Rapid-cycling Brassica Species: Inbreeding and Selection of Brassica napus for Anther Culture Ability and an Assessment of its Potential for Microspore Culture

A study was made to determine the feasibility of producing,by inbreeding and selection, lines of rapidcycling Brassicanapus L. with high or low potential for anther culture. In contrastto previous observations of B. campestris L., a self-incompatiblespecies, the anther culture potential of the plants of successiveinbred generations of B. napus remained uniform, and antherefficiency was poor, with a maximum of 0.476 embryoids producedfor each anther plated. This negative response to selectionmay have been due to an absence of variation with respect toanther culture ability in the base population, resulting fromthe self-fertility of the species. Cytological studies of culturedanthers of B. napus indicated that in each generation therewas a poor correlation between pollen induction and embryoidproduction. In an attempt to improve the yield of haploid embryoids of B.napus, isolated microspore culture was attempted, and was foundto be at least 60 times more efficient than anther culture,with as many as 32 embryoids being produced from each anther.In experiments designed to ascertain the reasons for such differences,an inhibitory effect of the anther wall on the anther embryogenesisof B. napus was observed, and embryoid yields were improvedby centrifuging buds prior to anther extraction to simulatethe effects of the centrifugation which is a component of themicrospore preparation procedure. Brassica napus, L. anther culture, microspore culture, inbreeding, selection     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Rapid-cycling Brassica Species: Inbreeding and Selection of B. campestris for Anther Culture Ability

A study was made to determine the feasibility of producing,by inbreeding and selection, lines of rapid-cycling Brassicacampestris with high or low potential for anther culture. Sincethe base population of rapid-cycling B. campestris is self-incompatible,inbreeding was achieved by a combination of bud-pollinationand the application of pollen to the cut surfaces of decapitatedstigmas. Three inbred generations were raised, and in each generationplants were selected for high or low potential for anther embryogenesis.The proportion of viable pollen present in anthers, as indicatedby a fluorochromatic reaction and a germination test, was alsodetermined at each stage. Lines of rapid-cycling B. campestriswith clearly defined high or low potential for anther embryogenesiswere isolated in these experiments. Within each line, however,continuous variation was always observed. Pollen viability andanther efficiency were not correlated. Although inbreeding depressioncaused a significant decrease in pollen viability over the threegenerations, there were no obvious deleterious effects on antherefficiency. In general, over the three generations of inbreeding,no segregation in plant morphological characters was observed,although many developmental abnormalities were seen in the 3rdinbred generation and there was a marked reduction in the numberof seeds set. No association between plant vigour and high orlow anther efficiency was noted. All plants regenerated fromanther embryoids of rapid cycling B. campestris were haploid.By treating anther-derived embryoids, axillary buds and wholeplants with colchicine, dihaploid plants were produced, butthese failed to set seed after self-pollination. The diploidnature of the plants was confirmed, however, when they producednormal seeds after cross-pollination with plants of the basepopulation. Brassica campestris, anther culture, inbreeding, selection     

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.     

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。     

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.     

Anther Culture in Nicotiana tabacum: The Role of the Culture Vessel Atmosphere in Pollen Embryo Induction and Growth

A comparison of four volumes of culture vessel atmosphere revealedthat this variable greatly influenced the induction and growthof pollen embryos from anthers of Nicotiana tabacum. The optimumfrequency of anthers producing embryos and plantlets was foundwith a culture atmosphere of 15 ml per anther, whereas the optimumnumber of plantlets was found with 5.5 ml per anther. A smallvolume (0.5 ml per anther) almost completely suppressed embryoinduction. Removal of specific components of the culture atmosphere(ethylene, carbon dioxide, oxygen) influenced the response ofthe anthers but did not produce a satisfactory explanation ofthe inhibition of pollen embryogenesis by the small cultureatmosphere volume. In particular, the influence of ethyleneabsorption on embryo induction and growth depended both on theculture atmosphere volume and on the stage of development ofthe pollen at the start of culture. Using anthers containingpollen at a stage after the first pollen grain mitosis. ethyleneabsorption was found to increase the survival of induced embryos.Treatment of anthers for 3 d with silver nitrate, a known antagonistof ethylene action, was not an efficient means of increasingthe yield of pollen plantlets.     

