变化;ATP酶的活性低于对照组。可能,马来酰肼对于花药中ATP酶等产生抑制作用,致使花药败育。     

Microspore embryogenesis induced through in vitro anther culture of almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis> Mill.)

Anther culture is one of the most widely used methods to induce gametic embryogenesis. The aim of this investigation was to induce microspore embryogenesis in almond (Prunus dulcis Mill.), through this technique. Anthers were cultured at the vacuolated developmental stage, and seven cultivars, two culture media and two temperature treatments were assessed. Although evidence of the microspore induction was observed in all the genotypes and treatments tested (symmetrical nucleus division and multinucleated structures), calli were produced merely by anthers cultured in the medium P and the regeneration of embryos was detected only in anthers of the cultivars Filippo Ceo, Lauranne and Genco, placed on medium P and subjected to the Control treatment (direct culture at 25?±?1?°C, without the hot thermal shock at 35?±?1?°C for 7 days). Characterization by SSR marker analysis of the embryo genotypes revealed that the regenerants had a single allele for each locus whereas the parent cultivar was heterozygous, indicating their development from haploid microspores. This study reports the evidence of gametic embryogenesis and, particularly, of microspore embryogenesis through in vitro anther culture, in almond, and, for the first time to our knowledge, the production of homozygous embryos.     

Cytological and ultrastructural changes induced in anther and isolated-microspore cultures in barley: Fe deposits in isolated-microspore cultures

To gain further insight into the role played by sporophytic anther tissues in the early stages of the androgenic process, we have compared the cytology and ultrastructure of barley embryogenic pollen grains obtained by anther culture with those obtained by isolated-microspore culture. The microspores behaved similarly in both culture systems but ultrastructural studies detected a significant difference: the presence of electron-dense deposits on the intine of embryogenic pollen grains generated by isolated-microspore culture compared to their absence in grains generated by anther culture. To discover the nature of these deposits, we applied proteinase K and EDTA treatments to ultrathin sections. We also subjected the deposits to X-ray microanalysis and found that they contained iron. Anthers and isolated microspores were cultured in media containing different concentrations of iron so as to evaluate the presence of these deposits on the intine. Deposits were not found in anther cultures at any iron concentration used or in microspore cultures when concentrations were lower than 40 mg/L. The Fe deposits on the intine appear to derive from an excess of Fe in the isolated-microspore culture medium which, if allowed to pass through the cell wall, could well be toxic to the embryogenic development of the microspores.     

The effect of sugars on niger embryogenesis and plant regeneration in anther culture

The influence of different sugars (sucrose, maltose, glucose and fructose, 0.05–0.5 M) on embryogenesis and plant regeneration from cultured anthers of niger [Guizotia abyssinica (L. f.) Cass.] have been studied. Among the different sugars tested, 0.2 M sucrose was the best for embryo induction and plant regeneration. Maximum of 57 embryos per 60 anthers were induced on embryo induction medium [Gamborg’s B5 medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2 μM kinetin (KIN)] containing 0.2 M sucrose. Embryo differentiation was achieved on B5 medium supplemented with 0.5 μM benzyladenine (BA) and 0.09 M sucrose. Embryo maturation was on B5 medium containing 10 μM abscisic acid (ABA) and 0.09 M sucrose. Embryo germination was achieved on B5 medium with 0.09 M sucrose. Embryos that were developed on B5 medium supplemented with 0.2 M sucrose showed highest frequency (68 %) of plant regeneration.     

Comparison of Anther and Microspore Culture in the Embryogenesis and Regeneration of Rye (Secale cereale)

Anther culture in solid and liquid medium and isolated microspore culture were compared in rye genotypes with potential agronomic characteristics. Some important factors influencing androgenic capacity were optimised. Three weeks cold pre-treatment of spikes and two days mannitol pre-treatment of anthers maximized callus and green plant yield in both culture methods. Intensity order of the culture methods in callus and green plant production was: isolated microspore culture, anther culture in liquid medium and anther culture in solid medium. Genotype ability of embryogenesis followed the same pattern in both cultivation methods. Kinetin (BA) with genotype dependent concentrations created the most effective regeneration conditions.     

Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA)

Summary The effect of the auxin phenylacetic acid (PAA) on wheat anther and on barley anther/microspore culture was investigated. With PAA the induction response was not usually significantly different from controls but a significantly higher number of green plants were produced in wheat anther and barley microspore culture. For wheat anther culture 100 mg/L PAA was beneficial. For barley microspore culture the optimum levels were from 1 to 100 mg/L, depending on genotype. In barley anther culture there were no improvements using PAA. In wheat anther culture, 145 green plants/100 anthers were obtained with cultivar VeeryS, while the average response from twelve F₁ hybrids in the breeding program was 332 green plants/100 anthers. At least 1000 green plants were obtained using isolated microspores from 100 anthers in barley cv. Igri. With cv. Bruce, regeneration occurred only when 100 mg/L PAA was used. The influence of PAA appears at the embryogenic phase of the culture system. The possible mechanisms by which PAA may improve regeneration are discussed.     

