Factors affecting variability in anther culture and in conversion of androgenic embryos ofSolanum phureja

The variation for embryo production in anther ofSolanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. Four clones ofS. phuyreja were grown in a greenhouse under a 16-h photoperiod. The temperature was monitored continuously. Buds (60 per day on 10 days) were collected and the anthers cultured in two groups of five flasks (30 anthers per flask). In the first group, each flask contained the 30 anthers from six buds; in the second group, each flask contained one anther from each of 30 buds. Significantly smaller coefficients of variation were observed for the second group, suggesting that variation for embryogenic capcity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature was examined by stepwise regression analysis. Embryogenic capacity of one clone was adversely affected by high temperatures (31–37°C) that occurred two and seven days before bud harvest. However, similarly high temperatures appeared to enhance the androgenic response of another clone. Conversion of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected conversion rate which ranged from 12.5% to 46.0%. Conversion rate declined on each serial subculture.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid, IAA-indole-3-acetic acid     

Genetic variation in monoploids of diploid potatoes and detection of clone-specific random amplified polymorphic DNA markers

Randomly amplified polymorphic DNA (RAPD) markers have been used to study the genetic variation among androgenetic monoploids of diploid Solanum species. Cluster analysis of pairwise genetic distances was used to construct a genetic relationship among anther donor and anther-derived potato plants. The clustering based on Rogers' distances resembled classifications based on parental origins and hybrid combinations. Six of the 32 RAPD primers used resulted in the selective amplification of DNA fragments which were polymorphic between the two S. phureja parental clones, 1.22 and A95. It should be possible to construct a genetic linkage map, without making crosses, using monoploids derived from a single heterozygous diploid clone and RAPD markers.     

Microsatellite marker analysis of an anther-derived potato family: skewed segregation and gene-centromere mapping.

Segregation patterns of polymorphic simple sequence repeat (SSR) primer pairs were investigated in monoploid potato families derived from anther culture. A total of 14 primers developed from the sequences in the database, as well as from a genomic library of potato, was used. Distorted segregation was observed for seven (50%) polymorphic loci among monoploids derived from an interspecific hybrid. Similar distortion was observed for only one of five loci that could be contrasted between the two monoploid families. Segregation distortion was less common in the sexually derived backcross population between the interspecific hybrid and either of its parents. One locus could be putatively linked to a lethal allele because it showed distorted segregation in both monoploid families, a group of 70 heterozygous diploids derived from unreduced gametes through anther culture, and a backcross population. These diploids were used to map the polymorphic SSR markers with respect to the centromeres using half-tetrad analysis. The majority of the SSR loci mapped more than 33 cM from the centromere, suggesting the occurrence of a single crossover per chromosome arm.     

CLUSTER ANALYSIS OF ANTHER-DERIVED PLANTS OF SOLANUM PHUREJA (SOLANACEAE) BASED ON MORPHOLOGICAL CHARACTERISTICS

The feasibility of grouping anther-derived plants of Solarium phureja according to ploidy based on their morphological characteristics was studied. Canonical discriminant analysis identified four characteristics (anther length, number of chloroplasts per pair of guard cells, leaf width, corolla width at widest diam) of nine measured as the most effective combination for diagnosing ploidy. Data for these characteristics from two sets of plants were subjected to two clustering techniques, one using the average linkage clustering (UPGMA of the NT-SYS programs) and the other using centroid sorting (SAS-Fastclus). Screening of anther-derived plants by cluster analysis proved to be an efficient means of separating monoploids from the other ploidy levels.     

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).     

