Exogenous Auxin Research Articles

Ectopic Expression of WINDING 1 Leads to Asymmetrical Distribution of Auxin and a Spiral Phenotype in Rice.

Ectopic expression of the rice WINDING 1 (WIN1) gene leads to a spiral phenotype only in shoots but not in roots. Rice WIN1 belongs to a specific class of proteins in cereal plants containing a Bric-a-Brac/Tramtrack/Broad (BTB) complex, a non-phototropic hypocotyl 3 (NPH3) domain and a coiled-coil motif. The WIN1 protein is predominantly localized to the plasma membrane, but is also co-localized to plasmodesmata, where it exhibits a punctate pattern. It is observed that WIN1 is normally expressed in roots and the shoot-root junction, but not in the rest of shoots. In roots, WIN1 is largely localized to the apical and basal sides of cells. However, upon ectopic expression, WIN1 appears on the longitudinal sides of leaf sheath cells, correlated with the appearance of a spiral phenotype in shoots. Despite the spiral phenotype, WIN1-overexpressing plants exhibit a normal phototropic response. Although treatments with exogenous auxins or a polar auxin transport inhibitor do not alter the spiral phenotype, the excurvature side has a higher auxin concentration than the incurvature side. Furthermore, actin filaments are more prominent in the excurvature side than in the incurvature side, which correlates with cell size differences between these two sides. Interestingly, ectopic expression of WIN1 does not cause either unequal auxin distribution or actin filament differences in roots, so a spiral phenotype is not observed in roots. The action of WIN1 appears to be different from that of other proteins causing a spiral phenotype, and it is likely that WIN1 is involved in 1-N-naphthylphthalamic acid-insensitive plasmodesmata-mediated auxin transport.

Genome-wide Identification and Expression of ARF Gene Family during Adventitious Root Development in Hot Pepper (Capsicum annuum)

Auxin response factors (ARFs) are transcription factors that activate or repress the expression of primary/early auxin response genes by binding to auxin-responsive elements (AuxREs) in their promoter regions. The ARFs play important roles in diverse developmental processes. To explore the ARF gene family in hot pepper (Capsicum annuum L.), we performed a genome-wide identification and expression analysis. In this study, 19 pepper ARF genes (CaARFs) clustered into three phylogenetic groups (I, II, and III) were comprehensively analyzed. Conserved domain analysis showed that all CaARFs contained a B3 DNA-binding domain and a middle domain, but two members lacked the carboxy-terminal dimerization (CTD) domain. The number of introns in CaARF genes ranged from 1 to 13 and the gene structure was similar among genes in the same phylogenetic group. Additionally, prediction of CaARFs promoter elements and putative targets for microRNAs suggested that the regulation of CaARFs may occur at both transcriptional and posttranscriptional levels. Most CaARFs were expressed in more than one tested tissue, and most CaARFs were identified as being responsive to exogenous auxin. Moreover, time-course transcription profiles of CaARFs revealed their roles in adventitious rooting of hypocotyl cuttings from pepper seedlings. Therefore, our results will provide a foundation for better understanding the regulatory mechanisms and molecular functions of CaARFs in hot pepper.

Interaction of stockplants shading and exogenous auxin on the propagation of Cola anomala K. Schum (Malvaceae) by cuttings

The establishment of an efficient protocol for vegetative propagation is still a challenge for Cola anomala, a priority target for domestication in West and Central Africa. The present study examined the rooting ability of C. anomala leafy stem cuttings in response to different levels of stockplants shading and different concentrations of exogenously applied indole 3-butyric acid (IBA). 900 cuttings were set in three blocks of a split plot experimental design in non-mist propagators. Each main plot contained three shading levels (0%, 50% and 75%), whereas five exogenous IBA concentrations (0, 1000, 2000, 5000 and 10,000mg/l) were tested at the subplot level. At each level, treatments were assigned at random to experimental units so as to have 3 blocks×3 levels of shading×5 IBA concentrations×20 cuttings. After 16weeks, cuttings were assessed for mortality and rooting. Results showed that stockplants grown under 50% and 75% shade resulted in a subsequent significantly lower cutting's mortality rate as well as significantly higher percentage of rooted cuttings and number of roots per rooted cutting, compared to stockplants grown under 0% shade. Although IBA concentration had no effect on mortality rate, it significantly affected rooting ability. Considering all levels of shading together, the percentage of rooted cuttings as well as the number of roots per rooted cutting increased with exogenous IBA concentration and reached their maximum values (100% and 6 respectively) at 10000mg/l IBA. With stockplants grown under high irradiance (0% shade), the maximum rooting percentage (100%) was obtained when cuttings were treated with 10,000mg/l IBA, whereas with stockplants grown under shade (50% and 75% shade) the IBA concentration necessary to achieve maximum rooting was reduced to 5000mg/l. These results indicate that stockplants shading increases the subsequent rooting ability as well as sensitivity to IBA of stem cuttings. This is valuable information to optimize the clonal propagation protocol for C. anomala and other species of the humid tropics of West and Central Africa, especially those considered to be difficult to propagate.