Embryogenesis and doubled haploid production from anther culture in gentian (<Emphasis Type="Italic">Gentiana triflora</Emphasis>)

The overall goal of this study is to develop an anther culture system to produce doubled haploid (DH) lines of gentian (Gentiana triflora), an ornamental flowering plant, for use in an F1 hybrid breeding program. Embryogenesis was induced from anther cultures incubated on half-strength modified Lichter (NLN) medium containing a high concentration of sucrose (130 g/l) and subjected to heat shock treatment. Among the various parameters investigated, anthers collected from buds 9–12 mm in length induced the highest frequency of androgenesis. Moreover, among three genotypes tested, cvs. Ashiro-no-Aki and Ashiro-no-Natsu produced 21.3 and 3.7 embryos per 100 anthers, respectively, whereas, cv. Lovely-Ashiro failed to produce embryos. Among a total of 427 embryos transferred to a regeneration medium consisting of Murashige and Skoog (MS) medium, 138 plants were regenerated. The ploidy levels of regenerants were determined by flow cytometry and chromosome counts, revealing the presence of 5% haploids, 25% diploids, and 70% triploids. Inter simple sequence repeat (ISSR) analysis using the 6PS line obtained following self-pollination of the diploid plant obtained from anther culture confirmed that the diploid plant was indeed a DH.     

Regeneration of androgenic embryos in apple (<Emphasis Type="Italic">Malus x domestica</Emphasis> Brokh.) <Emphasis Type="Italic">via</Emphasis> anther and microspore culture

Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed.     

Anther culture of maize and the visualization of embryogenic microspores by fluorescent microscopy

Summary Three maize genotypes previously shown in the literature to respond to anther culture were tested under various conditions. Studies indicated that embryogenic response ranged from 0 to 100 embryos per 1,000 anthers plated and was significantly lower without cold pretreatment of the anthers. Culture in liquid media tended to produce more embryos than in semi-solid as did the addition of activated charcoal to either liquid or solid culture media. Most results were confounded by plant-to-plant variation which tended to obscure significant differences. In one study, germination rate of androgenetic embryos averaged about 20%, but only 26% of those embryos that germinated completed their reproductive cycle and formed seed albeit through sibpollination since plants could not be selfed. Chromosome counts using root tip squashes indicated that regenerated plants were either haploid or diploid but plants scored as non-diploid yielded as much seed as scored diploids. This suggests that progeny can be recovered even from putative haploids, presumably as a result of sectoring in the developing ear. A DNA-specific fluorescent dye was used to visualize the presence of putative embryogenic microspores (PEMs) during the culture period. PEM counts were a function of time in culture and were apparently greater than the number of embryos obtained for a given treatment. The data indicate that, as previously reported for other species, both induction and survival phases also exist in maize anther culture.     

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     

Effect of Potato-2 Medium on Anther Culture of Interspecific Rice Hybrids

The effect of two culture media, potato-2 and N₆ supplementedwith kinetin and either 2,4-dichlorophenoxyacetic acid (2,4-D)or -naphthalene acetic acid (NAA) on anther culture responseof two interspecific rice hybrids was studied. While calluscould be successfully induced and plants regenerated from theF₁ of O. saliva x O. rufipogon, the other hybrid, O. salivax O. longistaminala did not respond to the anther culture. Nevertheless,some success in callus induction was achieved when anthers froma few selected F₂ plants were cultured from the latter cross.No interaction effects between the media (potato-2, N₆ and growthhormones (2,4-D and NAA) for anther response to callusing wereobserved. Potato-2 medium proved to be superior to N₆ in termsof increased anther response, early callus induction, multiplecalli formation and also overall green plant regeneration Oryza saliva L., O. rufipogon Griff., O. longistaminata A. Chev. et Roehr, interspecific hybrid, anther culture, potato-2 medium, N₆ medium     

Role of Sucrose in Microspore Embryo Production in Brassica napus ssp. oleifera

Dun well, J. M. and Thurling, N. 1985. Role of sucrose in microsporeembryo production in Brassica napus ssp. oleifera.—J.exp. Bot. 36: 1478–1491. One cultivar of winter oil seed rape (Brassica napus ssp. oleifera)and three cultivars of spring rape were used in a study of theeffects of sucrose on microspore survival and embryo inductionin cultured anthers. A preliminary study on the winter cultivar(Fiona) revealed that the osmotic pressure of the supernatantof anther homogenates was equivalent to a solution of 17% sucrose.A study of microspore survival and embryo induction in thiscultivar on media containing either 8 %, 12%, 16% or 20% sucroserevealed the highest survival (after 16 d) and the greatestnumber of anthers with induced embryos (after 42 d) occurredon the highest sucrose concentration. A subsequent study on three spring cultivars (Willi, Duplo andTower) examined microspore survival at 8 d and embryo induction(42 d) on media containing either 8 % or 16 % sucrose and againrevealed much higher survival and induction at the higher concentration.The variation in response between the cultivars was also reducedby culture at the higher sucrose concentration. The beneficialeffect of the 16% level occurred regardless of the growth environmentof the donor plants and of the stage of pollen development atthe start of culture. However, macroscopic embryos emerged onlyfrom anthers on the 8 % sucrose concentration, suggesting thattransfer of anthers from a high to a normal sucrose concentrationduring culture would ensure that full advantage was taken ofthe much higher initial survival on the higher concentration Key words: Brassica napus, sucrose, microspore embryo production     

