Anther Medium Research Articles

Improved Anther Culture Media for Enhanced Callus Formation and Plant Regeneration in Rice (Oryza sativa L.).

Anther culture technique is the most viable and efficient method of producing homozygous doubled haploid plants within a short period. However, the practical application of this technology in rice improvement is still limited by various factors that influence culture efficiency. The present study was conducted to determine the effects of two improved anther culture media, Ali-1 (A1) and Ali-2 (A2), a modified N6 medium, to enhance the callus formation and plant regeneration of japonica, indica, and hybrids of indica and japonica cross. The current study demonstrated that genotype and media had a significant impact (p < 0.001) on both callus induction frequency and green plantlet regeneration efficiency. The use of the A1 and A2 medium significantly enhanced callus induction frequency of japonica rice type, Nipponbare, and the hybrids of indica × japonica cross (CXY6, CXY24, and Y2) but not the indica rice type, NSIC Rc480. However, the A1 medium is found superior to the N6 medium as it significantly improved the green plantlet regeneration efficiency of CXY6, CXY24, and Y2 by almost 36%, 118%, and 277%, respectively. Furthermore, it substantially reduced the albino plantlet regeneration of the induced callus in two hybrids (CXY6 and Y2). Therefore, the improved anther culture medium A1 can produce doubled haploid rice plants for indica × japonica, which can be useful in different breeding programs that will enable the speedy development of rice varieties for resource-poor farmers.

Assessment of different anther culture approaches to produce doubled haploids in cucumber (Cucumis sativus L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.

Production and conversion of haploid embryos in chickpea (Cicer arietinum L.) anther cultures using high 2,4-D and silver nitrate containing media

Due to recalcitrant nature of chickpea (Cicer arietinum L.) to androgenesis, the production of double haploid plants has been only reported by Grewal et al. (Plant Cell Rep 28:1289–1299, 2009) using some physical stresses such as anther centrifugation and electrical shock. In the present study, we successfully obtained haploid plants from cultured anthers of two chickpea cultivars, Bivanij and Arman, using high 2,4-D and silver nitrate containing media without applying of these time and labor consuming stresses. For induction of androgenesis, different concentrations of 2, 4-D (0, 2, 5 and 10 mg/l) and silver nitrate (0, 5, 10, 15, 25 and 50 mg/l) were used in embryo development medium. In Bivanij cultivar, anther induction medium containing 10 mg/l 2,4-D and 15 mg/l silver nitrate produced the highest number of embryos (0.188) and regenerated plants (0.1) per each cultured anther, while the highest frequencies of embryos (0.1) and regenerated plants (0.075 and 0.063) were obtained from Arman cultivar when 10 mg/l 2,4-D was combined with 15 and 50 mg/l silver nitrate in anther culture medium, respectively. In second part of this study, different cold (4 °C for 4 and 7 days) and heat (30 °C for 10 days, 32 °C for 2 days and 35 °C for 8 h) pretreatments were applied on cultured anthers of Bivanij cultivar. Incubation of cultured anthers at 32 °C for 2 days significantly enhanced the rate of embryo formation up to 0.222 embryos per each anther, while the highest number of regenerated plants/anther (0.0332) was obtained when cold treated anthers at 4 °C for 7 days incubated at 30 °C for 10 days. Taken together, these results provide a good basis for large-scale generation of DH plants in this important legume species.

Induction of microspore-derived embryos by anther culture in selected pepper genotypes

Five pepper genotypes (A71, A269, A313, A109 and A74) and four different culture media were tested in this study carried out at the University of Cukurova, Turkey. The anthers were cultured at different periods in order to optimize the frequency of embryo production. Moreover, the embryos that were unable to complete their growth in the culture medium studied were placed in a medium containing 0.5 mgL-1abscisic acid for 10 days. At the end of the study, it was determined that embryo development varied with genotype, anther cultivation period and culture medium. The highest yield of embryos was obtained from A269, one of the genotypes tolerant to low temperature. The anthers cultured from April to May, gave the highest yields of embryos compared to anthers from the other periods. Most of the embryos were obtained from Medium III (MS medium containing 4 mg/L NAA, 1 mg/L BAP, 0.25% activated charcoal, 15 mg/L AgNO3, 30 g/L sucrose) and the Medium IV (modified MS medium containing 0.25% activated charcoal, 15 mg/L AgNO3, 4 mg/L NAA, 0.1mg/L BAP and with 0.5 mg/L ABA). There was no positive effect of abscisic acid on the mature embryos. Key words: Pepper (Capsicum annuum), genotype, anther culture, embryo quality.