Expression Profiling of Strawberry Allergen Fra a during Fruit Ripening Controlled by Exogenous Auxin.

Strawberry fruit contain the allergenic Fra a proteins, members of the pathogenesis-related 10 protein family that causes oral allergic syndrome symptoms. Fra a proteins are involved in the flavonoid biosynthesis pathway, which might be important for color development in fruits. Auxin is an important plant hormone in strawberry fruit that controls fruit fleshiness and ripening. In this study, we treated strawberry fruits with exogenous auxin or auxin inhibitors at pre- and post-harvest stages, and analyzed Fra a transcriptional and translational expression levels during fruit development by real-time PCR and immunoblotting. Pre-harvest treatment with 1-naphthaleneacetic acid (NAA) alone did not affect Fra a expression, but applied in conjunction with achene removal NAA promoted fruit pigmentation and Fra a protein accumulation. The response was developmental stage-specific: Fra a 1 was highly expressed in immature fruit, whereas Fra a 2 was expressed in young to ripe fruit. In post-harvest treatments, auxin did not contribute to Fra a induction. Auxin inhibitors delayed fruit ripening; as a result, they seemed to influence Fra a 1 expression. Thus, Fra a expression was not directly regulated by auxin, but might be associated with the ripening process and/or external factors in a paralog-specific manner.

Type-B ARABIDOPSIS RESPONSE REGULATORs Specify the Shoot Stem Cell Niche by Dual Regulation of WUSCHEL.

Plants are known for their capacity to regenerate the whole body through de novo formation of apical meristems from a mass of proliferating cells named callus. Exogenous cytokinin and auxin determine cell fate for the establishment of the stem cell niche, which is the vital step of shoot regeneration, but the underlying mechanisms remain unclear. Here, we show that type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), critical components of cytokinin signaling, activate the transcription of WUSCHEL (WUS), which encodes a key regulator for maintaining stem cells. In parallel, type-B ARRs inhibit auxin accumulation by repressing the expression of YUCCAs, which encode a key enzyme for auxin biosynthesis, indirectly promoting WUS induction. Both pathways are essential for de novo regeneration of the shoot stem cell niche. In addition, the dual regulation of type-B ARRs on WUS transcription is required for the maintenance of the shoot apical meristem in planta. Thus, our results reveal a long-standing missing link between cytokinin signaling and WUS regulator, and the findings provide critical information for understanding cell fate specification.

The role of expansin genes PtrEXPA3 and PnEXPA3 in the regulation of leaf growth in poplar

The genes of α-expansins of woody plants are of great interest for genetic engineering, since they can potentially be used to improve the tree growth parameters. In the flora of Russia, model woody plants for plant biotechnology are aspen (Populus tremula L.) and black poplar (Populus nigra L.). The objective of this study was to determine the role of α-expansin-encoding genes, aspen PtrEXPA3 and black poplar PnEXPA3, in the regulation and maintenance of woody plant growth. To achieve this goal, the PtrEXPA3 expression level were determined upon exogenous phytohormone treatment, the action of stress factors, and constitutive expression of the PnARGOS-LIKE gene. In addition, transgenic aspen plants with constitutive expression of the black poplar PnEXPA3 gene were generated, and their morphological analysis was carried out. The highest PtrEXPA3 mRNA level was detected in young intensely growing aspen leaves, and furthermore, expression of the gene was induced by exogenous cytokinins and auxins. In response to NaCl and constitutive expression of the PnARGOS-LIKE gene, the PtrEXPA3 mRNA level decreased. Transgenic aspen plants with constitutive PnEXPA3 expression were characterized by the decreased size of leaves, petioles, and internodes, as well as the increased size of leaf epidermal cells, while the stem size remained unchanged. Taken together, the data obtained enable the suggestion that the PtrEXPA3 and PnEXPA3 genes encode cytokinin- and auxin-regulated, leaf-specific expansins that are involved in the cell expansion.