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

Selection inherent in monoploid derivation mechanisms for potato

Monoploid potato (2n=1×=12) can be derived either paternally through anther or microspore culture (androgenesis) or maternally through crossing with a haploid-inducing pollinator (gynogenesis). Androgenic and gynogenic monoploid populations, derived from each of two Solanum phureja clones, were compared in both greenhouse and field studies, and their frequency of cells at the 1x, 2x and 4x DNA contents was estimated by flow cytometry of in vitro plantlets. In 15 of 17 comparisons of morphological data (plant height, number of main stems, vigor, leaf length and width, internode length, tuber number and tuber weight) where a significant difference was found due to derivation, the androgenic monoploids were agronomically superior to their gynogenic counterparts (13–18% greater leaf size, double to triple the total tuber yield). Only plant height was significantly greater (26–27%) in the gynogenic monoploids. Flow cytometry revealed that the gynogenic monoploids retained 5% more cells at the monoploid level, whereas androgenic monoploids exhibited 27% more endopolyploid cells at the tetraploid level. Received: 19 April 2000 / Accepted: 17 November 2000     

Evaluation of anther-derived Streptocarpus X hybridus and their progeny

Regeneration of plants by caulogenesis of anther derived callus of Streptocarpus X hybridus Concorde was obtained on MS basal medium with 2.0 mg/l NAA and 0.5 mg/l BAP. All regenerated plants were diploid and of somatic cell origin as determined by chromosome counts, flower color and morphological similarity to the anther donor plant, and flower color segregation among progeny of anther-derived plants. Variation expressed as deformed and dwarfed transient morphology was observed in 2 anther-derived plants. Variation was also observed among progenies of 5 anther-derived plants from the same anther donor plant for days to flowering, flowers per peduncle, peduncle length, and leaf area. Additional morphological variation including transient leaf variegation among seedlings and twisted or inverted leaves on mature plants was also observed among progeny of anther-derived plants.     

Variation of RuBP carboxylase activity among anther-derived plants of Solanum phureja

The effect of genotype and ploidy on RuBP carboxylase (EC 4.1.1.39) activity, chlorophyll content, leaf area, chloroplast ultrastructure and not photosynthesis among monoploid. diploid and tetraploid anther-derived plants of Solanum phureja Juz, and Buk. was studied. Within the monoploid group, RuBP carboxylase activity and concentration displayed a significant genotypic effect. For the diploids, variation among genotypes was significant for total protein content and maximum specific activity of RuBP carboxylase, and among the tetraploids for net photosynthesis and specific leaf weight. Ploidy effect was evident regarding net photosynthesis, leaf area and chlorophyll content. The different ploidy groups among the anther-derived plants surpassed the anther donor plant for all characteristics except maximum activity of RuBP carboxylase and net photosynthesis. For the latter only the tetra-ploid group was superior to the anther source plant. However, a monoploid genotype with an increase of 9% in maximum activity of RuBP carboxylase over the anther-donor plant was identified. Segregation of trails rind differential gene expression together with possible mutations during androgenesis are discussed as sources of variation.     

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. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of silver nitrate,colchicine, cupric sulfate and genotype on the production of embryoids from anthers of tetraploid wheat (Triticum turgidum)

Anther culture of four tetraploid wheat (Triticum turgidum) genotypes was studied using ten different culture medium treatments in a randomized block design with four replicates. Each replicate consisted of 2 pots with 3 plants. Anther donor plants were grown in a greenhouse with a 16 h day/8 h night at 25°C and 15°C, respectively. The first treatment which was considered as the control, was potato 2 medium modified by adding 0.5 g l^–1 glutamine and solidified by gelrite (4 g l^–1). The nine test treatments differed from the control by addition of 3 different concentrations of silver nitrate (1, 2.5 and 5 mg l^–1), colchicine (10, 100 and 200 mg l^–1) or cupric sulfate (2,5 and 10 mg l^–1). The study of about 2000 anthers per genotype and treatment showed that both genotype and treatment affected embryoid formation. The presence of cupric sulfate (10 mg l^–1) and silver nitrate (2.5 and 5 mg l^–1) usually increased the frequency of embryoid formation in 3 genotypes out of the 4 studied. On the contrary, colchicine had a significant and enegative effect on anther culture responses for three out of the four genotypes studied. Because of the large genotype x medium interaction, it is very difficult to identify the best medium for embryo production by all genotypes studied.     