Effect of genotype,induction medium,carbohydrate source,and polyethylene glycol on embryogenesis in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) anther culture

This study conducted two experiments involving in vitro anther culture of Zea mays L. The first experiment tested 46 maize genotypes, including inbred lines, single and three-way cross hybrids, and line A188 as control, in three different induction basal media (IMSS, N6 and YPm) for their androgenic responses. The results showed that the embryos were established 2–3 weeks after the anthers of the few responsive genotypes were cultured. Most responsive genotypes produced embryos in at least one of the three basal media; therefore, genotype is more important than the type of medium for androgenesis in maize. The mean number of anthers that developed to embryo ranged from 19 embryos per Petri dish in YPm medium for the cross (DH5 × DH7) genotype to 0 for some maize genotypes. In the second experiment, this research reports for the first time the effect of carbohydrates and polyethylene glycol (PEG) as a non-metabolized osmoticum on the embryogenesis anther culture of maize. The genotype DH5 × DH7 was used for this experiment, and the media were varied by altering sucrose, maltose, and PEG concentrations. Results showed that the maximum embryogenesis (32 embryos per Petri dish) was obtained by YPm basal medium supplemented with 60 gl^?1 sucrose + 0.0125 M PEG and 30 gl^?1 sucrose + 30 gl^?1 maltose + 0.0125 M PEG. The lowest rate of embryogenesis was observed in YPm basal medium with 60 gl^?1 maltose and 0.0125 or 0.025 M PEG. Sucrose or a high concentration of maltose was found to be necessary for embryogenesis in anther culture of maize. Therefore, the addition of low levels of PEG and/or different sugars in the experimental design appeared to improve the protocol currently available in the world, especially for anther embryo yield and haploid plant regeneration in maize.     

Comparison of media for their aptitude in wheat anther culture

Different media were evaluated with anthers of five spring wheat (Triticum aestivum L.) genotypes for their ability to produce embryos and green plants in anther culture. Our first experiment showed that the addition of a combination of 19 amino acids significantly increased the number of embryos and green plants obtained. The mean number of green plants per 100 anthers for the two genotypes in this experiment, HY320 and B723, went from 28.2 without amino acids in the medium, to 46.7 with addition of amino acids. Our second experiment with the genotypes HY320, Wim and Laval-19 showed that liquid medium with Ficoll is more efficient for anther culture (9.9 green plants/100 anthers) than solid (0 green plants), gelationous media (2.5 green plants/100 anthers) or liquid medium with Membrane Rafts (0 green plants; Hoechst Celanese Corp.). Our third experiment revealed that the effect of replacement of sucrose by maltose varied with the genotype of the donor plant. Maltose partially inhibited the androgenesis of three responsive genotypes, HY320, Wim and Reliance (40.3 green plants/100 anthers instead of 43.9 with sucrose), while maltose significantly increased the androgenesis of the recalcitrant genotype Laval-19 (10.8 green plants/100 anthers instead of 5.4 with sucrose). An amino acid x maltose interaction was also observed. Amino acids without maltose increased androgenesis, but the addition of maltose to the amino acid-enriched medium eliminated this positive effect of the amino acids.     

Shed Pollen Culture in Hordeum vulgare

Anthers of Hordeum vulgare cv. Sabarlis at the mid-unicellularpollen stage, pretreated in the excised spike for 14 d at 7°C, dehisce within 24 h of being floated on the surfaceof liquid medium. About half the pollen (1500 grains per anther)is liberated into the medium. If the anthers are then removedand the cultures re-incubated, calluses develop from the shedpollen in high yields. At low anther densities, 10p–20(1–3 x 10⁴ grains) per ml, medium preconditioned by anthersand supplemented with m-inositol (1000 mg 1^–1) is required,but at high densities, 120 anthers (2 x 10⁵ grains) per ml,preconditioning is less important, the cultured anthers themselveshaving a sufficient conditioning influence. Large-scale dissectionof anthers can be avoided by use of drops of medium, the volumebeing increased gradually as culture proceeds. Pollen remainingin the anthers after 3 d gives rise to calluses if isolatedmechanically and cultured in the inositol medium. The use ofshed pollen is seen as particularly valuable for culture inspecies whose anthers are small, tedious to dissect out anddifficult to process without severe damage.