Origin of Embryo-Like Structures in Soybean Anther Culture Investigated Using SSR Marker

The Satt418 microsatellite locus was examined in order to investigate the origin of embryo-like structures (ELS) obtained from soybean anther culture. Four heterozygous plants were used as anther donors. A total of 7000 anthers were placed on the induction medium under culture conditions known to trigger androgenic response. After 60 days of culture, upper portion of 216 ELS were carefully removed and transferred to a proliferation medium, in order to obtain sufficient tissue for DNA extraction. Callogenic masses originated from 114 ELS were screened for the Satt418 microsatellite locus. Heterozygous and homozygous ELS were identified, suggesting occurrence of somatic embryogenesis and androgenesis in the same system. This unexpected morphogenic response seems to be genotype-dependent.     

Enhanced post-germinative growth of encapsulated somatic embryos of Siberian ginseng by carbohydrate addition to the encapsulation matrix

Based on a protocol for microspore culture in apple (Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of in vitro androgenesis, both methods, via anther and microspore culture, were investigated using the same bud material. A comparison of the efficiency of embryo induction in anther and microspore cultures showed that microspore culture resulted in an increase up to 10 times, depending on the genotype. The regeneration route in microspore culture is similar to that of androgenic embryos via anther culture and showed adventitious shoot formation in most cases after a long period of secondary embryogenesis.Communicated by H. Lörz     

Colchicine Induced Embryogenesis in Maize

The present study involves in vitro androgenesis of Zea mays L. using anther culture. We tested combinations of single factors and their influence on microspore induction. Embryogenic induction of microspores within anthers in in vitro conditions was the best when combination of cold treatment, TIBA (0.1 mg l^–1) in media and colchicine (0.02% during first 3 days of culture) was applied, but colchicine alone can be factor, which can stimulate or initiate embryogenesis in anther culture of maize.     

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.     

Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem)

Callus culture has, to date, been reported only in a few species of Narcissus. We used anthers of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem) as explants for callus induction and plant regeneration. A high percentage of anthers at the early- to mid-uninucleate microspore stage were responsive on the basal MS medium supplemented with 0.5–1 mg l^–1 2,4-dichlorophenoxyacetic acid and 0.5–2 mg l^–1 6-benzyladenine under dark conditions. Calli were initiated from anther connective tissue or anther wall tissue, and no division of microspores occurred during callus formation, as determined by histological observation. Using 20 random amplified polymorphic DNA primers, we verified the genetic integrity of the anther-derived plants of Chinese narcissus with respect to the donor plants. These results suggest that anther culture in vitro can provide an efficient new micropropagation technique for Chinese narcissus as well as a new strategy for in vitro mass propagation of other daffodils.     

Induction of in vitro androgenesis in anther and isolated microspore culture of different spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) genotypes

This is the first report on isolated microspore culture—derived spelt wheat. The efficiency of anther- and isolated microspore was compared using four genotypes (‘Franckenkorn’, ‘GK Fehér’, ‘Mv Martongold’, ‘Oberkulmer Rotkorn’). In anther culture, genotype dependency was observed, and cold pre-treatment enhanced the efficiency of the method. In isolated microspore culture, the ovary co-culture supported the development of embryo-like structures. The presence of growth regulators (0.5 mg/l 2,4-D and 0.5 mg/l kinetin) were not essential for the induction of androgenesis, but these increased the production of embryo-like structures, green and albino plantlets. The low plant regeneration rate and high number of albinos hinder the practical application of isolated microspore culture while anther culture was efficient for in vitro green plantlets production in spelt wheat. The mean of green plantlets production was 41.45/100 anthers (from 20.93 to 83.07 depending on genotype). The phenomenon of albinism was mitigated in anther culture (3.48 albinos/100 anthers). Altogether, 1720 anther culture—derived green plantlets were produced from the four genotypes.     

