Why don’t crop plants grow as fast as they should? In optimal conditions, elite crop varieties routinely outperform those grown in the average field. The vast majority of this reduction in growth activity is due to abiotic stresses such as drought, heat, and nutrient limitation. Abiotic stress reduces plant growth by triggering a reduction of meristem size and causing premature differentiation of proliferating cells. Differentiated cells are no longer able to divide, and smaller meristems have a reduced capacity to restore growth when the abiotic stress passes. We have designed and evaluated a novel high-throughput screening system to identify compounds able to reduce or prevent this premature differentiation in order to retain modest growth capacity in stressful conditions and enable rapid recovery from stress. Such chemicals can be applied to crop plants using existing agricultural methods, and because there is no need for genetic modification, it is widely applicable to many different crop species. Using the novel technique of flow sorting followed by protoplast culture, we have developed a high-throughput automated confocal imaging method to screen chemicals for their effects upon cell differentiation. Meristem protoplasts isolated from the root tips of pROW1:GFP Arabidopsis plants were monitored for differentiation when exposed to different chemicals. To evaluate this system, a library of biologically active small molecules provided by Syngenta was screened against protoplasts and whole plants. Several compounds were identified with the ability to improve Arabidopsis root growth in in vitro growth conditions. Two subsets of these chemicals were identified: a subset of chemicals that improved stress tolerance through modulation of post-meristem differentiation, and a subset of chemicals that improve growth rate by increasing rates of cell division in the root apical meristem. This screening system is able to detect the subset of chemicals that was shown to affect postmeristem differentiation, but not the other subset. No false positives were detected. These results suggest that this single-cell screening system is a powerful, high-throughput method suitable for the detection of molecules for use in crop protection.

A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro é a base para a utilização da biotecnologia no melhoramento. Realizaram-se estudos com cinco cultivares de laranja doce (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin e Westin. Este trabalho objetivou a avaliação da eficiência de isolamento de protoplastos das cultivares de laranja doce; o estudo da eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes meios de cultura e avaliação da embriogênese somática em função da composição dos meios de cultura e concentração da fonte de carboidrato. As soluções enzimáticas testadas para o isolamento de protoplastos foram: 1. Grosser e Chandler (1987), composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R- 10 (Yakult Honsha) e 0,2% de pectoliase Y-23 (Seishin); 2. Grosser e Chandler (1987) modificado, composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha); 3. Solução enzimática composta de 4% de celulase Onozuka R-10 (Yakult), 1% macerase R-10. O plaqueamento dos protoplastos foi realizado em cinco densidades, 2 x 104, 5 x 104; 105; 2x 105 e 3 x 105 protoplastos . mL-1,nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou um maior rendimento no isolamento de protoplastos das cultivares Hamlin, Natal e Pera e solução enzimática 1 foi melhor para a cultivar Westin. A eficiência final de plaqueamento avaliada aos 90 dias foi superior nas densidades de de 3 x 105 e 2 x 105 protoplastos. mL-1 para as cultivares Hamlin, Natal e Lima Verde, e na densidade de 2 x 105 e 105 protoplastos. mL-1 para a cultivar Westin. A indução da embriogênese somática ocorreu em meio de cultura MT modificado com 500 mg.L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose e maltose, nas concentrações de 18, 37, 75, 110 e 150 mM à temperatura de 27 °C. A formação de embriões somáticos variou com o genótipo, sendo a cultivar Lima Verde e Westin apresentaram menor número de embriões somáticos. As melhores fontes de carboidratos foram a maltose, seguida pela lactose nas concentrações de 37 e 75 mM para a cultivar Pêra, 37 mM para a cultivar Natal e 37, 75 e 110 mM para a cultivar Hamlin. / Plant regeneration, by organogenesis or somatic embryogenesis from cell cultures and in vitro plant tissue culture is the basis for the use of biotechnology in plant breeding. Studies were conducted with five cultivars of sweet orange (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin and Westin. This work aimed to evaluate the isolation efficiency of protoplasts, to evaluate platting efficiency of protoplasts based on five densities of cells and different culture media and to evaluate somatic embryogenesis based on culture medium composition and concentration. The enzymatic solutions tested were: 1. Grosser and Chandler (1987): 1% de cellulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha) and 0,2% de pectoliase Y-23 (Seishin); 2. Grosser and Chandler (1987) modified: 1% de cellulase Onozuka RS (Yakult) and 1% macerase R-10 (Yakult Honsha); 3. Enzimatic solution containing 4% cellulase Onozuka R-10 (Yakult) and 1% macerase R-10. Protoplasts were cultured at densities of 2 x 104; 5 x 104; 105; 2 x 105 e 3 x 105 protoplasts.mL-1 in EME 0,7M, BH3 0,7M and BH3 + EME 0,7M, in the dark, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for the cultivars Hamlin, Natal and Pêra, and enzymatic solution 1 resulted in better protoplast isolation for cultivar Westin. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 105 e 2 x 105 protoplasts.mL-1 for Hamlin, Natal and Lima Verde, and at the density of 2 x 105 e 105 protoplasts.mL-1 for Westin. Somatic embryogenesis stimulation occurred in cultured medium MT (MURASHIGE AND TUCKER, 1969) modified with 500 mg. L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose and sorbitol at concentrations of 18, 37, 75, 110 and 150 mM, at 27 ± 1 ºC. Somatic embryos produced varied with the genotype, the smaller number of somatic embryos was observed in cultivars Lima Verde and Westin. The best source of carbohydrate were maltose, followed by lactose at concentrations of 37 and 75 mM for cultivar Pêra, 37 mM for cultivar Natal, and 37, 75 and 110 mM for cultivar Hamlin.

A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro é a base para a utilização da biotecnologia no melhoramento. Realizaram-se estudos com cinco cultivares de laranja doce (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin e Westin. Este trabalho objetivou a avaliação da eficiência de isolamento de protoplastos das cultivares de laranja doce; o estudo da eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes meios de cultura e avaliação da embriogênese somática em função da composição dos meios de cultura e concentração da fonte de carboidrato. As soluções enzimáticas testadas para o isolamento de protoplastos foram: 1. Grosser e Chandler (1987), composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R- 10 (Yakult Honsha) e 0,2% de pectoliase Y-23 (Seishin); 2. Grosser e Chandler (1987) modificado, composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha); 3. Solução enzimática composta de 4% de celulase Onozuka R-10 (Yakult), 1% macerase R-10. O plaqueamento dos protoplastos foi realizado em cinco densidades, 2 x 104, 5 x 104; 105; 2x 105 e 3 x 105 protoplastos . mL-1,nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou um maior rendimento no isolamento de protoplastos das cultivares Hamlin, Natal e Pera e solução enzimática 1 foi melhor para a cultivar Westin. A eficiência final de plaqueamento avaliada aos 90 dias foi superior nas densidades de de 3 x 105 e 2 x 105 protoplastos. mL-1 para as cultivares Hamlin, Natal e Lima Verde, e na densidade de 2 x 105 e 105 protoplastos. mL-1 para a cultivar Westin. A indução da embriogênese somática ocorreu em meio de cultura MT modificado com 500 mg.L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose e maltose, nas concentrações de 18, 37, 75, 110 e 150 mM à temperatura de 27 °C. A formação de embriões somáticos variou com o genótipo, sendo a cultivar Lima Verde e Westin apresentaram menor número de embriões somáticos. As melhores fontes de carboidratos foram a maltose, seguida pela lactose nas concentrações de 37 e 75 mM para a cultivar Pêra, 37 mM para a cultivar Natal e 37, 75 e 110 mM para a cultivar Hamlin. / Plant regeneration, by organogenesis or somatic embryogenesis from cell cultures and in vitro plant tissue culture is the basis for the use of biotechnology in plant breeding. Studies were conducted with five cultivars of sweet orange (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin and Westin. This work aimed to evaluate the isolation efficiency of protoplasts, to evaluate platting efficiency of protoplasts based on five densities of cells and different culture media and to evaluate somatic embryogenesis based on culture medium composition and concentration. The enzymatic solutions tested were: 1. Grosser and Chandler (1987): 1% de cellulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha) and 0,2% de pectoliase Y-23 (Seishin); 2. Grosser and Chandler (1987) modified: 1% de cellulase Onozuka RS (Yakult) and 1% macerase R-10 (Yakult Honsha); 3. Enzimatic solution containing 4% cellulase Onozuka R-10 (Yakult) and 1% macerase R-10. Protoplasts were cultured at densities of 2 x 104; 5 x 104; 105; 2 x 105 e 3 x 105 protoplasts.mL-1 in EME 0,7M, BH3 0,7M and BH3 + EME 0,7M, in the dark, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for the cultivars Hamlin, Natal and Pêra, and enzymatic solution 1 resulted in better protoplast isolation for cultivar Westin. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 105 e 2 x 105 protoplasts.mL-1 for Hamlin, Natal and Lima Verde, and at the density of 2 x 105 e 105 protoplasts.mL-1 for Westin. Somatic embryogenesis stimulation occurred in cultured medium MT (MURASHIGE AND TUCKER, 1969) modified with 500 mg. L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose and sorbitol at concentrations of 18, 37, 75, 110 and 150 mM, at 27 ± 1 ºC. Somatic embryos produced varied with the genotype, the smaller number of somatic embryos was observed in cultivars Lima Verde and Westin. The best source of carbohydrate were maltose, followed by lactose at concentrations of 37 and 75 mM for cultivar Pêra, 37 mM for cultivar Natal, and 37, 75 and 110 mM for cultivar Hamlin.

The major objective of this study was to produce synthetic genotypes of Brassica juncea from its parental species <i> B. rapa </i> and <i> B. nigra </i> via somatic and sexual hybridization. As prerequisites for somatic hybridization experiments, methods were developed to improve the culture of mesophyll and hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i>, to obtain reliable plant regeneration from mesophyll protoplast cultures of <i> B. nigra </i>, and to fuse protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. A modified Kao's medium (1977), was found suitable for the culture of mesophyll protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. At a density of approximately $110\sp5$ protoplasts/ml within a culture plate insert surrounded by culture medium, mesophyll protoplast cultures of <i> B. nigra </i> accessions R890, R1819, R3392 and U1218 and <i> B. rapa </i> cvs. R500 and Wong Bok formed colonies. Genotypic differences in cell division and colony formation were observed. Hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i> were successfully isolated from 6 day-old seedlings cultured in a modified Kao's medium (1977). With <i> B. nigra </i> accession R890 and <i> B. rapa </i> cv. R500, cell division and colony formation were optimal when hypocotyl protoplasts were cultured at a density of 0.5 to $1.010\sp5$ protoplasts/ml within a culture plate insert surrounded by a nurse culture of 4 to 6 day-old mesophyll protoplasts of <i> B. nigra </i>. Plant regeneration was obtained from mesophyll protoplast-derived calli of <i> B. nigra </i> accession R890 originally cultured in inserts; a shoot regeneration frequency of 8.1% was obtained on a medium containing the salts and vitamins of medium K3 (Nagy and Maliga 1976) with 3 g/l sucrose, 18.2 g/l mannitol, 2 mg/l ZR, 0.1 mg/l NAA, 10 g/l agarose, pH 5.6. For somatic hybridizatian studies, methods were developed to select out parental protoplasts using iodoacetic acid and to efficiently fuse protoplasts on the bottom of a petri dish using PEG. Twenty-nine plants were recovered from fusion experiments between mesophyll protoplasts of <i> B. nigra </i> accession R890 and hypocotyl protoplasts of <i> B. rapa </i> cv. Tobin. The somatic hybrid plants resembled natural <i> B. juncea </i>, had $2n=36$ chromosomes and had pollen viabilities ranging from 30 to 45%. Twenty-one plants, derived from one callus colony, possessed the mitochondrial and chloroplast genomes of <i> B. rapa </i>, as found in natural <i> B. juncea </i>. Eight plants, derived from another callus, had a novel cytoplasmic combination consisting of the mitochondrial genome of <i> B. rapa </i> and the chloroplast genome of <i> B. nigra </i>. Synthetic genotypes of <i> B. juncea </i> were also produced from reciprocal sexual crosses between <i> B. rapa </i> and <i> B. nigra </i>. Seventy-eight interspecific hybrid plants from the cross <i> B. rapa </i> x <i> B. nigra </i> and six hybrid plants from the reciprocal cross were identified by their morphology, pollen viability and chromosome number. The colchicine-induced allotetraploids resembled natural <i> B. juncea </i> in morphology, had 18 bivalents at metaphase I, and had between 35 and 70% pollen viability.

