Anther Induction Research Articles

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.

Proteomic and biochemical analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an important role of anti-oxidative enzymes

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenic embryos can produce completely homozygous, haploid or double-haploid plants. This study aimed to investigate changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis.

Efficacy of esterified glucomannan, sodium bentonite and humic acid to counteract experimental aflatoxicosis on antibody titers against Newcastle disease in broilers

Influence of ten different genotypes, age of trees and environmental temperature on induction of androgenesis and apperance albino horse chestnut embryos were studied. Efficiency of in vitro androgenesis via anther and microspore culture had been investigated. Microspores and anthers were used from the same closed flower bud. Androgenic response of different genotypes was measured and compared. Anther induction rates were from 5 to 37.6%, depending on genotype. The number of embryos per isolated anther varied between 0.5 to 5.0 embryos in anther culture, while in microspore culture varied between 3.0 to 27 embryos, depending on genotype. A microspore culture was 5 - 6 times efficient than anther culture for same genotype. Age of the trees had no influence on androgenesis induction. Temperature of about 4 - 5°C was optimal for androgenic embryo induction. Albino horse chestnut embryos phenomenon depended on genotype. The number of albino appearing in anther was higher than in microspore culture. The same correlation of appearing albino was observed between short and long day. Flow cytogenetic analysis of androgenic embryos originating from anther and microspore culture was done after a first generation of regenerants. All androgenic embryos, the first generation from microspore culture were haploid, while 50% of the regenerants originating from anther culture were haploid, and the other half diploid.

Impact of genotype, age of tree and environmental temperature on androgenesis induction of Aesculus hippocastanum L.

Influence of ten different genotypes, age of trees and environmental temperature on induction of androgenesis and apperance albino horse chestnut embryos were studied. Efficiency of in vitro androgenesis via anther and microspore culture had been investigated. Microspores and anthers were used from the same closed flower bud. Androgenic response of different genotypes was measured and compared. Anther induction rates were from 5 to 37.6%, depending on genotype. The number of embryos per isolated anther varied between 0.5 to 5.0 embryos in anther culture, while in microspore culture varied between 3.0 to 27 embryos, depending on genotype. A microspore culture was 5 - 6 times efficient than anther culture for same genotype. Age of the trees had no influence on androgenesis induction. Temperature of about 4 - 5°C was optimal for androgenic embryo induction. Albino horse chestnut embryos phenomenon depended on genotype. The number of albino appearing in anther was higher than in microspore culture. The same correlation of appearing albino was observed between short and long day. Flow cytogenetic analysis of androgenic embryos originating from anther and microspore culture was done after a first generation of regenerants. All androgenic embryos, the first generation from microspore culture were haploid, while 50% of the regenerants originating from anther culture were haploid, and the other half diploid.

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.

Doubled-haploid production in chickpea (Cicer arietinum L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15-20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4 degrees C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50-400 V with 25 microF capacitance and 25 Omega resistance, (3) anthers were centrifuged at 168-1,509g for 2-15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15-20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1-3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10-30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.

In vitro microspore selection in maize anther culture with oxidative-stress stimulators

In order to produce doubled-haploid maize plants tolerant of oxidative stress, in vitro microspore selection was carried out in anther culture with reactive oxygen species (ROS) progenitors such as paraquat, menadione, tert-butylhydroperoxide (t-BHP), and methionine combined with riboflavin. All the ROS progenitors reduced the anther induction, the formation of microspore-derived structures, and their regeneration potential. Abnormal cell divisions and progeny cell degradation could be observed during the development of microspores treated with ROS progenitors. Menadione and t-BHP influenced the microspore developmental pathway, as menadione induced the formation of embryoids, while t-BHP increased the proportion of calli in the microspore-derived structures. As the result of in vitro selection, 15, 10, 10, and 3 fertile doubled-haploid plants were obtained in cultures treated with paraquat, t-BHP, methionine combined with riboflavin, and menadione, respectively.