Haploid plant production in Zantedeschia aethiopica ‘Hong Gan’ using anther culture

This report describes advances in the anther culture of Zantedeschia aethiopica. Important factors for improvement as compared to the earlier procedure were: (1) using flowers from inflorescences developed at relatively low temperature during winter, (2) high temperature stress treatment at 32 °C for 2 days in the beginning of the culture, (3) use of Gamborg B5 as anther culture medium, and (4) addition of sucrose at high concentration of 8% in the culture medium. Plants were obtained via a callus phase. Frequency of anthers producing calli was around 4–5%. About 87% of the calli gave regenerants, of which 52% were haploid, 36% were diploid and the rest had other ploidy levels. In addition to chromosome counting, cytological examination of the microspore development and amplified fragment length polymorphism (AFLP) analysis of the regenerants showed that haploid as well as diploid plants originated from the microspores. Finally, 12 doubled haploid (DH) plants could be produced from each inflorescence. One quarter of the DHs equaled the original cultivar in growth vigor, while more than one third showed good fertility, indicating that inbreeding depression was not so severe in this heterozygous species. The improved protocol now enables production of sufficient number of DHs for application of haploid technology in genetic improvement and breeding of Z. aethiopica.

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).

The effect of potato extract used as additive in anther culture medium on culture responses in Triticum aestivum

In the present study, potato extract was used as an additive supplemented to a synthetic induction medium in anther culture of wheat (Triticum aestivum), attempting to make reasonable utilization of potato extract and improve the efficiency of this induction medium. The concentration of the potato extract in the medium was 5% (water extract from 50g of potato tubers for a litre of medium), the basic synthetic medium used was W14 medium, but the inorganic salts (except iron) and the vitamins were reduced to three fourths of the original strength of W14 medium. The experimental results showed that the potato-extract-supplemented W14 medium could elicit more pollen-derived calli (or embryoids) and these calli possessed greater green shoot regeneration ability compared with both the original W14 medium and a modified W14 medium in which the five major inorganic salts were each reduced to three fourths strength of the original W14 medium, and in turn the calli produced on the original W14 medium possessed greater green shoot regeneration ability compared with the modified W14 medium. However there were no significant differences in albino shoot differentiation frequency of the calli between the above-mentioned three media, it varied only with different genotypes of the anther-donors. This indicates that the medium components affecting green shoot regeneration would not certainly affect albino shoot regeneration as well.

EMBRYOGENESIS AND PLANT REGENERATION IN VITIS VINIFERA L BY ANTHER CULTURE

Different culture media were studied to obtain embryos from anther culture of two clones of the cultivar “Viosinho” from Vitis vinifera L., in order to regenerate plants from them. Six different culture media were used: five of them solidified with agar, ACM (anther culture medium) supplemented with 2,4 – D (1.0 and 1.5mg.L-1) and BAP (0.25 mg.L-1); ACM with NOA (1.0 and 1.5 mg.L-1) and BAP (0.25 mg.L-1); ACM with 2,4 – D (0.25 mg.L-1) and BAP (1.0 mg.L-1); one solidified with phytagel, and ACM with 2,4 – D (1.0 mg.L-1) and BAP (0.25 mg.L-1). In all the media tested, anthers with calli and embryos were produced and secondary embryogenesis obtained. After 10 weeks of in vitro culture, a non-significant effect (P>0.05) of clone and a significant effect (P<0.001) of culture medium composition were observed for all three parameters evaluated – namely the number of anthers with calli, the number of embryogenic calli/anther and the number of embryos/embryogenic calli. The highest number of anthers with calli was obtained in the medium containing 1.0 mg.L-1 NOA and 0.25 mg.L-1 BAP and the highest percentage of the embryogenic calli (35.2%) was observed in the medium containing 2,4 - D at 1.0 mg.L-1 and BAP at 0.25 mg.L-1. The ACM medium supplemented with 1.5 mg.L-1 2,4 - D and 0.25 mg.L-1 BAP proved to be the best for embryo production with 3.2 embryos formed per embryogenic calli.