Auxin, Cytokinin, and Ethylene Involved in Rice N Availability Improvement Caused by Endophyte Phomopsis liquidambari

Nitrogen (N) is one of the most limiting nutrients for rice yield. There is mounting evidence that the endophytic fungus Phomopsis liquidambari B3 can establish a mutualistic symbiotic relationship with rice, enhancing N uptake and metabolism in rice (Oryza sativa L.). To examine the mechanism underlying the effect of B3 on nitrogen accumulation and metabolism in rice plants, a pot experiment was conducted to examine the N and phytohormone levels in response to endophyte infection at four whole growth durations during exposure to different N levels. Our results showed that the contents of auxin, cytokinin, and ethylene in rice were significantly enhanced by B3 under low N levels at different growth durations; B3 symbiosis increased N accumulation and rice yield and induced the expression of some genes related to N uptake and metabolism. To further verify that B3 symbiosis enhances N use in rice by regulating phytohormones, we performed a hydroponic experiment in which exogenous phytohormones and their specific inhibitors were applied. The results showed that the application of exogenous auxin, cytokinin, and ethylene increased the rice content of nitrogen, and their inhibitors decreased the amount of nitrogen absorbed in rice. As expected, B3 infection alleviated the negative effect caused by inhibitors slightly. In summary, we conclude that P. liquidambari symbiosis may regulate the content of auxin, cytokinin, and ethylene to improve N use in rice.

Exogenous auxin improves root morphology and restores growth of grafted cucumber seedlings

The aim of this study was to investigate the effects of exogenously applied auxin over the rootstock cuttings on root morphology parameters and stand establishment rate of salt-stressed cutting grafted cucumber seedlings. For that purpose, before grafting, the cut ends of the rootstocks were soaked for few seconds into auxin solution (indole-3-acetic acid (IAA) or indole-3- butyric acid (IBA))at different concentrations and afterwards were grafted by the root pruned splice grafting (RPSG) method. Ten days after grafting, the grafted seedlings were transplanted into individual pots where two different levels of salt-stress (0 and 50 mM) were established. Root morphology parameters, as well as dry matter of the root system and the whole plants were successively analysed to randomly selected plants. Exogenous auxins improved root morphology parameters and restored root growth under salinity conditions. The best results were obtained through the application of IBA, which promoted a better rootstock – scion relationship, presumably due to faster development of phloem and xylem tubes, and the promotion of a vigorous root system which increases plant’s absorbing capabilities for water and nutrients under adverse soil conditions.

Phototropic bending of intact and wounded potato shoots

Intact and wounded shoots of in vitro cultured potato were investigated in situ to determine how their phototropic (PT) bending capacity was affected by water availability and exogenous auxin supplementation. Wounding strongly decreased PT bending but it recovered upon supplementation of water, auxin or both. Intact shoots required neither water nor auxin, while shoot segments required both. Shoot cuttings required only water, while, like shoot segments, decapitated shoots had a strong requirement for auxin. Water supplementation was beneficial in all treatments and PT bending was not affected in cultures that were submerged in water for a short period. Sucrose and inorganic salts present in the medium strongly affected PT bending capacity of cultures, favouring combinations with lower concentrations of both. Sucrose alone strongly promoted PT bending up to a concentration of 5%. Osmotic shock induced by the addition of small volumes of highly concentrated carbohydrate solutions (sucrose, glucose or sorbitol) induced a rapid but transient decline in PT bending capacity. These results indicate that water availability is a major factor that affects PT bending in potato plantlets.

