Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Selection of stable mutants from cultured rice anthers treated with ethyl methane sulfonic acid

To increase the frequency of stable mutants from cultured anthers of rice, the effects of EMS treatment on callus induction, plant regeneration and mutant induction were investigated according to the timing of treatment after anther inoculation on the medium. The frequency of callus induction was highest in anthers treated with 0.5% EMS 10 days after culture. Anthers treated directly at the initiation of culture exhibited a very low callus induction level, and the such calluses exhibited a poor plant regeneration capacity. The frequency of regeneration of green plants was significantly decreased by EMS treatments immediately after anther inoculation as compared with control. The frequencies of stable mutants were 20.7% and 12.0% in EMS treatments at 10 and 20 days, but unstable mutants were 43.1% and 52.6%, respectively. A total of 14 stable mutants, semidwarf mutants (4 lines), grain-shape mutants (2 lines) and glabrous mutants (8 lines) were selected from doubled haploid lines of the A₂ generation. The frequencies of callus induction, green plant regeneration and stable mutants were maximal in anthers treated with 0.5% EMS 10 days after culture.     

Inheritance of anther culture derived green plantlet regeneration in wheat (Triticum aestivum L.)

A study was set up to determine the inheritance and combining ability of the factors anther culture response and green plant regeneration. Reciprocal crosses were made between cultivar Ringo Sztar, showing high anther culture response and the cultivars Ciano 067 and Benoist H77022, showing a high level of green plant regeneration. Averaged over all genotypes, 23.0% of the anthers responded and a callus induction frequency of 77.8% was observed. Of all the embryos, 43.0% developed into plantlets, 25.6% of the regenerants being green, the result being that 3.3 green plants per 100 anthers were formed. Genotypic effects accounted for 57.7%, 86.3% and 77.5% of the total variance of anther culture response, callus induction frequency and embryo induction frequency, respectively. Additive and dominant gene action was detected for all characteristics, including green plant regeneration. No reciprocal differences were found for anther culture response, embryo induction frequency and green plant regeneration, indicating no cytoplasmic effects. A small but significant reciprocal difference was found for callus induction frequency. Embryo production was primarily correlated with anther culture response and not with the number of embryos produced per plated anther or per responding anther. Possible mechanisms for the inheritance of green plant regeneration are discussed.Abbreviations CIRA callus induction frequency per responding anther - ERA embryo induction frequency per responding anther - FHB fusarium head blight - MS-medium Murashige & Skoog (1962) medium - REML residual maximum likelihood     

Genetic studies of anther culture ability in rice (Oryza sativa)

Inheritance of three anther and culture characters, callus induction, green plant regeneration and culture efficiency was studied using incomplete diallel crosses with a gamete model. It was suggested that callus induction was mainly controlled by gametic additive effects and with less effect of the maternal effects. Green plant regeneration was mainly determined by maternal effects with less influence of gametic additive effects. Culture efficiency was controlled by gametic additive, maternal and cytoplasmic effects. Cultivar Lunhui 422 showed positive genetic effects for all three traits and was a very good parent for rice anther culture breeding. Significant positive heterosis was observed for callus induction. Both gametic additive and maternal correlations contributed to the significant genotypic and phenotypic correlations between callus induction and green plant regeneration suggesting these two traits to be linked.Abbreviations 2,4-d 2,4-dichlorphenoxyacetic acid - NAA -napthaleneacetic acid     

Application of activated charcoal and nanocarbon to callus induction and plant regeneration in aromatic rice (Oryza sativa L.)

The investigations of nanotechnology with the application on agricultural products also have been few reported, especially the plant regeneration. The effects of activated charcoal and nanocarbon on the callus induction and plant regeneration of aromatic rice were studied. Activated charcoal was added into the callus induction and regeneration medium. The presence of activated charcoal in the callus induction medium (100–500 mg L^?1), activated charcoal significantly reduced the percentage of the callus induction and biomass accumulation (fresh weight, dry weight and size). Whereas, the regeneration medium supplemented with 100 mg L^?1 of activated charcoal showed the highest percentage of plant regeneration (61.90%) and the ratio of the number of seedlings to the number of regenerated calli (RSR; 3.06) that derived from the callus induction medium (without activated charcoal). Moreover, the induced calli derived from the callus induction medium supplemented with nanocarbon at 5 mg L^?1 showed the highest percentage of callus induction (94.70%), the percentage of green spots (95.83%), the percentage of plant regeneration (60.42%) and the RSR (3.12) when transferred the calli into the regeneration medium (without nanocarbon). After that, nanocarbon was also added into the regeneration medium. The percentage of green spots (96.08%), the percentage of plant regeneration (62.75%) and the RSR (3.16) obtained from the regeneration medium supplemented with 20 mg L^?1 of nanocarbon showed the highest values. This experiment showed that the optimum concentration of activated charcoal and nanocarbon had potential to enhance the callus induction and plant regeneration frequencies in tissue culture medium of aromatic rice.     

