Embryo-like Structures Research Articles

Isolation and characterization of LEAFY COTYLEDON 1-LIKE gene related to embryogenic competence in Citrus sinensis

A member of the LEAFY COTYLEDON gene family encoding a HAP3 (heme activated protein 3) subunit of the CCAAT box-binding factor was isolated and termed as Citrus sinensis LEAFY COTYLEDON 1-LIKE (CsL1L). The deduced amino acid sequence shared a high similarity with LEAFY COTYLEDON 1-LIKE (L1L) in Arabidopsis thaliana, Phaseolus coccineus, Theobroma cacao, and Helianthus annuus. Quantitative RT-PCR results indicated that CsLIL was highly expressed in embryogenic callus, somatic embryos and immature seeds, but was rarely detected in non-embryogenic callus, vegetative and floral tissues. Ectopic expression of CsL1L in vegetative tissues could induce embryo-like structures, suggesting that CsL1L has the capability to transit cells from vegetative to embryogenic phase. Comparison of CsL1L expression in the newly formed and long-term subcultured embryogenic calli of W. Murcott tangor (C. sinensis × C. reticulata) and Hongkong kumquat (Fortunella hindsii Swingle) revealed that the potency of embryogenesis was related to the level of CsL1L expression. Sub-cellular localization analysis indicated that CsL1L was a nuclear protein in plant. A microsatellite in CsL1L was verified with polymorphism among the citrus species.

Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1

We isolated the full-length cDNA of PgCDPK2DS1 gene, the expression of which was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain an adult plant from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of the plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.

K. daigremontiana as a Model Plant for the Study of Auxin Effects in Plant Morphology

A large number of studies have been conducted in order to understand the mechanisms by which shoots, plantlets and vegetative propagules are produced. According to the first studies reported by Yarbrough [2, 3], in the fern species Camptosorus rhizophyllus (which has two different kinds of leaves), shoots are produced at the tip of long acuminate leaves, while in the species Tolmiea menziesii shoots are originated in a notch near the junction of the petiole and the leaf blade; in both cases, the shoots are produced from meristematic tissue and once they are mature are naturally detached from the leaf and fall to the ground originating a new plant. Also, in the orchid Malaxis paludosa, cells at the tip of mature leaves have an intense meristematic activity which originates embryo-like structures that once the mature leaf is wilted the small shoot detaches originating a new plantlet [4]. This phenomenon has been widely studied in plants of the Crassulaceae family, mainly in the genus Kalanchoe. According to the capacity to asexually produce such plants, species of this genus have been classified in four categories: a) plants which do not produce plantlets (K. marmorata, K. rhombopilosa, K. tomentosa and K. thyrsiflora), b) plants that spontaneously produce plantlets (K. daigremontiana), c) plants that produce plantlets by the action of an environmental stress (K. pinnata, K. fedstchenkoi, K. strepthantha, K prolifera and K. crenata) and d) plants that produce spontaneously plantlets by the action of stress and/or (K.

Wczesne etapy somatycznej embriogenezy w kalusie korzeniowym lędźwianu siewnego (Lathyrus sativus L.)

Callus cultures from root explants of Lathyrus sativus L. ‘Derek’ were tested for their morphogenic capacity. Primary explants (fragments of roots) were cultivated on three induction media. We obtained three lines of callus tissue among which we identified two non-embryogenic lines and one embryogenic line. Callus originally cultivated on modified MS medium supplemented with 0.05 mg*L-1 picloram, formed embryo-like structures upon transfer to media containing 0.1 mg*L-1 picloram or 0.9 mg*L-12,4-D. Histological examinations confirmed embryogenity of obtained structures. Previous studies had revealed that, notwithstanding efficient callus induction and proliferation, its capacity to differentiate shoots or somatic embryos is limited. Consequently, rhizogenesis was only form of complete organogenesis obtained in our experiments. However attempts to develop the methods for indirect plant regeneration in L. sativus would allow creation of new genetic variations required to improvement of this species.

Comparative analysis of in vitro anther- and isolated microspore culture in hexaploid Triticale (X Triticosecale Wittmack) for androgenic parameters

Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale.

Reprogramming in situ

Cellular reprogramming to a stem-cell state has now been achieved in tissues of genetically engineered mice. This work signals a future for regenerative medicine in which tissue fates might be manipulated in living organisms. See Article p.340 Manuel Serrano and colleagues show for the first time that reprogramming of somatic cells to pluripotency by the classic 'Yamanaka factors' Oct4, Sox2, Klf4 and c-Myc can be achieved in vivo. Analysis of induced pluripotent stem (iPS) cells induced in vivo from stomach, intestine, pancreas and kidney cells in mice shows that they are closer to embryonic stem cells than in vitro-generated iPS cells. The in vivo iPS cells also have the potential to generate embryo-like structures that express embryonic and extraembryonic markers, which suggests that they have totipotent features not found in conventional iPS or embryonic stem cells.