Expression of auxin and light-regulated arrestin-like proteins,G proteins and nucleoside diphosphate kinase during induction and development of wheat somatic embryos

The modulation of three signal transduction elements: arrestin-like proteins, G proteins and NDPK was assessed during the induction of wheat (Triticum aestivum L.) somatic embryogenesis under different auxin (2,4-D) and light conditions. Immunological approaches using specific antibodies, kinase activity measurement and [α-³²P]-GTP-binding assay were performed. The induction of embryogenic capacity by 2,4-D was characterised both by the increased expression of the classical 40-kDa arrestin-like form and by the appearance of an additional arrestin-like protein of 29 kDa. The 40-kDa arrestin-like soluble form was unaffected by light stimuli. On the other hand, the 29-kDa arrestin-like form, specific of the embryogenic tissue culture, was found to be light regulated. From embryogenic cultures grown under light or dark, different soluble G proteins from 22 to 48 kDa were detected by probing polyvinylidene fluoride (PVDF) blots with [α-³²P]-GTP. In addition, in the microsomal fraction from light-grown cultures, a polypeptide of 20 kDa was heavily labelled. Under light conditions, cell proliferation induced by 2,4-D stimulated the appearance of a 32-kDa nucleoside diphosphate kinase (NDPK) form in addition to the classical 16–18-kDa protein, without a significant change in the NDPK activity. The modulated expression of plant arrestin-like proteins, G proteins and NDPK molecules in response to auxin and light support the view that they play key roles in signalling cascades participating in plant development.     

Response of stem explants to screening and explant source as a basis for methodical advancing of regeneration protocols for chrysanthemum

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration, length of induction period and light quality on leaf regeneration of quince clone BA 29 were investigated. After 2, 4 or 6 days of induction with 2.5 mg l⁻¹ or 5.0 mg l⁻¹ 2,4-D, leaves were cultured under red, blue, red+blue, far-red+blue, white, far-red light or darkness conditions. Leaves thereby treated showed different responses, with respect to somatic embryogenesis, callus, red-nodular structures or roots. Callus production increased with increasing 2,4-D concentration and induction period, although it was not influenced by light quality; the only exception was far-red+blue light, which reduced callusing response. This result suggested involvement of the blue-absorbing photoreceptor system in the callus formation processes. A high regeneration of red-nodular structures with a meristematic appearance was also observed; from some histological characterizations, we presumed they were adventitious buds that were arrested at an early developmental stage. Red-nodular structures increased with decreasing 2,4-D concentration and induction period. In the regeneration of such structures, the blue-absorbing photoreceptor system appeared to have a negative effect but only at a low photoequilibrium value. In contrast, light quality which activated phytochrome induced an increment in regeneration, but the response did not vary for photoequilibrium values ranging from 0.43 to 0.86. For root regeneration, phytochrome seemed to be the only photoreceptor involved. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphogenesis in Isolated Microspore Cultures of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Androgenesis is a phenomenon in which microspores are made to bypass the sexual pathway and follow the sporophytic mode of development to generate new plants without the intervention of fertilization under specialized in vitro conditions. Microspore culture provides an ideal system, with a large, relatively uniform population of haploid cells, for use in mutant selection, genetic transformation and in studies on the molecular mechanism of induction of androgenesis and embryogenesis. This paper involves a study on establishing a reproducible and efficient protocol for microspore embryogenesis in various varieties of Brassica juncea. The genotype had a pronounced effect on androgenic response in microspore cultures. The cultivar Rajat exhibited the most response, producing around 3500 embryos/100 buds. The microspores of B. juncea cv. PR-45 from ed plants maintained at a day/night temperature of 10 °C/5 °C form embryos with suspensors with varied morphology. The microspore embryos germinated to produce plants with frequencies. These plants exhibited 52% survival and 74% fertility.     

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.     

Effect of growth regulators on microspore embryogenesis in coconut anthers

The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y₃ medium containing 66 μM 2,4-D), followed by maturation medium (Y₃ medium without growth regulators) and germination medium (Y₃ medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA₃), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).     

Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

An isolated microspore culture provides an excellent system for the study of microspore induction and embryogenesis, provides a platform for an ever-increasing array of molecular studies, and can produce doubled haploid (DH) plants, which are used to accelerate plant-breeding programs. Moreover, isolated microspore cultures have several advantages over anther culture, wherein presence of the anther walls can lead to the development of diploid, somatic calli and plants. Although protocols for isolated microspore culture vary from laboratory to laboratory, the basic steps of growing donor plants, harvesting floral organs, isolating microspores, culturing and inducing microspores, regenerating embryos, and doubling the chromosomes, remain the same. Over the past few years, a large proportion of the research reports on isolated microspore culture have focused on cereal and Brassica species. For some of these species, isolated microspore culture protocols are well established and routinely used in laboratories around the world for developing new varieties, as well as for basic research in areas such as genomics, gene expression, and genetic mapping. Although these species are considered highly responsive to microspore culture, improvements in efficiency are still being made. However, with many species, isolated microspore culture is simply not yet efficient enough at producing DH plants to be cost-effective for breeding programs. There has been a recent resurgence of haploidy research with response being reported in some species once considered recalcitrant. Future research programs aimed at elucidating pathways involved in microspore induction and embryogenesis will be of benefit, as will novel approaches to improve the efficiency of microspore culture for DH production. With many species, anther culture has proven to be more effective than isolated microspore culture, necessitating more research to clarify the contribution of the anther wall to embryogenesis. The development of molecular markers for use in determining the gametic origin of regenerated plants, irrespective of their ploidy, would also be beneficial. In this review, we aim to provide an overview of the basic isolated microspore culture protocol with an emphasis on recent progress in several crop species.     