Universiẗat, Diss.--Halle, 1994.

Contamination of soils with heavy metals is one of the serious environmental problems threatening human being. Heavy metals are considered as the special hazard of soil pollutants because of the adverse effects on the plant growth, the amount, activity of useful microorganisms in soils and the quality of food. Regard to the persistent and toxicity, the heavy metals are toxic when we consider different kinds of pollutants in soils. In the soil, zinc (Zn), cadmium (Cd), lead (Pb) and mercury (Hg) toxicities frequently occur than the other metals because of their precipitation and sorption by the soil. It is a very dangerous situation because when these metals are taken up by plants, they can be transported to the food web and food chains. In the present study, the accumulation of four heavy metals (mercury, zinc, lead and cadmium) in the whole grain of spring accessions of emmer, einkorn and common spring wheat cultivars and potato (Solanum tuberosum) is reported. Heavy and essential elements were monitored in potato cultivars in the exact field experiments and in hydroponically grown plants. The elements were determined by methods FAAS, ET AAS, and AMA (Advance Mercury Analysis). Statistical analyses were performed using SPSS 9.0 with the Tukey HSD (Honestly Significant Difference) test (alpha equal to 0.05). In our study, the concentration of heavy metals decreased in the order zinc (Zn) > lead (Pb) > cadmium (Cd) > mercury (Hg) in the wheat grain. The comparison between three varieties of investigated wheat revealed that the emmer wheat was rich in zinc content (62.12 mg kg-1 dry matter), while the spring wheat had the lowest average concentration of zinc in the grain (40.99 mg kg-1 dry matter). Generally, the values of lead concentration in grain wheat varieties were low (ranging from 0.1268 mg kg-1 dry matter to 0.2950 mg kg-1 dry matter). The concentrations of mercury in four typical growth stages of wheat (boot stage 10, heading stage 10.2 1/4 of head emerged, leaf-stage 10.2 and stage ripening 11 according to Feekes) were also determined. It has been shown that the concentrations of mercury in different wheat varieties were absorbed differently at different growth stages of plant. Stage 10.2 and leaf stage 10.2 showed the high mercury content (0.0152 mg kg-1 dry matter and 0.0214 mg kg-1 dry matter, respectively). Among individual varieties significant differences were determined. Amounts of toxic and potentially toxic elements detected in investigated potato tubers are characterized by a large variability within investigated groups. Performing statistical analysis (one way ANOVA) showed that there were no significant differences between two investigated groups of samples (samples from Uhříněves and Valečov in the year 2013 and 2014) considering either one of investigated metals. Measurable levels of mercury were found in smallest amounts in all investigated potato samples comparing to other metals (Cd, Pb). Plant cells compared to animal cells are characterized by the formation of cell walls. Plasma membrane or cell membrane is a biological active membrane separating the interior of cell from the outside environment. An adjusted method for isolation of protoplasts was developed and adapted for isolation of protoplasts from plant material (potatoes). In our experiment, the plants were grown hydroponically in the Research Institute of Plant Crops Prague-Ruzyně. If we examine the plant membrane, one option is to remove the cell wall by means of special mixture enzymes. Protoplasts were released in the dark at 25 degrees of Celsius for 18 hours. The 70 and 90 microns sieve was used to filter and then centrifugation for 5 minutes at 100 x g. All the steps were carefully carried out to prevent the damage or breakage of protoplasts.