Reprogramming in vivo produces teratomas and iPS cells with totipotency features

Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

Phytosulfokine alpha enhances microspore embryogenesis in both triticale and wheat

Isolated microspore culture (IMC) has been used to develop doubled haploid lines and to generate homozygous lines in a single generation for varietal development. Phytosulfokine has been previously used in promoting cell growth and embryo development in various systems. In this study, phytosulfokine alpha (PSK-α) supplemented IMC induction medium was evaluated in triticale and wheat cultivars, and the production of embryo-like structures (ELS), green and albino plants were recorded. In addition, the contribution of ovary co-culture was also evaluated in IMC on a NPB99 + 10F induction medium. Over a range of concentrations up to 10−7M, PSK-α yielded more ELS and green plants in wheat and triticale cultivars at the highest dose, when compared to the control. It also minimized albinism in wheat cultivars, but not in triticale. Interestingly, 10−7M PSK-α also supported the formation of a large number of embryos and a few green plants in the absence of nursing ovaries.

Induction medium osmolality improves microspore embryogenesis in wheat and triticale

The objective of this study was to determine the effect of induction medium osmolality on embryogenesis and green plant production in wheat and triticale. Isolated microspores of wheat and triticale were subjected to a range of osmolality (300–500 mOsm kg−1) using mannitol. In both species, the maximum number of embryo-like structures (ELS) and green plants were obtained at 350 mOsm kg−1 when the induction medium was supplemented with 9.1 g L−1 of mannitol. A sharp decline in microspore response was observed at higher osmolality. These results demonstrate the effect of osmolality on induction of ELS and production of green plants indicating that the process of microspore embryogenesis can be improved in wheat and triticale by increasing osmolality of the induction medium to 350 mOsm kg−1.

Induced Expression of AtLEC1 and AtLEC2 Differentially Promotes Somatic Embryogenesis in Transgenic Tobacco Plants

Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene’s induction of somatic embryogenesis.

Characterization of polarity development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in Brassica napus L

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical–basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-013-0530-y) contains supplementary material, which is available to authorized users.

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Cefotaxime (100mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production. Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F1 hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.

Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4 ± 1.0 after 6 wk on basal medium (1/2 strength Murashige and Skoog) with 2.5 μM kinetin and 0.5 μM 6-benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 μM indolebutyric acid and 2 μM α-naphthaleneacetic acid for 7 wk, followed by transfer to medium (without growth regulators) for a further 7 wk. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 μM indolebutyric acid and 20 μM α-naphthaleneacetic acid for 1 wk then placed in Rockwool plugs (under propagation house conditions) for another 7 wk resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after 7 wk. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5 μM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5 μM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1 μM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after 2 wk on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further 4 wk on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6μM IBA and 0.3μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9μM BAP and 0.1μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.

Wuschel overexpression promotes somatic embryogenesis and induces organogenesis in cotton (Gossypium hirsutum L.) tissues cultured in vitro

This work shows that overexpression of the WUS gene from Arabidopsis enhanced the expression of embryogenic competence and triggered organogenesis from some cells of the regenerated embryo-like structures. Agrobacterium-mediated genetic transformation of cotton was described in the late 1980s, but is still time consuming and largely genotype dependant due to poor regeneration. To help solve this bottleneck, we over-expressed the WUSCHEL (WUS) gene, a homeobox transcription factor cloned in Arabidopsis thaliana, known to stimulate organogenesis and/or somatic embryogenesis in Arabidopsis tissues cultured in vitro. The AtWUS gene alone, and AtWUS gene fused to the GFP marker were compared to the GFP gene alone and to an empty construct used as a control. Somatic embryogenesis was improved in WUS expressed calli, as the percentage of explants giving rise to embryogenic tissues was significantly higher (×3) when WUS gene was over-expressed than in the control. An interesting result was that WUS embryogenic lines evolved in green embryo-like structures giving rise to ectopic organogenesis never observed in any of our previous transformation experiments. Using our standard in vitro culture protocol, the overexpression of AtWUS in tissues of a recalcitrant variety did not result in the production of regenerated plants. This achievement will still require the optimization of other non-genetic factors, such as the balance of exogenous phytohormones. However, our results suggest that targeted expression of the WUS gene is a promising strategy to improve gene transfer in recalcitrant cotton cultivars.