Induced androgenesis in tomato (Lycopersicon esculentum Mill). II. Factors affecting induction of androgenesis

The influence on androgenesis of donor plant growth conditions, anther size and developmental stage of the microspore, medium composition and different anther treatments prior to culture was investigated in L. esculentum Mill. cv Roma and its hybrids. Growth conditions of donor plants affected the induction of tomato androgenesis. Anthers isolated from plants grown in the greenhouse during winter at high humidity and in short days possessed higher androgenetic ability than those grown in the field. The physiological state and age of the donor plants also influenced the processes investigated. Regarding the developmental stage of microspores, the period from prophase to telophase II is optimal for tomato anther implantation. More then 20 culture media were tested. Two, based on Murashige and Skoog medium were selected as most favourable for callus induction, organogenesis and regeneration. The effect on callus induction of 2ip in combination with indole-3-acetic acid (IAA) was greater than that of zeatin and IAA. Zeatin promoted entire plant regeneration. A highly significant interaction between genotype and medium was observed. Temperature and gamma ray treatments of anthers enhanced callus production, shoot formation and plant regeneration. Treatments at 4 °C (48 h) and 10 °C (9 days) stimulated these processes. Combined treatment of anthers with 4 Gy and 10 °C for 9 days was the most efficient. Received: 5 September 1997 / Revision recieved: 5 June 1998 / Accepted: 15 June 1998     

Influence of 2,4-D, IAA, and duration of callus induction in anther cultures of spring wheat

Culture media and environmental factors may significantly influence the yield of haploid plants from anther cultures. Our objectives were to identify a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) concentrations which produce the maximum number of haploid plants, and to evaluate the effects of duration in induction medium on calli induction, plant regeneration, and green plant production from anther cultures in spring wheat. Significant (P ≤ 0.01) plant growth regulator concentration effects (2,4-D and IAA) were observed on the number of calli, green plants and albino plants produced, and on direct plant regeneration. Addition of 2,4-D to the induction medium resulted in significantly (P ≤ 0.01) higher means for all anther culture components compared to IAA> While addition of 2,4-D significantly (P ≤ 0.01) reduced plant regeneration, it substantially increased green plant percentage at a 0.3-mg l⁻¹ concentration of IAA. Use of response functions to estimate the maximum effective 2,4-D × IAA combination implied that higher 2,4-D levels in the induction medium should be investigated, and that the optimum hormone combination differs for plant regeneration and green plant percentage. Significant (P ≤ 0.01) effects of duration on callus induction medium were observed for plant regeneration and green plant percentage.     

Micrografting and <Emphasis Type="Italic">ex vitro</Emphasis> grafting for somatic embryo rescue and plant recovery in avocado (<Emphasis Type="Italic">Persea americana</Emphasis>)

To improve plant regeneration from oat (Avena sativa L.) anther culture, the effects of induction medium supplements and culture conditions were studied. Significantly better plant regeneration rates were obtained with cultivars Lisbeth (naked type) and Aslak when a medium containing W₁₄ salts and vitamins, supplemented with 2,4-d, BAP, Ethephon, l-cysteine and myo-inositol, was used for induction in the dark compared with a medium containing only 2,4-d and kinetin. Genotypes reacted differently on the light during the induction phase. Use of dim light significantly decreased the green plant regeneration rates in cv. Lisbeth, while in cv. Aslak the difference was not so clear. Up to 30 green plants per 100 anthers were recovered from Aslak × Lisbeth progeny and in total, over 500 oat regenerants were produced. With these numbers, acceptable rates of DH-production for cultivar breeding and genetic study purposes are approached. The agronomic performance of some DH lines was compared with that of the plants derived from commercial seeds of the same cultivars in the field experiment. A few differences were found, but generally DH lines yielded the same or more as the commercial cultivars.     

Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem)

Callus culture has, to date, been reported only in a few species of Narcissus. We used anthers of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem) as explants for callus induction and plant regeneration. A high percentage of anthers at the early- to mid-uninucleate microspore stage were responsive on the basal MS medium supplemented with 0.5–1 mg l^–1 2,4-dichlorophenoxyacetic acid and 0.5–2 mg l^–1 6-benzyladenine under dark conditions. Calli were initiated from anther connective tissue or anther wall tissue, and no division of microspores occurred during callus formation, as determined by histological observation. Using 20 random amplified polymorphic DNA primers, we verified the genetic integrity of the anther-derived plants of Chinese narcissus with respect to the donor plants. These results suggest that anther culture in vitro can provide an efficient new micropropagation technique for Chinese narcissus as well as a new strategy for in vitro mass propagation of other daffodils.     

Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light

Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing‐induced flashing light regimes. In this study, photosynthetic efficiency and growth of the green microalga Chlamydomonas reinhardtii were measured using LED light to simulate light/dark cycles ranging from 5 to 100 Hz at a light‐dark ratio of 0.1 and a flash intensity of 1000 µmol m⁻² s⁻¹. Light flashing at 100 Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time‐averaged light intensity (i.e., 100 µmol m⁻² s⁻¹). The efficiency and growth rate decreased with decreasing flash frequency. Even at 5 Hz flashing, the rate of linear electron transport during the flash was still 2.5 times higher than during maximal growth under continuous light, suggesting storage of reducing equivalents during the flash which are available during the dark period. In this way the dark reaction of photosynthesis can continue during the dark time of a light/dark cycle. Understanding photosynthetic growth in dynamic light regimes is crucial for model development to predict microalgal photobioreactor productivities. Biotechnol. Bioeng. 2011;108: 2905–2913. © 2011 Wiley Periodicals, Inc.     

Reciprocal effects in anther cultures of wheat hybrids

This study was conducted to determine the reciprocal effects for anther culture response in wheat (Triticum aestivum L.) using a set of 4 × 4 full diallel crosses. Both reciprocal and nuclear genetic effects were highly significant for anther culture response and useful for selection and breeding purposes. General combining ability (GCA) effects were predominant for all investigated anther culture traits. Also, significant differences for specific combining ability (SCA) effects were detected between reciprocal crosses. Although significant reciprocal differences for responding anther, callus number and green plant regeneration were recorded in some reciprocal crosses, there were no significant reciprocal differences for albino plant regeneration. The use of one parent as male or female could lead to change at the production of green plants from the F₁ hybrids and screening of inbred lines for response to anther culture, without reciprocal effects, could decrease the utilization of breeding material.     

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.     

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.     

Cold pretreatment to enhance green plant regeneration from rye anther culture

Cold pretreatment of detached tillers of rye, Secale cereale, was tested under two light regimes. The ryes included two spring and two winter cultivars. Significant increases in green plant regeneration were recorded in each experiment when cold pretreatments of two to four weeks were applied. Dim light during the stress period improved green plant regeneration for two of the four cultivars tested. The highest regeneration rate, 30.6 green plants per 100 plated anthers, was reached following three weeks at +4 °C under dim light, for spring rye Jo02. Starvation stress applied to plated anthers in mannitol medium suppressed anther response. This revised version was published online in June 2006 with corrections to the Cover Date.     

甲基磺酸乙酯对水稻花药离体培养效应的研究

本文以粳稻“105”、籼稻“华03”和“鄂早6号”为实验材料,研究了不同浓度化学诱变剂EMS处理对离体培养水稻花药中花粉细胞脱分化与再分化和培养初期花药呼吸作用的影响。结果表明,低浓度EMS能显著提高花药愈伤组织诱导率,高浓度EMS则有明显的抑制作用。EMS对培养初期花药呼吸作用的影响与花药愈伤组织诱导率之间存在明显的平行关系。     

Obtention par gynogenèse in vitro de plantes haploïdes chlorophyliennes chez le blé dur

Morphogenic expression leading to green plant formation in unfertilized ovary culture (gynogenesis) was studied in durum wheat. After 4–5 weeks of culture in the optimal conditions tested, a creamy white callus grew out of the base of the ovary from which it was detached. After sub-cultures on a pre-regeneration then a regeneration medium, green zones appeared followed by shoot formation. A genotypic effect has been demonstrated. A study of various components of the induction media based on that of Murashige and Skoog showed that 2,4-dichlorophenoxyacetic acid was necessary for callus formation and green plant regeneration. Other factors, such as a thermal pretreatment (5–15 d at 5 °C) and an initial period of darkness (2–5 weeks) followed by light (16 h daily) were also favorable for gynogenesis. All plants formed were green and chromosome counts in root tips of regenerated plants showed the haploid number n = 14.     

Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of KNO3 concentration in callus induction medium for wheat anther culture

KNO₃ concentration was found to significantly affect the anther culture of wheat (Triticum aestivum L.). When KNO₃ was increased from 0 to 15 mM (in cultivar Jinghua 1) or from 10 to 15 mM (in cultivars 2531-10, Xiaoyan 759 and Norin 10), the callus induction frequency increased significantly. When KNO₃ was increased further above 20 mM, the callus induction frequency decreased significantly in all the tested cultivars. The subsequent frequency of green plantlet regeneration increased significantly, and the ratio of green to albino regenerants increased sharply when KNO₃ concentration increased. Further experiments found that the decrease of callus induction frequency in the medium with too much KNO₃ might be caused by NO₃ ^- ion alone, while the effect of KNO₃ on green plantlet regeneration might be caused by both K⁺ and NO₃ ^- ions, and that the effects of NO₃ ^- concentration were independent of NH₄ ⁺ concentration in the medium.     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.