Inbred lines for hybrid crop production have been a mainstay of plant breeding. Biotechnological approaches to hasten the process are available including anther culture to halve the genome and protoplast fusion to create hybrids between incompatible partners. We applied these techniques to potato to evaluate their potential for breeding highly heterozygous, cross-pollinating species. Four families of monoploids (2n=1x=12), developed from diploid hybrids with diverse genomic constitutions but heavily favoring Solanum phureja, a primitive cultivated potato, were used in electrofusion experiments to create intermonoploid somatic hybrids (SH). The "monoploid sieve" results in the survival of only those gametes free of lethal and deleterious genes but generates sterile sporophytes, necessitating protoplast fusion for SH development. From six intermonoploid electrofusion combinations, 276 plants were regenerated over 6-9 months. Fusion conditions were optimized. Ploidy was determined by flow-cytometry and SH confirmed by microsatellite analysis. Field evaluations over three years revealed that intermonoploid SH were inferior to cultivars. Dihaploids derived by anther culture of a tetraploid intermonoploid SH were reduced in vigor with an increase in homozygosity, while 2x X 2x sexually derived populations had better yield than the SH, suggesting that producing SH introduced or eliminated factors required for productivity. Molecular analysis of the SH was conducted to examine genomic stability through protoplast isolation and plant regeneration. Sequence specific amplified polymorphism (S-SAP) represents a hybrid system incorporating amplified fragment length polymorphism (AFLP) technology in conjunction with the use of a defined genomic sequence, e.g., retrotransposon display (RD) when the defined sequence is anchored into a consensus sequence of a retrotransposon such as the long terminal repeat (LTR) sequence of Tst1. Parental monoploids, SH and various Solanaceae were evaluated by RD. Fluorescently-labeled retrotransposon-based primers were used in the ALFexpress automated fragment analyzer system. Eleven probes from RD were created for Southern blot analysis and used to verify taxonomic relationships between selected Solanaceae. Blots of intermonoploid somatic hybrids confirmed hybridity and occasional loss of genomic fragments. No activation or replication of retrotransposons was detected. Sequencing of inter-retrotransposon amplified polymorphism (IRAP) and S-SAP fragments revealed that all fragments had the expected Tst1 retroelement and/or the AFLP adaptor sequence. BLAST analysis identified 4 of the 17 fragments sequenced as part of the chloroplast genome, a tobacco anther-specific gene, repetitive DNA, and the phytochrome F gene. / Ph. D.