Global transcriptome profiling analysis of ethylene-auxin interaction during tomato fruit ripening

Auxin-ethylene interactions are crucial for fruit ripening processes. However, the molecular basis of the regulatory network of auxin-ethylene interaction during ripening is still not very clear. To reveal the potential molecular mechanism of ethylene-auxin interplay in tomato (Solanum lycopersicum L.) fruit ripening, global transcriptome profiling analysis was performed on cherry tomato fruit treated with auxin, ethylene or the combination of the two hormones. The results showed that ethylene modulated auxin transport, metabolism and signaling processes by affecting the expression patterns of genes encoding auxin carrier proteins, aldehyde dehydrogenase and primary auxin-responsive proteins. Most genes involved in ethylene biosynthesis and signaling were regulated 7days after treatment with exogenous auxin. Furthermore, the expression levels of mitogen-activated protein kinase (MAPK) and of ubiquitination-related genes were altered in auxin-treated fruit, suggesting that auxin regulates ethylene metabolism and signaling via complicated mechanisms. The potential interaction points in auxin-ethylene crosstalk were also identified and a model was proposed. Our analyses provide a global insight into the ethylene-auxin interaction and predict the potential regulators in the crosstalk of the two hormones during the fruit ripening process.

Shoot stem cell specification in roots by the WUSCHEL transcription factor.

The WUSCHEL homeobox transcription factor is required to specify stem-cell identity at the shoot apical meristem and its ectopic expression is sufficient to induce de novo shoot meristem formation. Yet, the manner by which WUS promotes stem-cell fate is not yet fully understood. In the present research we address this question by inducing WUS function outside of its domain. We show that activation of WUS function in the root inhibits the responses to exogenous auxin and suppresses the initiation and growth of lateral roots. Using time lapse movies to follow the cell-cycle marker CYCB1;1::GFP, we also show that activation of WUS function suppresses cell division and cell elongation. In addition, activation of WUS represses the auxin-induced expression of the PLETHORA1 root identity gene and promotes shoot fate. Shoot apical meristem formation requires a high cytokinin-to-auxin ratio. Our findings provide evidence for the manner by which WUS specifies stem-cell identity: by affecting auxin responses, by reducing the cell mitotic activity and by repressing other developmental pathways. At the meristem, the stem-cells which are characterized by low division rate are surrounded by the highly proliferative meristematic cells. Our results also provide a model for WUS establishing the differential mitotic rates between two cell populations at the minute structure of the meristem.

The Physcomitrella patens exocyst subunit EXO70.3d has distinct roles in growth and development, and is essential for completion of the moss life cycle.

The exocyst, an evolutionarily conserved secretory vesicle-tethering complex, spatially controls exocytosis and membrane turnover in fungi, metazoans and plants. The exocyst subunit EXO70 exists in multiple paralogs in land plants, forming three conserved clades with assumed distinct roles. Here we report functional analysis of the first moss exocyst subunit to be studied, Physcomitrella patens PpEXO70.3d (Pp1s97_91V6), from the, as yet, poorly characterized EXO70.3 clade. Following phylogenetic analysis to confirm the presence of three ancestral land plant EXO70 clades outside angiosperms, we prepared and phenotypically characterized loss-of-function Ppexo70.3d mutants and localized PpEXO70.3d invivo using green fluorescent protein-tagged protein expression. Disruption of PpEXO70.3d caused pleiotropic cell elongation and differentiation defects in protonemata, altered response towards exogenous auxin, increased endogenous IAA concentrations, along with defects in bud and gametophore development. During mid-archegonia development, an abnormal egg cell is formed and subsequently collapses, resulting in mutant sterility. Mutants exhibited altered cell wall and cuticle deposition, as well as compromised cytokinesis, consistent with the protein localization to the cell plate. Despite some functional redundancy allowing survival of moss lacking PpEXO70.3d, this subunit has an essential role in the moss life cycle, indicating sub-functionalization within the moss EXO70 family.