CHLOROPLASTS OF MONOPLOID AND DIPLOID OENOTHERA HOOKERI

Comparison of leaves from diploid Oenothera hookeri plants with leaves of their monoploid derivatives revealed the following differences: the leaves of the diploids are almost twice as thick as those of the monoploids; the diploids have only one-fifth as many cells per unit leaf area as do the monoploids; leaf tissue of the diploids has an average of 36.3 chloroplasts per cell compared with 27.4 for the monoploids; chloroplasts of the diploids average 4.68 μm in length, those of the monoploids, 3.74 μm; chloroplasts from the diploids contain about seven times more chlorophyll than those of the monoploids and have an average of seven thylakoids per granum compared with an average of only three for the monoploids; the average chlorophyll a/b ratio for the diploid chloroplasts is 2.98 compared with 3.48 for the monoploids.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.     

Monoploids in Zea mays L. following crosses with untreated and X-rayed pollen

Monoploids in Zea mays L. occur spontaneously among individual diploid seedlings. Plants with the gametic chromosome number have also been detected among members of multiple seedlings of maize and numerous other species of angiosperms. Previous reports disclosed that Xirradiation of the pollen successfully stimulated reduced parthenogenesis in some other angiosperms, but the results of X-ray treatment were inconclusive in maize. Therefore, a tester stock of maize homozygous for lg _land gl _l was crossed with pollen from inbred CI3A, carrying the dominant alleles. The pollen was exposed to 0, 1000, 2000, and 4000r units of X rays. chromosome counts were made from root tips of plants exhibiting both recessive phenotypes to establish the frequencies of monoploids in the control and X₁ populations.Monoploids were more abundant among the individual seedlings from crosses with untreated pollen than in the X₁ populations. X irradiation of the pollen is not a feasible method for the induction of monoploids in maize. The X-ray treatments greatly increased the frequency of multiple seedlings, and deficiencies were numerous among them. The members of a set of multiple seedlings were always genetically identical, and no monoploid members occurred. It is concluded that the induced deficiencies caused atypical development resulting in zygotic or embryonic cleavage.Scientific Article No. A2070, Contribution No. 5023 of the Maryland Agricultural Experiment Station, Department of Botany.     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture

The objective of this study is to induce the nuclear DNA duplication of anther-derived embryos of cork oak (Quercus suber L.) to obtain doubled-haploid plants. Anther culture of this species produces a low percentage (7.78%) of spontaneous diploids, as assessed by flow cytometry. Therefore, three antimitotic agents, colchicine, oryzalin and amiprophos-methyl (APM), were applied in vitro to anther-derived cork oak haploid embryos from six genotypes at different concentrations and for different treatment durations. Antimitotic toxicity was determined by embryo survival. Efficiency in inducing chromosome doubling of haploid embryos was evaluated by flow cytometry measurements and differences were observed between treatments. Nuclear DNA duplication and embryo survival of cork oak haploid embryos was most efficiently induced with oryzalin 0.01 mM for 48 h. Around 50% diploid embryos were obtained. The rate of chromosome duplication induced by APM 0.01 mM was also acceptable but lower than that induced by oryzalin, regardless of the duration of the treatment. Colchicine 1.3 or 8.8 mM was the least efficient, with the induction of necrosis and only a small rate of nuclear DNA duplication.     

In vitro induction of tetraploid in pomegranate (Punica granatum)

Tetraploid plants were obtained in pomegranate (Punica granatum L. var. `Nana') by colchicine treatment of shoots propagated in vitro. Shoots cultured on MS medium supplemented with 10 mg l^–1 colchicine, 1.0 mg l^–1 BA and 0.1 mg l^–1 NAA for 30 days produced tetraploids at a high frequency of 20%. No tetraploids were detected by treating the shoots in 5000 mg l^–1 colchicine for 114 h. Shoots treated by 5000 mg l^–1 colchicine for 96 h produced three morphological mutants with narrow leaves, which were later confirmed as mixoploids that separated into diploids and tetraploids after further subculture. In vitro tetraploid plants had shorter roots, wider and shorter leaves than the diploid ones. Tetraploid pomegranate plants grew and flowered normally in pots, but possessed flowers with increased diameter and decreased length compared to diploids. The number of pollen grains per anther was higher in tetraploids, but the viability of pollen decreased significantly.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> 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°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω 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.