Genetic improvement of the potato through classical breeding has been limited by its tetraploid nature, the narrow genetic variability within cultivars, and interploidy barriers between tetraploid cultivars and diploid germplasm. Breeding at reduced ploidy levels has been proposed as a solution to these problems. Because of sterilities, somatic hybridization via protoplast fusion has been considered an alternative to sexual polyploidization for resynthesizing superior diploids from selected monoploids, and tetraploids from selected diploids and dihaploids. Successful application of somatic hybridization largely depends upon protoplast culturability and regenerability of a plant. The ability of callus formation and plant regeneration from protoplasts varies among plants. To understand the genetic basis for this variation, the mode of inheritance for protoplast culturability, defined as the ability to develop calli from cultured protoplasts, was studied in the diploid potato species, Solanum phureja. Based upon data from F₂ as well as from F₁ and backcross progenies, it was found that protoplast culturability in this potato species was controlled by two unlinked loci with dominant effect. In addition, there was quantitative variation for protoplast plating efficiency among culturable genotypes. Male sterility in cultivars of Solanum tuberosum ssp. tuberosum results from nuclear-cytoplasmic interactions. 'Donor-recipient' protoplast fusion and regeneration were conducted between a sterile S. tuberosum ssp. tuberosum cultivar, Russet Burbank, and fertile selections of S. tuberosum ssp. andigena which have a non-sensitive cytoplasm and were used as the cytoplasmic donor. Sixteen regenerated plants possessed nuclear background and chloroplast DNA of Russet Burbank. However, two of these regenerants had improved pollen stainability. The possible causes for the improvement of pollen stainability are discussed. In the last chapter, allelic polymorphism in a monoploid population derived from anther culture of a clone of S. phureja was assessed by isozyme electrophoresis. Fourteen monoploids and their anther donor were examined for six enzymes. No allozyme variation was detected in these plants. However, genetic variability among these monoploids was manifested by variations in some growth characters and general morphology. The limitation of enzymatic markers in detecting allelic polymorphism in these monoploids is discussed. / Ph. D.

With the current state of climate change and world peak oil on the horizon, it is important to focus our research efforts on alternative sources of energy. Ethanol obtained from the digestion of biomass (bioethanol) and oil harvesting from algae (biodiesel) are two promising fields of study for transportation fuel production. However, in their current state of development, neither option is capable of reasonably replacing the transportation fuel demand for this country. The land demand needed is too large for either process to become a viable option, albeit the land demand for biodiesel is considerably smaller than that of bioethanol. Therefore, when moving forward with alternative transportation fuel, harvesting oil from algae is a more promising option. Therefore, I investigated oil producing green algae to better understand algal growth, the algal cytoskeleton, and tried to establish a methodology to genetically manipulate algae. I developed a microgrowth assay in order to investigate algal growth and proliferation, while at the same time using considerably less material and space. This assay can directly monitor algal growth in response to media contents, and overcomes many of the limitations of existing microassays due to its use of solid media agar and fluorescent imaging. I also investigated algal genetic manipulation with the intention of creating a standard operating procedure, which could lead to further investigation of how to increase lipid output and increase lipid harvesting cycles through studying lipid production and cell division. Electroporation and PEG mediated transformation were the two chief methods investigated for nuclear transformation. Lastly, I performed an algal kinesin phylogenetic study to characterize the currently available algal kinesin superfamily, providing insight to proteins that are important for cell division as well as other functions within this superfamily. Kinesins 5, Kinesin 7s Class II and Class V, and Kinesin 14 Class I were identified to be important for algal cell division, while Kinesin 8, 12, 11, and some orphan kinesins will require further investigation due to their unknown plant function. Overall, this research provides a foundation for future algal studies required for optimal oil production necessary for a more sustainable future.

PIN-FORMED (PIN) proteins are plant-specific secondary transporters acting in the efflux of plant signaling molecule auxin from cells. Their asymmetrical localization within cells determines the directionality of auxin flow and thereby influences plant development. The activity of PIN proteins is regulated at multiple levels; however the primary step in the regulation of PIN proteins takes place at the level of gene transcription. Therefore the main focus of this diploma thesis is the characterization of the transcriptional regulation of PIN proteins, namely PIN4 protein. The observation of plants carrying transcriptional fusion consisting of various lengths of PIN4 promoter and green fluorescent protein (GFP) showed which part of PIN4 promoter is essential for binding transcription factors and for the start of transcription. This part of PIN4 promoter was used as bait for transcription factors in yeast one hybrid screens. Altogether, 24 transcription factors were identified in which the most numerous were transcription factors from GATA and APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) families. To verify the interactions between identified transcription factors and PIN4 promoter, the protoplast transient expression assay was used. Protoplasts isolated from Arabidopsis thaliana leaves and tobacco BY-2 cell...