Genome-wide identification and expression analysis of ClLAX, ClPIN and ClABCB genes families in Citrullus lanatus under various abiotic stresses and grafting

BackgroundAuxin plays an important role in regulating plant growth and development as well as in the response of plants to abiotic stresses. Auxin is transported by three kinds of major protein families, including the AUXIN RESISTANT 1/LIKE AUX1 (AUX⁄LAX) influx carriers, the PIN-FORMED (PIN) efflux carriers and the ATP binding cassette B/P-glycoprotein/Multidrug-resistance (ABCB/MDR/PGP) efflux/condition carriers. The biological function of several auxin transporter genes has been well characterized in Arabidopsis thaliana. However, their function in response to exogenous auxin and abiotic stresses in watermelon (Citrullus lanatus. L) remained unknown.ResultsHere, the latest updated watermelon genome was used to characterise the ClLAX, ClPIN and ClABCB family genes from watermelon. The genome-wide analysis of the ClLAX, ClPIN and ClABCB family genes, including chromosome localisation, gene structure, and phylogenic relationships, was carried out. Seven ClLAXs, 11 ClPINs and 15 ClABCBs were mapped on 10 watermelon chromosomes. The expression profiles of the ClLAX, ClPIN and ClABCB genes under exogenous indole-3-acetic acid and various abiotic stresses (salt, drought, and cold stresses) treatments were performed by quantitative real-time PCR (qRT-PCR). The transcriptional level of majority ClLAX, ClPIN and ClABCB genes were changed by abiotic stresses in both shoots and roots. We also analysed the expression levels of ClLAX, ClPIN and ClABCB genes in graft response.ConclusionAnalysis of the expression patterns of ClLAX, ClPIN and ClABCB genes under salt, drought, cold treatment and grafting response helps us to understand the possible roles of auxin transporter genes in watermelon adaptation to environmental stresses.

The small auxin-up RNA OsSAUR45 affects auxin synthesis and transport in rice.

This research is the first to demonstrate that OsSAUR45 is involved in plant growth though affecting auxin synthesis and transport by repressing OsYUCCA and OsPIN gene expression in rice. Small auxin-up RNAs (SAURs) comprise a large multigene family and are rapidly activated as part of the primary auxin response in plants. However, little is known about the role of SAURs in plant growth and development, especially in monocots. Here, we report the biological function of OsSAUR45 in the model plant rice (Oryza sativa). OsSAUR45 is expressed in a tissue-specific pattern and is localized to the cytoplasm. Rice lines overexpressing OsSAUR45 displayed pleiotropic developmental defects including reduced plant height and primary root length, fewer adventitious roots, narrower leaves, and reduced seed setting. Auxin levels and transport were reduced in the OsSAUR45 overexpression lines, potentially because of decreased expression of Flavin-binding monooxygenase family proteins (OsYUCCAs) and PIN-FORMED family proteins (OsPINs). Exogenous auxin application rapidly induced OsSAUR45 expression and partially restored the phenotype of rice lines overexpressing OsSAUR45. These results demonstrate that OsSAUR45 is involved in plant growth by affecting auxin synthesis and transport through the repression of OsYUCCA and OsPIN gene expression in rice.

Effects of exogenous auxin on pigments and primary metabolite profile of postharvest tomato fruit during ripening

Auxin is an important plant hormone and plays crucial roles in regulating fruit ripening. The delay of ripening after auxin treatment has been found in tomato and other fleshy fruit. However, the influence of auxin on metabolites alteration during tomato ripening period has not been extensively studied. To investigate the impact of exogenous auxin on tomato fruit quality, pigment metabolism, primary metabolite profiling, and the expression of selected ripening-related transcription factor genes were analyzed. The results showed that exogenous auxin significantly interfered the accumulation and conversion of pigments, total phenolics and flavonoids but did not largely influence the final content of these compounds in full ripe tomato fruit. Dramatic changes on the content of primary metabolites were induced by auxin during tomato ripening period and the alterations were not able to be completely restored at the end of ripening. The contents of citric acid, threonic acid and succinic acid were increased whereas alanine and aspartic acid accumulation was repressed in auxin-treated fruit. The expression patterns of transcription factor genes related to ripening were also disturbed by exogenous auxin. The present study provided an overall insight on how auxin regulates pigment and primary metabolite accumulation during ripening stage and offered useful information for further investigation of auxin impact on fruit quality.

