Influence of Preculture Variables on Microspore Embryo Production in Brassica napus ssp. oleifera cv. Duplo

The effect of four preculture variables on microspore embryoinduction and growth were examined: (1) the source of the budselected for culture (apical or axillary inflorescence; (2)the method of harvest (single harvest of whole inflorescenceor sequential harvest of individual buds; (3) the length ofthe bud (2, 3 or 4 mm); and (4) the application of a 4 ^°Cpretreatment to the bud after harvest. Microscopic and macroscopicanalysis of every anther used for culture permitted an assessmentof the following parameters: (1) the percentage of induced buds;(2) the number of induced anthers per induced bud; (3) the numberof productive buds (with macroscopic embryos) as a percentageof the induced buds; (4) the degree of induction per inducedanther (an estimate of the number of microspores in which initialembryogenic divisions had commenced); and (5) embryoid survival(the number of embryos as a proportion of the degree of induction). The product of parameters 1 and 2 gave the number of inducedanthers and all five parameters were components of the finalyield - the number of embryos produced per bud cultured. It was found that the maximum number of induced buds (67·0per cent) occurred with 2 mm sequentially harvested non-pretreatedbuds. Overall, the values decreased with increasing bud lengthand were lower for pretreated and axillary buds. In contrast,the two other estimates of induction - number of induced anthersper induced bud and degree of induction per induced anther -both had maximum values from 3 mm sequentially harvested, pretreatedbuds from apical inflorescences. The highest final yield ofembryos per cultured bud (44·9) was found with 2 mm non-pretreatedbuds taken from a single harvest of the apical inflorescence.The study therefore confirmed that the different componentsof the final embryo yield are differentially affected by thefour preculture variables tested. These variables must be controlledif reproducible results are to be achieved. Brassica napus, tape, anther culture, pollen, microspore, haploid     

Genotype,plant, bud size and media factors affecting anther culture of cauliflowers (Brassica oleracea var. botrytis)

Summary Eleven F₁ hybrid cultivars of cauliflower, representing a range of maturity types, were examined for their responsiveness to anther culture. Embryos were produced from each of the cultivars tested, and the mean embryo yield varied from 82.2 embryos per 100 anthers cultured for cv Dova to 0.6 embryos for cv Serrano. Variation between genotypes and between plants within a genotype was significant, both in terms of embryo yield and percentage responsive anthers. Autumn and winter maturing cauliflowers were generally more responsive than summer types. Embryo yields were enhanced by culturing anthers on solid rather than on liquid media. An increase in concentration of 2,4-Dichlorophenoxyacetic acid (2,4-D) from 0.1 to 0.3 mg/l also increased embryo yield. Embryo yield was doubled when anthers were cultured on solid media containing 0.3 mg/l 2,4-D compared to liquid media containing 0.1 mg/l 2,4-D. Although bud size alone did not have a significant effect on embryo production, genotype x bud size and plant x bud size (within genotype) interactions were significant. Estimation of the variance components demonstrated that, apart from the residual plate-to-plate variation, variation between plants was the largest source of variation, accounting for approximately 30% of total variance. Plant x bud size (within genotype) interaction accounted for 18% of total variance and genotypic differences for approximately 8%.     

Effect of temperature shock and elevated incubation temperature on androgenic embryo yield of diploid potato

Using three diploid tuber-bearing Solanum clones as anther donors, experiments were conducted on the effect of high temperature shock and elevated incubation temperature during anther culture on androgenic embryo production. Five incubation treatments were tested on two clones and three treatments were repeated in a second experiment on one of the same clones and an additional one. In the first experiment, temperature treatment, genotype, date of culture initiation, and their interactions were all significant sources of variation. A treatment combining a high temperature shock (35 °C for 12 h) with elevated incubation temperature (30/20 °C) yielded 11 times as many embryos (44 per flask) as the control 20 °C (4 per flask). By conducting several replications per day of bud collection, the significant variation due to experimental dates was separated from experimental error to provide a more sensitive test of treatment effects. Temperature shock (35 °C 12h) during anther culture did not appear to influence the subsequent conversion rate of androgenic embryos.     

