Anther culture of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis>: callus formation and the development of multicellular and embryo-like structures

Androgenesis is an important technique to generate double haploid plants. Anther and microspore cultures are the methods to induce haploid embryogenesis. For culture initiation, it is necessary to select anthers with the appropriate developmental stage of microspores. For lupins, limited reports about the establishment of initial cultures for androgenesis are available. In this study, different parameters of anther culture of three genotypes of Lupinus angustifolius were investigated. For all genotypes, a considerable correlation was observed between the buds and the anthers, depending on their location in the inflorescences. Buds from the central segment of inflorescences had yellowish green anthers that contained the maximum number of microspores at uninucleate stage. Cytological investigation shows that the anthers containing these microspores were the most responsive to induction. Two types of developmental pathways were observed for microspores. In case of cold pre-treated and untreated inflorescences, microspores developed into multicellular and embryo-like structures, respectively. Effects of different factors showed significant differences among: genotypes, pre-treatment, growth regulators (GRs) and genotypes × GRs interaction. Among three genotypes, Emir showed the highest number of multicellular and embryo-like structures on MS medium + 2.0 mg/l 2,4 D + 0.5 mg/l Kinetin (Kin). For all genotypes, anthers produced calli on MS medium containing 2.0 mg/l 2,4 D + 0.5 mg/l Kin. These calli continued their growth on regeneration medium (MS + 2.0 mg/l BA + 0.5 mg/l NAA) and produced roots. Taken together, these results provide a good basis for further research towards the development of haploid plants for L. angustifolius.     

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     

High frequency androgenesis from isolated microspores of maize

Anthers from a highly androgenic genotype of maize (139/39-02), when cultured in a modified, liquid YP medium, dehisced within 2–7 days resulting in a stationary suspension of microspores. After 12–15 days, the microspore suspension was found to contain multicellular masses which went on to produce macroscopic embryo-like structures within 20–25 days of culture initiation. Embryogenic callus could be obtained by transferring microspore-derived embryos onto a modified N6 medium supplemented with 2.5 mg/l dicamba and 0.1 mg/l 2,4-D. Subculture onto hormone-free medium resulted in plant regeneration. Over 400 embryo-like structures per 100 anthers cultured have been obtained from liquid induction medium as compared to 55 embryos per 100 anthers cultured on an agar-solidified medium. Approximately 5–25% of these embryo-like structures went on to produce callus from which plants could be recovered. Mechanical isolation of microspores from anthers precultured for 0, 3, and 7 days also resulted in embryo production and plant regeneration. This represents the first report of plant recovery from isolated maize microspores. The use of a liquid induction medium applied to a highly androgenic genotype allows for the production of large numbers of microspore-derived plants and provides a single, haploid cell regeneration system for maize.     

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     

Improved isolated microspore culture efficiency in medium with maltose and optimized growth regulator combination in japonica rice (Oryza sativa)

The influence of maltose and growth regulators on microspore culture response was investigated in japonica rice. High frequency of callus induction of isolated microspores was obtained with liquid medium containing MS salts, 100 mg l^–1 myo-inositol, 1 mg l^–1 thiamine-HCl, 500 mg l^–1 glutamine, 60 g l^–1 maltose, and several growth regulators. The effect of maltose on promoting callus formation was associated with keeping a high proportion of swollen microspores after 5 day preculture and increasing the microspore division rate on the 3rd day after culture initiation. No significant effect of maltose in place of sucrose on plantlet regeneration was seen in regeneration medium. Among the growth regulators tested, the combination of auxin 2,4-dichlorophenoxyacetic acid (1 mg l^–1), naphthaleneacetic acid (1 mg l^–1), and cytokinin (6-benzyl-aminopurine 1 mg l^–1) in the medium proved to be much better for callus formation than in the other media, and the percentage of callusing microspores of that medium reached 0.86%. Indole-3-acetic acid (0.5 mg l^–1) and kinetin (2 mg l^–1) in regeneration medium were beneficial for green plantlet differentiation. The results also showed that the frequencies of microspores initial division, callus formation and green plant regeneration varied among genotypes no matter what kind of growth regulator and sugar were used. Xiushui 117 was the best variety for callusing followed by 02428 & Taipei 309. Taipei 309 showed a good ability for green plantlet regeneration.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 6-BA 6-benzylaminopurine - KT kinetin - IAA indole-3 acetic acid     