Genome-wide analysis of auxin transport genes identifies the hormone responsive patterns associated with leafy head formation in Chinese cabbage

Auxin resistant 1/like aux1 (AUX/LAX), pin-formed (PIN) and ATP binding cassette subfamily B (ABCB/MDR/PGP) are three families of auxin transport genes. The development-related functions of the influx and efflux carriers have been well studied and characterized in model plants. However, there is scant information regarding the functions of auxin genes in Chinese cabbage and the responses of exogenous polar auxin transport inhibitors (PATIs). We conducted a whole-genome annotation and a bioinformatics analysis of BrAUX/LAX, BrPIN, and BrPGP genes in Chinese cabbage. By analyzing the expression patterns at several developmental stages in the formation of heading leaves, we found that most auxin-associate genes were expressed throughout the entire process of leafy head formation, suggesting that these genes played important roles in the development of heads. UPLC was used to detect the distinct and uneven distribution of auxin in various segments of the leafy head and in response to PATI treatment, indicated that the formation of the leafy head depends on polar auxin transport and the uneven distribution of auxin in leaves. This study provides new insight into auxin polar transporters and the possible roles of the BrLAX, BrPIN and BrPGP genes in leafy head formation in Chinese cabbage.

Effect of modeled microgravity on radiation-induced adaptive response of root growth in Arabidopsis thaliana

Space particles have an inevitable impact on organisms during space missions; radio-adaptive response (RAR) is a critical radiation effect due to both low-dose background and sudden high-dose radiation exposure during solar storms. Although it is relevant to consider RAR within the context of microgravity, another major space environmental factor, there is no existing evidence as to its effects on RAR. In the present study, we established an experimental method for detecting the effects of gamma-irradiation on the primary root growth of Arabidopsis thaliana, in which RAR of root growth was significantly induced by several dose combinations. Microgravity was simulated using a two-dimensional rotation clinostat. It was shown that RAR of root growth was significantly inhibited under the modeled microgravity condition, and was absent in pgm-1 plants that had impaired gravity sensing in root tips. These results suggest that RAR could be modulated in microgravity. Time course analysis showed that microgravity affected either the development of radio-resistance induced by priming irradiation, or the responses of plants to challenging irradiation. After treatment with the modeled microgravity, attenuation in priming irradiation-induced expressions of DNA repair genes (AtKu70 and AtRAD54), and reduced DNA repair efficiency in response to challenging irradiation were observed. In plant roots, the polar transportation of the phytohormone auxin is regulated by gravity, and treatment with an exogenous auxin (indole-3-acetic acid) prevented the induction of RAR of root growth, suggesting that auxin might play a regulatory role in the interaction between microgravity and RAR of root growth.

The Use of Auxin Quantification for Understanding Clonal Tree Propagation

Qualitative and quantitative hormone analyses have been essential for understanding the metabolic, physiological, and morphological processes that are influenced by plant hormones. Auxins are key hormones in the control of many aspects of plant growth and development and their endogenous levels are considered critical in the process of adventitious root induction. Exogenous auxins are used extensively in the clonal propagation of tree species by cuttings or tissue culture. Understanding of auxin effects has advanced with the development of increasingly accurate methods for auxin quantification. However, auxin analysis has been challenging because auxins typically occur at low concentrations, while compounds that interfere with their detection often occur at high concentrations, in plant tissues. Interference from other compounds has been addressed by extensive purification of plant extracts prior to auxin analysis, although this means that quantification methods have been limited by their expense. This review explores the extraction, purification, and quantification of auxins and the application of these techniques in developing improved methods for the clonal propagation of forestry trees.

Auxin synthesis gene tms1 driven by tuber-specific promoter alters hormonal status of transgenic potato plants and their responses to exogenous phytohormones