Variations in response to high temperature treatments in anther culture of Brussels sprouts

The level, time of application and duration of the high temperature treatment necessary for embryo production from Brussels sprouts anther culture were examined. The effects of 29, 32, 35, and 38°C given for 24 h immediately following removal of the anthers from the bud, were tested on different cultivars, on different plants within the cultivars and on different occasions for each plant. Most embryos were produced following 32 and 35°C, very few following 30°C and none following 38°C. Although there was a tendency for some cultivars to respond better to one or other of the two more favourable temperatures, this varied considerably between individual plants. Plant to plant variation was also seen in the overall level of the response, although responsiveness tended to decline with successive samplings of the same plant. Experiments with cultivars Hal and Gower suggested that high temperature was required for at least 12 h after anther removal, but beyond that time the optimum period varied from plant to plant. If the excised anthers were held at 25°C for 16 h or more with Hal or 24 h or more with Gower before being exposed to the high temperature treatment, embrogenesis tended to be reduced. It is suggested that apparent non-responsiveness in anther culture may result to a large extent from the specific conditions that are used during the anther culture process.     

A STUDY OF THE RELIABILITY OF SYNCHRONY IN THE DEVELOPMENT OF POLLEN MOTHER CELLS OF LILIUM LONGIFLORUM AT THE FIRST MEIOTIC PROPHASE

Lilium longiflorum anthers have been used by a number of investigators as a source of supposedly synchronous cells for studying genetic, chromosomal and molecular events of meiotic prophase I. Because of questions raised by the literature as to the reliability of such synchrony, new baseline data were obtained for L. longiflorum ‘Croft’ in preparation for experiments requiring homogeneity or synchrony. Homogeneity was found to be reliable at the same level of locules of the same anther and of anthers from the same bud up to the onset of diplotene. Synchrony deteriorates rapidly from diplotene on. Less than half the anthers examined had PMC's at the same stage from apex to base. Hence experiments requiring complete homogeneity within anthers would call for rigorous monitoring of each anther included in the sample. Synchrony between buds of the same length was insufficient to allow bud length to be used as a reliable index of meiotic stage.     

The influence of various physical parameters on anther culture of broccoli (Brassica oleracea var. italica)

Embryo formation by cultured broccoli (Brassica oleracea L. var. italica) anthers was best in the pH range of 5.5 to 5.8. Manipulation of the initial medium pH showed, however, that embryos could be recovered throughout the entire pH range tested. Experiments designed to test the influence of anther density on embryo production exhibited an apparent population effect. Comparison of anthers cultured with and without filaments showed a significantly lower level of embryo formation with filaments attached. The importance of anther orientation with the adaxial surface up was also demonstrated. Detailed studies of the effect of temperature on anther response showed the importance of 35°C treatments. Other temperatures and a variety of temperature manipulations were either comparatively ineffective or inhibitory. The duration of 35°C exposure required for optimal response varied widely between 18 and 48 h. Wide variation in plant to plant response was observed despite attempts to optimize the manipulation of physical parameters. Individual plants were identified that reliably formed many thousands of embryos, whereas other plants failed to form embryos under all tested conditions.     

Indifference of potato anther culture to colchicine and genetic similarity among anther-derived monoploid regenerants determined by RAPD analysis

Effects of colchicine on androgenesis of diploid potato (Solanum phureja Juz. & Buk.) and ploidy of anther-derived plants were examined in three experiments. In the first, no significant difference was found for mean embryos per anther of an interspecific potato clone after application of five colchicine treatments (0, 25, 50, 100 and 200 mg l^-1) for 24 h to freshly excised anthers containing late uninucleate microspores. The same colchicine treatments were applied to six hybrid potato families in the second experiment. Families differed for number of embryos per anther and embryo regeneration frequency; however, androgenic response did not differ significantly among colchicine treatments. The 312 regenerated plants included 233 (75%) monoploids. The third experiment examined durations (0, 90 s vacuum infiltration, 24, 48 and 72 h) of high colchicine treatment (200 mg l^-1) on anther culture of seedlings representing one family. Mean embryos per anther, though not statistically significant, ranged from 0.96 to 1.90 for 48 h colchicine and 90 s vacuum infiltration, respectively. There were 126 plants regenerated of which 62% were monoploid. Frequency of monoploid plants regenerated from colchicine treatments did not differ significantly. RAPD analysis was conducted on 26 anther-derived monoploids of one family, based on common flasks of origin. The 13 decamer primers revealed 54 polymorphic loci. These were used to characterize the monoploids genetically. From one flask, two pairs of monoploids among six examined were genetically indistinguishable. Examination of a second and third flask revealed, six of seven and three of seven monoploids that were genetically indistinguishable. These data suggest the regeneration of genetic clones within flasks and may indicate the occurrence of secondary embryogenesis during anther culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