Viability,cell division and microcallus formation of barley microspores in culture

Barley microspores were viable when cultured in a sugarless medium. Adding 2g of glucose to 1l of this medium resulted in a significant reduction in the frequency of viable microspores. The frequency of viable microspores was further reduced when 50g of cellobiose, glucose, maltose, melezitose, raffinose or sucrose were added to 1l of the culture medium containing 2g/l glucose. Adding 50g of melibiose, Ficoll, polyethylene glycol (PEG) or a combination 50g each of Ficoll and PEG to 1I of the medium containing 2g/l glucose had very little effect on the viability of the microspores.Up to 66% of the viable microspores were able to divide and many of these developed into microcalli in the basal medium complemented with melibiose, maltose, melezitose, raffinose, Ficoll, PEG or a combination of Ficoll with PEG. Sucrose, cellobiose and glucose added in large quantities inhibited cell division in microspores or destabilized the microspores and only very few of them developed into microcalli.The microcalli in the PEG, Ficoll, Ficoll-PEG and melibiose media were smaller in size than those grown in the melezitose, maltose and raffinose media. Sustained cell division and microcallus formation were observed in a medium with melibiose or maltose as sole source of sugars.Abbreviations 2.4-D 2,4-dichlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - PEG Polyethylene glycol     

High frequency embryogenesis in immature zygotic embryos of sunflower

In the present investigation, nutritional requirements for induction of a high frequency of well formed somatic embryos (SEs) from zygotic embryos (ZEs) of sunflower were assessed. Variables like genotype, embryo size (0.5–10 mm), sucrose concentration (30–240 g l⁻¹), carbohydrate source (sucrose, glucose, maltose), agar strength (0.2–1.0%), basal media (MS, Gamborg, Nitsch, White), photoperiod (light/dark) and temperature (20–36°C) were tested. All these variables except photoperiod had significant effect on the frequency of embryogenesis. Highest frequency of embryogenesis was facilitated by Gamborg basal salt media, 120–210 g l⁻¹ sucrose, 0.8–1.0% agar, smaller sized embryos (0.5–2 mm) and incubation temperature of 28–32°C. In addition to these, growth regulator combinations (2,4-D, 2,4-D+kinetin, BA+NAA) in varying concentrations were tried. Media supplemented with 2,4-D promoted direct embryogenesis, BA+NAA facilitated formation of single/multiple shoots while there was no response on 2,4-D+kinetin supplemented media. Zygotic embryos with well differentiated embryos were transferred to growth regulator free half strength MS medium for whole plantlet development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Embryogenesis and plant regeneration of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) through isolated microspore culture

We report high frequencies of embryo production and plant regeneration through isolated microspore culture of hot pepper (Capsicum annuum L.). Microspores cultured in modified NLN medium (NLNS) divided and developed to embryos. Globular and heart-shaped embryos were observed from 3 weeks after the beginning of culture, and many embryos reached the cotyledonary stage after 4 weeks of culture. These cotyledonary embryos developed to plantlets after transfer to solid B5 basal medium. We also optimized conditions for embryo production by varying the pretreatment media, the carbon sources, and culture densities. Heat shock treatment in sucrose-starvation medium was more effective than in B5 medium. Direct comparisons of sucrose and maltose as carbon sources clearly demonstrated the superiority of sucrose compared to maltose, with the highest frequency of embryo production being obtained in 9% (w/v) sucrose. Microspore plating density was critical for efficient embryonic induction and development, with an optimal plating density of 8 × 10⁴–10 × 10⁴/ml. Under our optimized culture conditions, we obtained over 54 embryos, and an average of 5.5 cotyledonary embryos when 10 × 10⁴ microspores were grown on an individual plate.     

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

 To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l^–1α-naphthaleneacetic acid and 1 mg l^–1 N⁶-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid. Received: 3 September 1998 / Revision received: 25 November 1998 / Accepted: 5 December 1998     

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.     

Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus

Various components of culture media were tested to characterize factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. It was found that plantlet regeneration from rice callus was affected by concentrations of gelling agents, osmoticum, and the combination of hormones in the regeneration medium. High concentrations (4–6 g/l gellan gum, 10–16 g/l agar) of gelling agents promoted regeneration frequency. However, the total number of plantlets decreased with gellan gum concentrations above 4 g/l. Addition of sorbitol (15–75 g/l) promoted plantlet regeneration. However, the addition of mannitol was inhibitory and no regeneration was observed at concentrations above 30 g/l. This difference in the effects on regeneration suggests that sorbitol had another function besides as a osmoticum. High regeneration frequency was obtained with combinations of NAA (0.05–0.5 g/l) and kinetin (0.5–2 mg/l). However, higher concentrations (2 mg/l) of NAA are preferred to increase the total number of regenerated plantlets.     

The effect of cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis

The effect of stress pretreatments on embryo induction in anther cultures of selected genotypes of Avena sativa and A. sterilis was tested. A heat pretreatment of isolated anthers at +32°C for 5 days was best for the A. sativa line WW 18019 and for A. sterilis line CAV 2648. Genotype dependency may exist since in ‘Stout’ heat pretreatment did not increase embryo production. For A. sterilis 13 green and three albino regenerants were produced, of which five plants (haploids) survived transfer to the greenhouse. For A. sativa, 30 various differentiation media/treatment combinations were used in an attempt to regenerate plants from embryos, with no success. Seven day cold treatment of cut tillers increased slightly the response level in ‘Stout’ and was routinely used in subsequent experiments. Maltose proved to be better then sucrose as a carbon source for the genotypes tested. Fourteen percent maltose promoted the highest induction in A. sterilis, but the quality of embryos was improved in the presence of 10% maltose for both species. Sub-optimal carbohydrate levels did not enhance embryo induction in oats. This revised version was published online in June 2006 with corrections to the Cover Date.     

An efficient androgenic embryogenesis and plant regeneration method through isolated microspore culture in timothy (Phleum pratense L.)

A method employing isolated microspore culture was established for the androgenic embryogenesis of timothy (Phleum pratense L). Embryos/calli were obtained and green plants regenerated. The induction medium was PG-96 (1.0 mg l⁻¹ 2,4-D, 0.1 mg l⁻¹ Kinetin) supplemented with 6% maltose monohydrate. Timothy microspore culture was genotype-dependent, among 12 genotypes, 6 produced embryos/calli and 4 produced green plants. Macerating the spikes with a blender and purifying the microspores at a mannitol/maltose monohydrate interface gave a relatively high percentage of cell vitality. The optimum microspore developmental stage was from the very late uninucleate stage to the binucleate stage. Heat shock promoted the initiation of microspore culture. Over 150 regenerated green plants were obtained; in a random sample of 32 of these 65.6% were doubled haploids (6n=42). Albinism was a problem in plant regeneration (9.3–22%). This paper is the first to describe timothy androgenic embryogenesis by isolated microspore culture. Received: 9 September 1999 / Revision received: 6 December 1999 / Accepted: 13 December 1999     

Improvement of maize (Zea mays L.) anther culture responses by algae-derived natural substances

This is the first report on the beneficial effect of microalgal and cyanobacterial biomass on anther cultures of maize (Zea mays L.). Investigations were made on the cytokinin- and auxin-like activity and content of terrestrial and fresh-water living microalgal and cyanobacterial strains. The influence of media supplemented with biomass from four selected strains on the anther induction, the frequency of microspore-derived embryo-like structures, and regeneration capacity in anther cultures of maize was also studied. The addition of cyanobacterial and microalgal biomass to the induction and regeneration media in concentrations of 1 or 2 g/L improved the androgenic response, and was able to reduce the quantity of the synthetic auxin 2,4-dichlorophenoxy-acetic acid (2,4-D) required, or replace it completely.     