Influence of plant preservative mixture (PPM)TM on adventitious organogenesis in melon, petunia, and tobacco

The influence of PPMTM on somatic embryogenesis in melon, adventitious shoot organogenesis in petunia, and androgenesis in tobacco was studied by culturing explants in regeneration media supplemented with 0, 2, 5 or 10 ml l−1 PPM for 8–12 wk. The percentage of melon cotyledon explants that produced callus and somatic embryos and the number of embryos per explant were reduced when incubated in embryo initiation and embryo development media containing more than 5 ml l−1 PPM. Less PPM was required to inhibit petunia shoot organogenesis. The number of shoots and number of buds per Petri dish were reduced 3–6.9-fold when leaf explants were incubated in shoot regeneration medium containing more than 2 ml l−1 PPM. In contrast, the addition of up to 10 ml l−1 PPM to tobacco anther culture medium had no effect on androgenesis. Our results suggest that the influence of PPM on plant regeneration depends on the plant species. We recommend that experimenters examine a range of PPM concentrations when using it for the first time on an untested plant species.

Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants

Three interspecific diploid potato hybrids between selections of Solanum phureja Juz. & Buk. and S. chacoense Bitt. were used in anther culture experiments to construct a monoploid family. Different aspects of the anther culture process were affected by the treatments, such as: growing conditions of donor plants, ways of preparing the anther culture medium, and conditions of anthers in culture. Genotype and date of culture initiation were among the most significant sources of variation. Significant improvements in anther culture response were achieved by growing plants at 30°C and by a heat shock of 35°C for 12 h given to anthers in culture, which gave an increase of up to 40% in embryo yield. However, the heat shock reduced the plant regeneration rate. The majority of regenerated plants was diploid, suggesting that there were several recessive lethal alleles in heterozygous status in the anther-donor. Among the regenerants, the homozygotes could be successfully identified by simple sequence repeat analysis, using eight polymorphic primer pairs.

Screening diploidSolarium genotypes responsive to different Anther culture conditions and ploidy assessment of Anther-derived roots and plantlets

Twenty-three diploid (2n=2x=24) potato clones were screened for response to four different anther culture media. Anther derived tissues (calli/embryos) were obtained from seven of the clones in liquid culture media. Anther culture response of these seven clones was further quantified on five additional media. Since clonal responses are specific to culture conditions (Tiainen, 1992), the seven genotypes somewhat differed from each other in the magnitude of the response to each of the media. In agreement with Heberle-Bors (1985) a more realistic assessment of the responsiveness was possible when pollen counts per anther were used to express the yield of anther-derived material. A relatively higher level of exogenous cytokinin (6-benzyladenine) to that of auxin (3-indoleacetic acid) proved to be favourable for the inducton of calli/embryos. Use of a semi-solid medium saved space and was convenient for culture observations therefore it was found more conducive for the genotypic screening. Anther derived tissues that regenerated to produce root tissue and/or plantlets were further multiplied on a root culture medium and a plantlet medium. Liquid culture conditions on orbital shakers were conducive for the growth of root explants. Anther derived material (roots and plantlets) originating from clone 9507–04 revealed differences in growth vigor. Flow cytometric estimation of ploidy in regenerated roots and plantlets, revealed that clone 9507–04 produced 44% monoploid roots and 77% monoploid plantlets. Seventy-three percent anther-derived plantlets from clone 6028–02 were monoploids.

Influence of anther pretreatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars

Different pretreatments were given to anthers of barley before culturing, and their effects assessed on the frequency of embryos and green doubled haploid plants produced. Mannitol pretreatment was better than cold pretreatment for some low responding cultivars. Optimal concentration of mannitol for pretreatment depended on cultivar. Low responding genotypes needed a higher concentration of mannitol than responsive ones. The addition of Ficoll to liquid medium increased the number of embryos and green plants. The influence of the growth regulators 2,4-D and TIBA was assayed using ten cultivars of barley grown in Spain. The anti-auxin TIBA gave good embryo production with some of the low responding cultivars. Two row-type cultivars always produced higher number of embryos and green plantlets than six row-type. The application of these modifications to 10 F1 hybrids with potential agronomic value, allowed the production of almost 1000 doubled haploid plants from only 3500 anthers. Up to two doubled haploid plants per flower were produced from the cross Monlon × Sonja.