Morpho-histological and ultrastructural study on direct somatic embryogenesis of Capsicum chinense Jacq. in liquid medium

Somatic embryo-like structures were produced from the hypocotyls of aseptic plants of Capsicum chinense. Different concentrations of 2,4-dichlorophenoxyacetic acid (0, 4.5, 9.05 μM), several exposure times of the explant to this auxin (15, 30, 45, 60 days) and the development of somatic embryos cultured in a solid and/or liquid medium were evaluated. As a result, a novel system of regeneration via direct somatic embryogenesis in liquid medium was established, with an efficiency of 1.77 × 104 somatic embryos per liter of medium. Critical stages of embryogenesis, including cellular acquisition of morphogenetic competence, suspensor formation, and development and maturation of somatic embryos, were identified by histological analysis and scanning electron microscopy. Our results show a promising new outlook on the in vitro regeneration of this species. Contrary to what has been reported to date for the Capsicum genus, it is a species of plants with higher embryogenic potential in vitro.

The auxins centrophenoxine and 2,4-D differ in their effects on non-directly induced chromosome doubling in anther culture of wheat (T. aestivum L.)

Doubled haploid technologies have become key tools for plant breeding. Using these techniques, the speed and efficiency of plant improvement processes can be significantly enhanced. Anther culture-based technologies have the potential to regenerate large numbers of doubled haploid plants without colchicine treatment. In an attempt to elucidate the influence of phytohormones on non-directly induced chromosome doubling, two synthetic auxins, 2,4-D and centrophenoxine, were tested in a wheat anther culture approach. Whereas the induction of androgenic embryo-like structures (ELSs) was efficient for both auxins, we observed a significantly higher frequency of chromosome doubling when using 2,4-D than when using centrophenoxine. When 2,4-D was added to the induction medium, a positive correlation between the size of ELSs and their ploidy level was detected by flow cytometry. The morphological selection of ELSs, a process that was included in routine operations of the method without significantly extending the input of time and effort, facilitates the production of fertile DH plants with a frequency of 60 %. Our findings may contribute to a more efficient production of doubled haploid wheat plants using a colchicine-free anther culture approach.

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA3 (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA3 was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.

Evaluation of different embryogenic systems for production of true somatic embryos in Arabidopsis

Somatic embryogenesis (SE) in Arabidopsis was induced using various systems, including auxin treatment of in vitro cultured explants (immature zygotic embryos, IZEs) and transgenic plants overexpressing embryogenesis-related transcription factors, e.g. LEC2 together with the GUS reporter gene under control of the auxin-induced DR5 promoter. The study indicated that the SE-systems used gave different embryogenic capacities for the production of true embryos. The highest ratio of true embryos (75 %) was found among embryo-like structures in transgenic seedlings overexpressing LEC2. Analysis of in vitro induced SE systems indicated that in somatic embryos produced in response to exogenous auxin treatment the formation of root poles is frequently disturbed. A lack of a properly formed root meristem was observed in 35–80 % of in vitro induced somatic embryos, in dependence on auxin concentration and duration of treatment.

Anther culture of Lupinus angustifolius: callus formation and the development of multicellular and embryo-like structures

Androgenesis is an important technique to generate double haploid plants. Anther and microspore cultures are the methods to induce haploid embryogenesis. For culture initiation, it is necessary to select anthers with the appropriate developmental stage of microspores. For lupins, limited reports about the establishment of initial cultures for androgenesis are available. In this study, different parameters of anther culture of three genotypes of Lupinus angustifolius were investigated. For all genotypes, a considerable correlation was observed between the buds and the anthers, depending on their location in the inflorescences. Buds from the central segment of inflorescences had yellowish green anthers that contained the maximum number of microspores at uninucleate stage. Cytological investigation shows that the anthers containing these microspores were the most responsive to induction. Two types of developmental pathways were observed for microspores. In case of cold pre-treated and untreated inflorescences, microspores developed into multicellular and embryo-like structures, respectively. Effects of different factors showed significant differences among: genotypes, pre-treatment, growth regulators (GRs) and genotypes × GRs interaction. Among three genotypes, Emir showed the highest number of multicellular and embryo-like structures on MS medium + 2.0 mg/l 2,4 D + 0.5 mg/l Kinetin (Kin). For all genotypes, anthers produced calli on MS medium containing 2.0 mg/l 2,4 D + 0.5 mg/l Kin. These calli continued their growth on regeneration medium (MS + 2.0 mg/l BA + 0.5 mg/l NAA) and produced roots. Taken together, these results provide a good basis for further research towards the development of haploid plants for L. angustifolius.