Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal

Summary Medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components), combined with preculture exposure to activated charcoal (AC) were evaluated for effects on maize anther culture response. The addition of AC to filter sterilized medium had no effect on the number of embryo-like-structures (ES) produced. For autoclaved medium, pre-culture AC treatment resulted in a 3-fold increase in ES yield over medium lacking AC. When AC was included, autoclaved medium was more productive than filter sterilized medium. Autoclaved media without AC gave lower response than filter sterilized medium. Separate sterilization of sucrose or FeEDTA was beneficial for media autoclaved in the absence of AC. However, when all components were autoclaved together in the presence of AC, there was no advantage to separate sterilization. The maximum ES frequency (224.6 ES/100 anthers) was obtained with the genotype ETH-M 52 cultured in autoclaved medium which had been exposed to AC (5 g/L) for 96 h prior to culture initiation. It is supposed that the higher ES frequencies observed with AC-treated, autoclaved media were due to the availability of glucose and fructose following heat-induced hydrolysis of sucrose and the AC-mediated adsorption of inhibitory compounds produced during autoclaving.     

Effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis

The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving. Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1% activated charcoal, added before autoclaving. In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolyzed.     

Assessment of the changes of 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

The rate of adsorption of 2,4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal (AC) from liquid and semi-solid tissue culture media was determined using 2-[¹⁴C]-2,4-D. In liquid medium 99.5% of the added 2,4-D (10^-4 M) was adsorbed by AC (2.5 gl^-1) within 5 days of preparation of the medium. Higher 2,4-D levels of reduced AC concentrations increased the level of available 2,4-D in the medium and extended the period necessary for the level of 2,4-D in the medium to become stabilized. In semi-solid medium the rate of adsorption of 2,4-D by AC was considerably reduced. A stable ratio of gel/2,4-D:AC/2,4-D was only reached after 10 to 20 days, depending on the 2,4-D concentration used. Low pH levels and maintenance of the medium at higher temperatures (20–30°C) accelerated the adsorption of 2,4-D by AC. In vitro tissue cultures of coconut palm showed marked differences in their growth response according to the age of the medium used and the associated variations in 2,4-D concentrations.Abbreviations AC activated charcoal - BAP 6-benzylamino-purine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Cytological analysis of early microspore divisions and embryo formation in cultured soybean anthers

Young inflorescences of two Brazilian soybean cultivars (IAS 5 and RS 7) were subjected to 4 °C pretreatment for 0, 5, and 10 days. Cytological examinations of the t in vitro anthers were done during the first four weeks of culture. The cold pretreatment had no clear effect on the frequencies of symmetrical-binucleate microspores or multinucleate grains. The multinucleate grains might originate either by symmetrical or assymmetrical division. The best medium for callus and embryo induction was B5 long containing 2.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ benzyladenine. The frequency of anthers/calli with embryos was about 2% in all cultivars. Histological analysis of such embryos showed that they were similar to zygotic embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid     

Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

Histological study of embryogenesis and organogenesis from anthers ofVitis rupestris du Lot cultured in vitro

Summary Anthers ofVitis rupestris du Lot were cultured in vitro at the uninucleate stage of the microspore, in order to investigate the histology of embryogenic and organogenic processes in this genotype. Microspores divided in the anther loculi resulting in the formation of globular structures with a ruptured exine. Somatic embryogenesis and, occasionally, caulogenesis and rhizogenesis occurred in calli produced from all anther tissues except the endothecium. The initial cell of the embryoid was surrounded by a jacket layer when situated deep within the callus. When the embryoid's initial cell was situated in the peripheral callus, a cutinized wall was present and the three-celled proembryoid was almost always segmented, showing the same embryonal type as the zygotic proembryo. Root differentiation and elongation and cap differentiation occurred during the growth phase in liquid medium. The mature root was diarch and contained cells with calcium-oxalate raphides, as seen in vivo. No starch or tannin deposition was ever observed in the mature embryoids.Abbreviations 6-BAP 6-benzylaminopurine - CC3 Nitsch and Nitsch (1969) basal medium - CS cross section - 2,4-D 2,4-dichlorophenoxy-acetic acid - LS longitudinal section     

The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 M -naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 M 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 M 2,4-D and acclimated to greenhouse conditions.     

Regeneration of Oxalis triangularis ssp. triangularis from suspension cells cultured in three different systems (solid, liquid-flask and bioreactor cultures)

In attemps to establish in vitro cultures of Oxalis triangularis ssp. Triangularis, the explants of leaves, petioles, bulb scales and suspension cells derived from regenerated bulbs were examined using solid (petri dish), liquid-flask and bioreactor cultures. Only bulb-derived suspension cells were able to regenerate in all culture systems. The liquid-flask and bioreactor cultures supported organogenesis and yielded larger amount of buds than solid culture. The inclusion of AC in the culture medium delayed bud initiation but promoted plantlet development by minimising callusing of the buds. Morphological differences in regenerated plantlets affected by AC, such as the length and diameter of the petiole, leaf unfolding and the development of a red colour development on leaves and petioles, varied with the culture systems. Upon transfer to pots normal plants were recovered from buds regenerated in various culture systems. Received: 18 August 1998 / Revision received: 27 October 1998 / Accepted: 20 November 1998     

Differentiation in Lilium bulbscales grown in vitro. Effects of activated charcoal, physiological age of bulbs and sucrose concentration on differentiation and scale leaf formation in vitro

Organ differentiation and growth of tissue cultured bulbscales of Lilium auratum Lindl. was investigated. Benzyladenine stimulated bulb and bulbscale differentiation but inhibited root formation. Addition of activated charcoal to the medium negated the effect of BA on differentiation, while the growth of bulbs was markedly stimulated. Organ formation was also influenced by the physiological age of bulbs. From bulbscales of a 3-week-old bulb, a large number of bulbs and roots was formed but callus was not produced. A high sucrose concentration (90 g/1) reduced the subsequent sprouting after planting in soil. Scale leaf formation was also regulated. Sucrose (30 g/1) stimulated scale leaf formation but 90 g/1 were inhibitory.