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus induction in culture of Oenothera hookeri and Oenothera picensis anthers

In the present work we try to determine optimum conditions for callus induction in anther culture of Oenothera hookeri and O. picensis. The anther callus yield was increased when the anthers were cultured on modified MS medium supplied with 2 mg dm^-3 2,4-D and 2 mg dm^-3 NAA, in both species. In O. hookeri, best results were obtained when anthers were excised from 7.2 - 9 mm buds at the stage of vacuolated microspores, then pretreated at 4 °C for 2 d and grown under 16-h photoperiod. The response to anther culture of O. picensis was generally very poor compared with that of O. hookeri. The higher yield of calli was obtained when anthers were excised from 6.2 - 8 mm buds at the stage of vacuolated microspores and grown under continuous light. The cold pretreatment of buds decreased anther response in this species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Haploid plantlets derived by anther culture of Cucurbita pepo

This work was conducted to study the effect of sucrose and 2,4-D combinations on induction of haploid plants of a summer squash cultivar through anther culture; therefore, sucrose was used at 30, 60, 90, 120 and 150 g l⁻¹and 2, 4-D was used at 0.1, 1.0, 2.5 and 5.0 mg l⁻¹on solid MS anther culture medium. Anthers at the mid or late uninucleate microspore stage without filament were excised from sterilized buds and plated on 20 different induction media. The most plantlets resulted from the induction medium supplemented with 150 g l⁻¹sucrose and 5 mg l⁻¹2, 4-D. Root tips from 20 plantlets were cytologically examined under a light microscope. The results revealed ten diploid (2n>= 2x= 40) and ten haploid (2n= x= 20) plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Meiotic metaphase I to telophase II as the most responsive stage during microspore development for callus induction in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) anther cultures

The generation of homozygous doubled haploid lines through induction of androgenesis is a promising alternative to the classical inbreeding and selection programs. However, this technology is poorly developed in tomato, where doubled haploid tomato plants have only been obtained through anther culture. Despite the fact that anther culture is routinely used in a number of economically interesting crops, there are still many drawbacks that prevent tomato breeders from adopting this technique, and improvements in methodology are required. One key issue is the correct identification of the optimal stage for anther excision and culture. In this paper we characterise in vivo microsporogenesis in tomato, defining the different microspore stages and relating them to the length of the donor flower bud. In parallel, we cultured anthers of these stages to obtain embryogenic callus, and followed the microscopic development of the callus contained within the anther. Our data suggest that the stage with the highest response, in terms of callus generation, is meiosis. In particular, we propose the window from metaphase I to telophase II, including tetrad cellularisation, as the timeframe where induction can be accomplished in tomato anther cultures.     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.     

Culture conditions for induction of green plants from barley microspores by anther culture methods

Summary With barley a large variation in frequency of plant formation from microspores of spikes from the same plant has been observed. The highest frequency of plant formation was obtained when culturing anthers in the dark on a high Ficoll medium containing 2,4-D and kinetin to induce proembryo (or callus) formation. Subsequently the proembryos or calli were cultured in dim light on a high Ficoll-high sugar medium containing IBA and kinetin. Finally the embryos were transferred to a starch agar medium. A maximum of 13 green plants were obtained from microspores of a single anther.The ratios of green to albino microspore derived plants varied from 91 to 19 depending on culture conditions. Under anaerobic conditions, lactic acid and other organic acids may have damaged the organelles in the cells resulting in the formation of albino plants. Thus, direct embryogenesis by using a well-buffered, high Ficoll-high sugar medium and proper aeration are essential for obtaining high frequency of green plants from microspores.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA 3 indolylbutyric acid     

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     

Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.