Increased induction and chromosome doubling of androgenetic haploid rye

Summary Further progress of studies aimed at increasing production of androgenetic Secale cereale plants via the culture of anthers is described. Two culture media initially developed for rice and wheat anther culture have been shown to have pronounced influence on rye. It has been possible to increase the average percentages of responsive anthers (i.e. those producing embryoids or calluses) from 0.26% to 10% with a maximum in certain experiments of over 40 %. Of nearly 400 plants produced in 1976, 1/4 are green and can be grown further by transfer to potting compost; 3/4 are albino. Stable green haploid lines were present amongst the plants, and after vegetative propagation of the lines representative samples have been treated with colchicine resulting in diploid, triploid and tetraploid plants. The influence of the genetic background of the donor plants on the success rate of anther culture and on the percentage of albino formation is discussed.     

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea

Summary An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL^–1 BA and 0.1 mgL^–1 NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL^–1), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.     

Effects of n-butanol on barley microspore embryogenesis

Doubled haploid (DH) production is an efficient tool in barley breeding, but efficiency of DH methods is not consistent. Hence, the aim of this study was to study the effect of n-butanol application on DH barley plant production efficiency. Five elite cultivars of barley and thirteen breeding crosses with different microspore embryogenesis capacities were selected for n-butanol application in anther and isolated microspore cultures. Application of 0.1 % n-butanol after a mannitol stress treatment in anther culture significantly increased the number of embryos (up to almost twice) and green plants (from 1.7 to 3 times) in three low-responding cultivars: Albacete, Astoria and Majestic. No significant differences on microspore embryogenesis efficiency were observed in medium and high responding cultivars. The application of n-butanol treatment to isolated microspores from cold treated spikes in thirteen spring breeding crosses with a low or very low androgenetic response did not have a significant effect on the overall number of green plants. Nevertheless, an increase in the number of green plants was observed when 0.2 % n-butanol was applied in four out of seven low-responding crosses. Therefore, application of n-butanol could be routinely applied to anther cultures using mannitol treatment, in low-responding material. However, further studies are needed to determine optimal conditions in protocols using cold treatment and isolated microspore cultures.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

Stimulation of embryogenesis and haploid production in Brassica campestris anther cultures by elevated temperature treatments

Summary Culture of Brassica campestris anthers at 35°C for one or three days prior to culture at 25°C significantly stimulated the yield of microspore-derived embryos. More than 100 plants were regenerated from cultured embryos and haploids were identified amongst them. The haploid frequency was greater than 70% if all small-flowered sterile plants were considered to be haploid. The yield of microspore-derived plants in B. campestris is approaching the level where anther culture may be utilized as a practical breeding tool.     

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.     

Plant regeneration from carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) anther culture derived embryos

The research concerned of the regeneration of plants from embryos obtained from anther cultures of seven carrot (Daucus carota L.) cultivars. The aim was to determine the influence of the regeneration medium on the efficiency of the regeneration process. The optimization of the adaptation of the obtained plants was also carried out. Embryogenesis occurred on four of the tested media: B5 and MS without hormones, MS with charcoal, and MS with 1 mg dm⁻³ BA and 0.001 mg dm⁻³ NAA. Embryos obtained from the anther cultures produced secondary embryos, from which the regenerations of plants was observed. Secondary embryos were formed most extensively on the B₅ medium without hormones. The efficiency of the regeneration process depended on the cultivar. Most of the secondary embryos were formed by androgenetic embryos of the cultivar ‘Feria F₁’. The highest number of plants (102) regenerated from one embryo during 12 weeks of culture was also obtained in case of the cultivar ‘Feria F₁’. Secondary embryogenesis and plant regeneration from embryos allow to omit the difficult stage of root induction applied when plants are regenerated form shoots' explants. This makes the plant regeneration process quicker and easier. The plants regenerated by the conversion of embryos are better adapted to the ex vitro conditions than those obtained in the two-stage organogenesis involving the regeneration of shoots and in second stage roots induction.