Haploid plantlets derived by anther culture of Cucurbita pepo

This work was conducted to study the effect of sucrose and 2,4-D combinations on induction of haploid plants of a summer squash cultivar through anther culture; therefore, sucrose was used at 30, 60, 90, 120 and 150 g l−1and 2, 4-D was used at 0.1, 1.0, 2.5 and 5.0 mg l−1on solid MS anther culture medium. Anthers at the mid or late uninucleate microspore stage without filament were excised from sterilized buds and plated on 20 different induction media. The most plantlets resulted from the induction medium supplemented with 150 g l−1sucrose and 5 mg l−12, 4-D. Root tips from 20 plantlets were cytologically examined under a light microscope. The results revealed ten diploid (2n>= 2x= 40) and ten haploid (2n= x= 20) plants.

Effect of amino acids, growth regulators and genotype on androgenesis in barley

The effects of an amino acid mixture and of plant growth regulators added to the FHG barley anther culture medium were examined using three barley cultivars (Cadette, Leger, and Igri) grown in two environments (growth cabinet and glasshouse). ‘Leger’ and ‘Igri’ were known as responsive, and ‘Cadette’ as recalcitrant to androgenesis. Our first experiment showed that the amino acid-supplemented medium was best for embryogenesis and regeneration of ‘Cadette’ and ‘Igri’ in both environments, and if ‘Leger’ in the growth cabinet. The addition of ABA and TDZ did not improve embryogenesis and plant regeneration, and PAA decreased them in the growth cabinet. The addition of the amino acid mixture in the FHG medium also reduced the percentage of albino plants in the growth cabinet, but growth regulators did not improve the percentage of albino plants, and in some cases increased it. In the growth cabinet, disregarding media, ‘Leger’ produced more embryos than ‘Cadette’ and ‘Igri’, and Leger' and ‘Igri’ produced more green plants than ‘Cadette‘. Percentages of albino plants were higher or ‘Cadette’ than for ‘Igri’ or ‘Leger’. In a second experiment, we compared seven hybrids with their parents for androgenic responsiveness. Hybrids had a higher ability to generate green plants than expected based upon the weighted average reflecting the contribution of each parent.

Enhanced anther culture efficiency of indica rice (Oryza sativa L.) through modification of the culture media

AbstractThe production of microspore‐derived green plants from anther culture of indica rice is generally very low compared with japonica cultivars. A modified anther culture medium, consisting of a higher KNO3 content (31 mm) and casein hydrolysate (CH, 500 mg/1) but without ammonium salts, was tested in comparison with a medium consisting of the widely‐used N6 medium nitrogen background, using four indica × indica F1 hybrids as test materials. Green plant regeneration frequency was at least three‐fold higher in the microspore‐calli derived from the former medium than in those derived from the modified N6 medium. More than 700 microspore‐derived plants were raised in the field. Another study was carried out using indica × japonica and indica × javanica F1 hybrids. The results indicated that a medium with higher (3.5 mm) ammonium sulphate may induce a higher frequency of anthers with microspore‐calli but not necessarily lead to a larger number of green‐plant regenerating calli. Subsequently, using the indica cv. ‘IR‐43’ as the test material, use of a lower level (1.75 mm) of (NH4)2SO4, in addition to KNO3 (31 mm), was found to be better than CH (500 mg/l) for anther‐response as well as green plant regenerability of the derived microspore‐calli. Nitrate‐nitrogen or ammonium‐nitrogen alone elicited poor response. Twenty‐five media involving combinations of KNO3 (20–34 mm) and (NH4)2SO4 (1–3 mm) were tested for their effects on anther response. Combinations involving KNO3 (31–34 mM) and (NH4)2SO4 (2.0–2.5 mm) were found superior not only for achieving greater anther response but also, for subsequent green‐plant regeneration. This contrasts with the 28 mm of KNO3 and 3.5 mm of (NH4)2SO4 in the widely‐used N6 medium developed for japonica rice. Other than potassium nitrate and ammonium sulphate, and potassium phosphate to some extent, the levels of other inorganic salts tested did not make any significant difference to the process of anther response. Based on these results, modified media with three levels of ammonium sulphate were tested for anther culture efficiency of indica × japonica and indica × javanica derivatives (F3s). Microspore‐calli derived from a medium of a lower (1.75 mm) level of (NH4)2SO4 showed a higher regeneration potential overall than those derived from a higher (3.5 mm) level. A revised medium has been suggested, on the basis of these results, for the realization of improved anther culture efficiency and, consequently, improved feasibility of using doubled haploids in genetic and breeding research with indica rice.

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.