Effect of selected factors on the effectiveness ofCapsicum annuum L. anther culture

The primary aim of the study was to establish the effectiveness of induced androgenesis in in vitro anther culture of two pepper (Capsicum annuum L.) breeding lines--ATZ1 and PO, and a hybrid between these two lines (ATZ1 x PO)F1. Anther culture was maintained according to the method developed by Dumas de Vaulx et al. (1981) with some modifications. The experiment revealed that the effectiveness of androgenesis ranged from 4 %; for the ATZ1 line to 1.5 %; for the (ATZ1 x PO)F1 and strongly depended on the developmental stage of flower buds, as well as the conditions for anther culture maintenance. The development of androgenic embryos was successfully induced only in anthers which originated from the flower buds with petals equal or slightly longer than sepals and there was a clear relationship between the length of the period of anther induction on CP medium and the level of kinetin in R1 regeneration medium.

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures.

The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10-3-10-5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M2 doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M2 doubled-haploid lines carrying mutations per line analysed was 3.8% when 10-4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10-5 and 10-4 M sodium azide were applied to freshly isolated microspores from model cultivars.

Colchicine, an efficient genome-doubling agent for maize ( Zea mays L.) microspores cultured in anthero

The construction of maize genotypes with high haploid induction capacity made it possible to study the effect of colchicine on maize androgenesis in vitro. Anther cultures of three hybrids were treated with 0.02% and 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine added to the induction medium had no negative influence on the androgenic responses (anther induction, induction of structures of microspore origin and their regeneration ability) of the genotypes examined. However, significantly higher fertility was observed in plants originating from colchicine-treated microspores, especially at 0.03%. Cytological examinations showed that colchicine treatment before the first microspore division efficiently arrested mitosis and resulted in homozygous doubled-haploid microspores. Under the experimental conditions, the antimitotic drug had no later effect on the division symmetry of the microspore nucleus, and unequal divisions remained dominant. Callus formation from the induced microspores seemed to be more typical (ranging between 60–70%), but embryo frequency was increased by approximately 10%, especially at the higher colchicine concentration. These results suggest that the mechanism of colchicine action in premitotic maize microspores may differ from that previously observed in wheat.

Genetic analysis of anther and leaf disc culture in two clones of Solanum chacoense Bitt. and their reciprocal hybrids

Two diploid clones of self-incompatible Solanum chacoense Bitt. with androgenetic ability were tested for anther and leaf disc culture response together with eight of their reciprocal F1 hybrids. Large differences were found among genotypes in frequency of anther induction as well as in the phase of plant regeneration. Anthers harvested in June showed a significantly higher percentage of response (17.5%) at the induction phase than those collected in July (13.8%) or August (12.7%). The lowest induction frequency was observed in May (7.3%). By contrast, plant regeneration from induced anthers did not vary during this time. Genotypic differences were also observed in leaf disc response. The two parental clones and two of their hybrids failed to produced any shoots. Among the remaining genotypes, two had only sporadic occurrence of shoot formation, two gave an intermediate response (15% and 24% of their discs carried shoots), whereas the discs of the two remaining genotypes responded well to culture (68% and 77%). The genetic analysis performed on the reciprocal hybrids revealed that a positive significant correlation existed between anther induction and leaf disc response (Spearman's r=0.82; p=0.01). This suggests that, under our conditions, these two aspects of tissue culture might share a common system of genetic control. Estimates of broad sense heritabilities, for leaf disc culture, 83% were obtained and the number of effective factors involved in the control of tissue culture response, indicated a relatively simple genetic control. Finally, considering the potentialities opened by the use of RFLP analysis, it might be possible to find probes that are linked with genes involved in tissue culture competence.

Effect of a Modified Potato Medium on Anther Culture of Wheat1

Modification of a potato extract medium was undertaken to increase pollen embryogenesis from anthers of a hard red spring wheat (Triticum aestivum L. cv. Chris). Whole potato tubers (Solatium tuberosum L. cv. Kennebec) were separated into peeled tubers and peel from which extracts were prepared. A 3 ✕ 3 latin square design with three replications in time was used to compare media prepared from: (i) extract of whole potato tubers, (ii) extract of the peel only, and (iii) extract of peeled tubers. A 60% increase in anther induction resulted from the peeled tuber extract medium compared to the whole potato medium. Removal of the peel from tubers probably reduces the concentration of compounds that may be inhibitory to wheat embryogenesis during anther culture. Anther source, defined by spike section (basal, middle, and distal), was a second variable in the experiment but did not significantly affect embryogenesis.