Ectopic auxin overproduction in transgenic potato leads to enhanced productivity accompanied with concerted and occasional changes in hormonal status, and causing altered response of transformants to exogenous auxin or cytokinin. Previously, we generated potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 driven by tuber-specific patatin gene promoter (B33-promoter). Here, we studied the endogenous hormonal status and the response to exogenous phytohormones in tms1 transformants cultured in vitro. Adding indole-3-acetic acid (IAA) or kinetin to culture medium affected differently tuberization of tms1-transformed and control plants, depending also on sucrose content in the medium. Exogenous phytohormones ceased to stimulate the tuber initiation in transformants at high (5-8%) sucrose concentration, while in control plants the stimulation was observed in all experimental settings. Furthermore, exogenous auxin partly inhibited the tuber initiation, and exogenous cytokinin reduced the average tuber weight in most transformants at high sucrose content. The elevated auxin level in tubers of the transformants was accompanied with a decrease in content of cytokinin bases and their ribosides in tubers and most shoots. No concerted changes in contents of abscisic, jasmonic, salicylic acids and gibberellins in tubers were detected. The data on hormonal status indicated that the enhanced productivity of tms1 transformants was due to auxin and not mediated by other phytohormones. In addition, exogenous cytokinin was shown to upregulate the expression of genes encoding orthologs of auxin receptors. Overall, the results showed that tms1 expression and local increase in IAA level in transformants affect both the balance of endogenous cytokinins and the dynamics of tuberization in response to exogenous hormones (auxin, cytokinin), the latter reaction depending also on the carbohydrate supply. We introduce a basic model for the hormonal network controlling tuberization.

МОРФОГЕНЕЗ ЯРОВОЙ ТВЕРДОЙ ПШЕНИЦЫ В КУЛЬТУРЕ ЗРЕЛЫХ ЗАРОДЫШЕЙ

<p>Mature wheat embryo is a convenient type of explants because of its unlimited availability at any time of the year. But the regenerative capacity of the calli derived from mature embryos is low due to the peculiarities of their hormonal status. A high-performance protocol for culturing these explants is necessary to develop to use them in various areas of applied plant biotechnology. Induction and maintenance of a high rate for unorganized growth in plant cell cultures take place on a nutrient medium with high levels of an exogenous auxin, but the presence of a cytokinin is required to induce differentiation processes. We have carried out a study of the various morphogenetic processes in mature embryo cultures of three spring durum wheat genotypes, depending on the time of their cultivation on the callus induction medium. Mature embryos were cultured in the dark at 26 ± 1 °Con Murashige & Skoog (MS) medium containing MS basal salts and vitamins supplemented with 0.7% agar, 3% sucrose, as well as 2 mg L<sup>-1</sup> 2,4-dichlorophenoxyacetic acid (2,4-D) (callus induction medium). For morphogenesis induction a part of calli was transferred every five days to a differentiating medium of the same composition of salts and vitamins supplemented with 0.5 mg L<sup>-1</sup> 2,4-D and 0.5 mg L<sup>-1</sup> kinetin. Cell cultures were grown in the light at 22 – 24 °C with a 16-hour photoperiod. Six variants of time intervals for callus proliferation on the induction medium have been studied (5, 10, 15, 20, 25, 30 days). A variant of cell culturegrowing without transferring to the differentiating medium was examined too. Frequencies of callus induction, morphogenesis induction and regeneration capacity (relatively morphogenetic calli) were calculated.<strong></strong></p><p>We found active callus induction was visible on the 5th – 7th day after placing explants on the MS inducing medium. The greatest level of callusogenesis (92.3%) was discovered under incubating cultures on the original medium for 30 days. After the short-term cultivation of explants on the initiating medium (for five days) new calli on the differentiating medium were not initiated. In this variant, proliferation of the before induced cell clusters was taking place. This resulted in a low frequency of callus formation (44.3%). Development of the primary callus on the inducing medium for 20 – 30 days helped to keep the competence in somatic tissues of mature embryos and generated the largest number of morphogenetic structures of different qualities. The way of morphogenesis depended on the time interval for cell culture growing on the initial medium. Rhizogenesis decreased by 25% after increasing the incubation period to 15 days. This was followed by active nodular structure formation in calli and plant regeneration. For Oasis variety and 12S2-24 line the most effective variant for the realization of regenerative capacity of morphogenetic calli was to incubate cultures on the induction medium for 15 – 20 days and then to transfer them to the differentiating medium. For Pamyati Yanchenko variety the best variant was to grow calli on the induction medium for 25 days. We have shown the significant effects of a genotype and cultivation conditions at different developmental stages of mature embryo cultures from durum wheat. The specificity of a variety began to manifest after 5 – 10 days staying on the induction medium.</p>