Rapid‐cycling Brassica species: anther culture potential of B. campestris L. and B. napus L.

summaryA study was made of the production of embryoids by cultured anthers of rapid‐cycling populations of Brassica campestris L. and Brassica napus L. In the case of B. campestris, best results were obtained when anther donors were grown at 24 °C with continuous light; anthers were excised from 1.6–2.00 mm buds with the pollen at the mid‐uninucleate stage of development and were cultured on a solidifed Brassica anther culture medium of Keller &amp; Armstrong (K), and when incubation was at 35 °C for 1 day, followed by 30 °C for 1 day and then continuous incubation at 25 °C. The response to anther culture of B. napus was generally very poor compared with that of B. campestris but the best results were obtained when anther donors were grown at 18 °C with continuous light; anthers were excised from 2.1–2.5 mm long buds with the pollen at the mid‐uninucleate stage of development, were cultured on a liquid formulation of K medium, and incubation was at 35 °C for 1 day, followed by 30 °C for 6 days and then continuous incubation at 25 °C.

Genotype-specific response of cultured broccoli (Brassica oleracea var. italica) anthers to cytokinins

Anther culture medium was prepared with different types and concentrations of cytokinins to gain greater insight into the control of embryo formation during Brassica oleracea L. var. italica (broccoli) anther culture. The independent addition of the four cytokinins tested had widely divergent effects dependent upon cytokinin concentration and the genetic background of the test plants. All cytokinins were generally inhibitory at high concentrations, however, individual plants showed significant stimulation of embyro formation at typical physiological levels. The influence of cytokinins was highly cultivar-specific, some lines were stimulated, others inhibited and still other test lines were largely unaffected. Although the addition of cytokinins was needed for embryo formation for some plants, in no instance were cytokinins able to replace the inductive effect of high-temperature treatments.

Improvement of Anther Culture Methods for Haploid Production in Wheat

The yield of haploid production in wheat (Triticum aestivum L.) anther culture has been extremely low with the currently employed methods. The objective of this study was to determine whether variation in anther culture media would affect the yield of haploids. After a 48‐h cold pretreatment, anthers from six spring wheat cultivars and one winter wheat cultivar were cultured on liquid and agar‐solidified media, and in some cases the liquid medium was supplemented with Ficoll (Sigma Chemical Co., St Louis, MO). Liquid culture significantly increased callus induction, which averaged 98.3 calli per 100 anthers for the seven cultivars tested, compared with 5.1 calli per 100 anthers for agar culture. Although the calli from liquid media usually had lower plantlet regeneration than calli from agar‐solidified media, regeneration ability was improved by adding 200 g/L Ficoll 400 to the liquid induction medium, or by changing the plant growth regulator composition of the regeneration medium. By employing the liquid culture method and the modified regeneration medium, it is now more feasible to use doubled haploids in plant breeding practice and genetic research.

Varietal differences in green plant regeneration ability of wheat pollen embryoids.

The present study was undertaken in order to clarify the effect of the culture media and the genotypes on the regeneration of green plantlets from pollen embryoids by anther culture of Japanese wheat cultivars. Pollen embryoids of 4 cultivars produced on Potato-2 medium were transferred onto two kinds of regeneration media (Table 1). On both media, embryoids grew into calli and then regenerated green or albino shoots and/or roots (Fig. 1 b and c). In rare cases, embryoids germinated (Fig. 1 a). The frequencies of green plantlets regenerated from embryoids differed with their genotypes even in the regeneration medium from 2.8% in Nanbukomugi to 33.5% in Chinese Spring. Moreover, the ability of embryoids to regenerate green plantlets was not affected by anther culture media, Potato-2 and C1 medium (Table 2). The effects of abscisic acid (ABA) on the embryoid formation and green plantlet regeneration were studied in anther culture of 7 cultivars (Table 3). ABA at a low concentration (0.1 and 0.5mg/l) had a stimulatory effect on the embryo formation in some cultivars which were genotypes of a rather low ability in embryoid formation, while ABA had a suppressive effect in other cultivars. However, ABA did not show a stimulatory effect on the regeneration of green plantlets in all cultivars tested. In conclusion, the ability of the green plantlet regeneration depended on genotypes regardless of the anther culture and regeneration media tested in this study: Norin 12 and Chinese Spring showed relatively high frequency of the green plantlet regeneration, while Hachimankornugi, Kitakarnikornugi and Norin 61 did not regenerate green plantlets. On chromosome constitutions of the regenerated green plants almost 659 were haploid (2n=21) and about 35% were diploid (2n=42) (Table 4, Fig. 2).