A study of factors affecting anther culture of cauliflower (Brassica oleracea var. botrytis)

Experiments on three autumn-heading cauliflower genotypes (2 hybrids and a genotype selected from a population) were conducted to study different factors affecting anther culture. Culture conditions of the donor plants proved to be important: the best results were obtained during spring in a greenhouse where the temperature was maintained between 10 and 20°C. Overall winter and spring seemed more suitable than summer and early autumn for culture establishment. The optimal bud development stage depended on the genotype: for the hybrid 702, the greatest number of embryos for 100 plated anthers was obtained at the uninucleate pollen stage of the microspores; for V23.2 and 703, the optimal stage of the buds corresponded to the first mitotic division. Sucrose proved to be the best carbon supply for embryogenesis with an optimal concentration of 140 g l^-1. The addition of a cytokinin (BAP) in the medium led to lower embryo production, and this negative effect increased when the hormone concentration in the medium increased. The use of liquid medium and a dark incubation period immediately after the high temperature treatment were favourable for embryogenesis.     

Haploid plants from in vitro anther culture of the leguminous tree,Peltophorum pterocarpum (DC) K. Hayne (Copper pod)

The development of haploid callus, embryos and plantlets from cultured anthers and the various factors affecting androgenesis in Peltophorum pterocarpum (Copper pod), a tropical legume tree is reported. A pretreatment of flower buds at moderate temperature of 14°C for 8 days was most effective for callus production. The colour of the anther was found to be a reliable and efficient indicator for identification of suitable stage of anther for culture. The frequency of anthers which produced callus and shoots was highest when anthers were cultured at mid or late-uninucleate stage. A high sucrose concentration of 10% is a specific media requirement for androgenesis. The haploid nature of the embryos, callus and regenerated plants (n=14) were confirmed by chromosome count.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid - BAP bezylaminopurine     

Production of androgenic plants through Pollen embryogenesis in anther cultures ofBrassica carinata A. Braun

Pollen embryogenesis occurred in anther cultures of two genotypes ofBrassica carinata A. Braun. Pretreatment of anthers at 35°C for 3 or 6 days was essential for the induction of androgenesis on growth regulator-free culture medium. A combination of sucrose and glucose was better than sucrose alone. None of the pollen embryos germinated normally. Full plants were raised through adventitious bud differentiation from their hypocotyl.     

Stimulation of androgenesis in white cabbage (Brassica oleracea var. capitata) anthers by low temperature and anther dissection

Anther culture was performed on two local cultivars, Ljubljansko and Varadinsko, and the F₁ cv. Krautman (Bejo-Zaden). The effects on androgenesis of hot and cold temperature treatments and different dissections of anthers were evaluated. In contrast to cv. Krautman, cvs. Ljubljansko and Varadinsko produced more embryos after cold pretreatment of flower buds (4°C, 48 h) than after standard treatment (35°C, 24h). Simultaneous cutting of the anther tip and removal of the filament gave the best results in comparison to other tested dissections. Microscopical observations of sectioned anthers revealed enhanced embryo development near the cut ends of the anthers. Ploidy analysis revealed the presence of haploids among embryos resulting from cold treatment (4°C, 48 h), treatment at elevated temperature (35°C, 24 h), and among embryos resulting from dissections of anther tips.     

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.     

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.     

Genetic and non-genetic factors affecting anther culture of Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Eight inbred lines of Brussels sprouts and ten F₁ hybrids derived from them were tested for their response to anther culture. From 5–19 plants per genotype were tested, and each plant was tested on 3–6 separate occasions. Results from the inbred lines were broadly similar to those from the F₁ hybrids, despite the inbreds producing fewer buds and having a higher frequency of anther deformities. The maximum embryo yield from an inbred line was 215 embryos per 100 anthers, and from a hybrid was 275. From estimation of the variance components it was calculated that, for both inbreds and hybrids, about half the total variation was genetic whereas variation due to plants within genotypes and to occasions within plants were each about 13% of the total. The narrow sense heritability of responsiveness to anther culture (estimated by the proportion of variation between inbred lines which was genetic) was 0.48, and there was partial dominance for this character. In three cases the hybrid outyielded the better inbred, and this heterosis may well be due to dispersed dominant genes.     

Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin

Embryos of Picea abies (L.) Karst were pulse-treated with water or cytokinin for 2 h and then cultured on medium lacking cytokinin. Adventitious buds developed on cytokinin-treated embryos, but not on water-treated embryos. The general appearance and the surface morphology were similar on water and BA (benzyladenine)-treated embryos after 3 days. The epidermal cells were elongating after 6 days on water-treated embryos, while they were dividing on cytokinin-treated embryos. Furthermore, the cells surrounding the stomata had started to proliferate on BA-treated embryos. This was the first micromorphological sign of bud initiation. During the second week prominent meristemoids developed from these cells. A stoma was observed on the top of each meristemoid. The variation in developmental pattern of meristemoids among different embryos as well as within each embryo was small. However, during the subsequent development of bud primordia and buds, the morphological variation was significant. The meristemoids continued to develop into cone-shaped bud primordia, which successively changed shape during the transition to adventitious buds. The epidermal cells divided and the epidermis did not rupture during the formation of adventitious bud primordia. The epidermis was identified as the protoderm of the bud primordium.     

The influence of bud length,age of the tree and culture media on androgenesis induction inAesculus carnea Hayne anther culture

Statistical analyses of the data revealed very significant differences in androgenesis induction ofA. carnea Hayne anther culture depending on the bud length, nutrient medium composition and age of the parental tree. Significant mutual influence of all these factors was also observed. The highest number of androgenic anthers was obtained when 4 mm long buds were used. Older trees (60 and 100 yrs) gave a higher number of androgenic anthers than the younger ones (20 and 40 yrs). MS medium supplemented with 2,4-d and Kin (1 mg l^–1, each) was the most favourable for androgenesis induction. Pollen embryos (haploids and aneuploids) were formed by the division of uninuclear microspores.The highest percentage of germinated embryos and further synchronous development of the shoot and root was achieved in MS medium supplemented with IAA, GA₃ (1 mg l^–1) and activated charcoal (1%). When other germination media were used, malformations of androgenic embryos were observed.Abbreviations AC activated charcoal - H casein hydrolysate - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - GA₃ gibberelic acid - Kin 6-furfurylaminopurine - MS Murashige and Skoog - T thidiazurone - N phenyl-N'-1,2,3-thiadiazol-5-ylurea - Z zeatin-6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine     

A study of factors affecting embryo yields from anther culture of cabbage

In cabbage (Brassica oleracea var. capitata), a thermal shock treatment of 24 h at 35 °C at the start of the culture period resulted in higher embryos per 100 anthers (30.0) compared to a treatment of 48 h. Similarly , a chilling treatment of 24 h at 4 °C resulted in a higher embryo yield (6.0) per 100 anthers compared to a treatment of 48 h. However, the embryo yields were significantly higher (p> 0.01) in thermal shock than chilling treatments in all experiments. Treatments of 6 days at either 35 °C or 4 °C gave no embryos. The most responsive cultivar was the F₁ hybrid , Hercules, in all experiments. Although anther culture was successful in the other genotypes, the open pollinated ones, the highest number of embryo yields per 100 anthers was obtained in the hybrid. High temperature treatment before culture had a beneficial effect on the embryo yields. The responsiveness of anthers to addition of increasing concentration of silver nitrate (AgN0₃) (the ethylene inhibitor) to the culture medium, showed a progressive increase in the embryo yields in all the genotypes. Since embryos were also formed in the absence of silver nitrate, probably, due to a greater genotype × medium interaction, it is noted that the presence of silver nitrate in the medium may not be essential for cabbage anther culture as reported earlier. The findings of this study may be recommended for large production of cabbage embryos in culture.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

抗根肿病大白菜的花药培养

大白菜是中国北方的重要蔬菜之一,根肿病是危害大白菜生产世界性病害,利用花药培养可以大大加速抗根肿病大白菜杂交育种工作的进程。对33份抗根肿病大白菜品种(品系)进行花药培养,有24个品种诱导出胚,品种诱导率为72.7%,以东方皇冠×C11的诱导率最高,为1.86胚/蕾。对大部分基因型来说,在培养基中添加0.4 g/L谷氨酰胺的诱导效果最好。研究还发现生长在温室中的供体植株更容易诱导出胚状体。变绿的子叶型胚转到B5+0.2 mg/L BA+0.1 mg/L NAA+0.1%活性炭的胚分化培养基上可诱导生芽。