Effect of genotype,growth regulators,carbon source,and pH on shoot induction and plant regeneration in tomoto

Summary Ten cultivars of tomato were screened for their ability to produce shoots and shoot primordia on media containing a range of 6-benzyladenine (BA) concentrations (0.0–4.0 mgl⁻¹; 0.0–17.7 μM) in combination with indole-3-acetic acid (IAA; 0.0, 0.1, 0.2 mgl⁻¹; 0.0, 0.57, 1.14 μM, respectively). Both genotypes and growth regulator levels showed significant differences at α=0.05. However, their interaction was not significantly different. A comparison of the mean number of shoots produced six cultivar classes, with UC82, UC97-3, Castlerock, Red Rock, and Peto86 producing the highest means. There were four cultiver frequency classes, the highest including cultivars Peto Rock (93.7%), Peto86 (92.3%), and Strain B (90.7%). Growth regulator mean comparisons produced eight classes, the highest included three different BA/IAA combinations, with media containing 2.0 mgl⁻¹ (8.87 μM) BA and 0.2 mgl⁻¹ (1.14. μM) IAA giving the highest mean and frequency (94.1%). The effect of carbon source was studied using glucose, fructose, maltose, and sucrose at 3.0% concentration. Three high regenerating genotypes and four media were used in combination with each sugar. Sugars were significantly different with maltose giving the highest number of shoots (31.6) followed by glucose (20.9). Growth regulator means, sugar-growth regulator interaction, and cultivar-sugar interaction were significant. A three-way interaction was nonsignificant. The effect of maltose and sucrose concentration (1.5–9.0%) showed the positive effect of maltose over sucrose in inducing shoots and in reducing loss rates of shoots on the regeneration media. Maltose at 1.5, 3.0, and 4.5% was the most effective. Six pH values (4.0–6.5) were used to test their effect on shoot induction in three cultivars (Peto86, UC97-3 and Castlerock). pH effects and cultivar-pH interaction were not significant.     

Anther culture in Helianthus annuus L., influence of genotype and culture conditions on embryo induction and plant regeneration

Summary Production of microspore-derived embryos from cultured anthers is now a well established technique for the isolation of homozygous lines in many crop plants. We describe here a culture method for embryo induction and plant regeneration from anthers of four sunflower genotypes. For preliminary experiments, anthers of uninucleate microspores were cultured on four types of basal media viz., Murashige and Skoog's MS, Gamborg's B5, Nitsch and Nitsch, and White's W, supplemented with 1.0 mg/l 2,4 dichlorophenoxy acetic acid and 0.5 mg/l 6-benzylaminopurine and 40 g/l sucrose. MS basal medium, being more responsive for embryo induction, was used for further experimentation. To optimise the culture requirement MS basal medium was supplemented with 0.2–2.0 mg/l 2,4 dichlorophenoxy acetic acid and 0.5 and 1.0 mg/l 6-benzylaminopurine. The effect of cold pretreatment, hormone regime and sucrose concentration were tested for embryogenic efficiency. Genotype had a significant effect on the capacity of embryo induction. Addition of silver nitrate (2.5 mg/l), an ethylene inhibitor, stimulated embryo germination. Plantlets were obtained (10–15%) from embryos of only one genotype.Abbreviations 2,4-D 2,4 dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - IAA indole-3-aceticacid - BAP 6-benzylaminopurine - KN Kinetin - ABA abscisic acid - GA₃ gibberellic acid     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora

The response of five Coffea canephora Pierre genotypes with regard to somatic embryogenesis was tested on media containing silver nitrate (AgNO₃) and different carbohydrates (sucrose, fructose, maltose and glucose). The presence of AgNO₃ caused only small modifications to the ionic equilibrium of the media. At concentrations between 30–60 M, AgNO₃ improved embryo yield for the genotypes evaluated, while higher doses negatively affected the regenerative capacity. The substitution of maltose, glucose or fructose for sucrose produced different responses depending on the genotype. Fructose significantly increased somatic embryo production in genotypes N91 and N128, while maltose was highly effective for N75. In addition, more synchronous embryo development was observed in genotype N91 when glucose was used instead of sucrose.     

Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized first, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose. Received: 5 May 1998 / Received revision: 3 August 1998 / Accepted: 8 August 1998     

Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.)

 Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2–4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium. Received: 1 December 1997 / Revision received: 15 February 1999 / Accepted: 26 February 1999