Aluminium effects on growth and Al,Ca, Mg,K and P levels in triticale,wheat and rye

Summary The effects of A1 on the growth and mineral composition of different cultivars of triticale (X Triticosecale, Wittmack), wheat (Triticum aestivum L.) and rye (Secale cereale L.) growing in 1/5 strength Steinberg solutions containing 0 or 6 ppm A1 were evaluated after 32 days. Aluminum increased the concentrations of P and K in the roots and K in the tops of most of the cultivars tested. A1 tolerant triticale retained a lower concentration of Mg in the roots and tops than the A1 sensitive triticale, when subjected to A1 stress. In addition, A1 treatments resulted in smaller increases in root P for the A1 tolerant triticale than for the A1 sensitive cultivars.The concentration of root Ca and P of the A1 tolerant wheat cultivars were significantly below that of the more sensitive plants. Aluminum tolerance in rye appeared to be associated with lower Ca and higher Mg concentrations in the tops. The accumulation of P and A1 in the roots was characteristic of sensitivity in triticale, wheat and rye.     

Effect of soil salinity and water stresses on growth and content of nitrogen,chloride and phosphate of wheat and triticale

Summary Three wheats and one triticale were grown, up to flowering stage, in pots on calcareous soil adjusted to a range of salinities (S₁=3.5, S₂=6, S₃=8.5, and S₄=11 mmhos/cm, 20°C, soilpaste extract) by adding solution consisting of 3∶2∶1 of Na-, Ca- and Mg chlorides in chemical equivalent amounts. Moisture in the pots was kept at 100% (W₁), 40% (W₂) and 20% (W₃) of the available water. The vegetative growth, nitrogen and phosphate were affected by S and W treatments, chloride was affected only by S. The interaction S×W affected only dry weight. Varietal effect was observed between wheat as a group and triticale. Multiple quadratic regression equations of these properties on salinity and water revealed that the higher the available water the wider the range of tolerable salinity. Triticale was relatively more tolerant to water stress. Salinity increases Cl and decreases N, whereas water stress enhances N accumulation to a certain extent. However, in triticale at S₃ and S₄ the effect of water stress on N was overshadowed by the excessive salinity. This did not occur for the wheat (Florence). P trends were described. R² for P was low (0.7435–0.3603) which made interpretations rather difficult.     

The Effect of Parental Genotypes of Rye Lines on the Development of Quantitative Traits in Primary Octoploid Triticale: Plant Height

When breeding the primary spring octoploid triticale derived from crosses of various inbred rye lines to wheat Chinese Spring, the effects of the rye genotype and growth conditions on the plant height and proportion of the first, second, and final (pedicle) internodes to the entire stem length were studied. Two triticale groups were examined: homozygotes for the dominant (Ddw1) and recessive (ddw1) alleles of the gene responsible for short stem in rye. In the short stem triticale lines carrying the Ddw1 alleles, the plants were 20 cm shorter on average than those in the ddw1-carrying lines, and the distribution of the two triticale groups overlapped significantly. In both groups, the lines significantly differing in plant height could be differentiated, because of allelic diversity of the additional genes controlling this trait along with the Ddw gene. In most triticale lines, especially in theDdw1-carrying ones, the plant height was much reduced under unfavorable growth conditions. At the same time, a short-stem line was isolated, which is characterized by ecological plasticity, like the maternal wheat cultivar. In the triticale studied, the stem structure depended on the short-stem rye genotype. The two-year study showed that in the triticale carrying the dominant allele of this gene, the first internode is significantly extended, whereas the upper (pedicle) internode is reduced, which increases plant lodging resistance. The differences revealed between the rye lines as well as their effect on the quantitative triticale traits are discussed in view of a variant of the hybridological analysis, which had been previously proposed for identification and mapping of the correspondent rye genes.     

A comparison of physical distribution of recombination in chromosome 1R in diploid rye and in hexaploid triticale

Summary Polymorphism for six C-bands on chromosome 1R was used to study the frequency and distribution of recombination along the chromosome in a diploid rye (Secale cereale L.) and in a hexaploid triticale (X Triticosecale Wittmack) derived from it. In rye, the total recombination frequency in five segments of chromosome 1R was 93.7%. Recombination was concentrated in the distal regions of both chromosome arms and was infrequent in the proximal regions. In hexaploid triticale the total recombination frequency in the same chromosome was reduced to 51.7%. In both backgrounds the distal half of the long arm showed similar recombination frequencies, 51.4% and 45.7% for rye and triticale, respectively. The remaining about two-thirds of the chromosome length showed 42.3% recombination in rye but only 6% recombination in triticale. The results demonstrate that the genetic background in which mapping is performed not only affects the total amount of recombination, but also its distribution along the chromosome length.     

Comparative studies on the activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in the leaves of triticale and wheat infected with Stagonospora nodorum

The activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase, constitutive and induced by Stagonospora nodorum were examined in the 10 – 14 day old seedlings of three triticale and two wheat cultivars under controlled environmental conditions and in flag leaves of two triticale cultivars in the field. Two S. nodorum isolates of different virulence were used. Both the constitutive and induced activities in triticale and wheat depended on genotype and in triticale the effect of growth conditions was also evidenced. The constitutive activities of chitinase, β-1,3-glucanase and peroxidase were several fold lower in flag triticale leaves in plants from the field than in the seedlings, growing under controlled conditions, but induction in the infected flag leaves was significantly more pronounced. In triticale genotypic differences in the response to infection were revealed only upon inoculation by S. nodorum isolate of higher virulence. The enzymatic activities increased several fold during successive days after the infection except for phenylalanine ammonia lyase. Induction of this enzyme was only transient and the activity decreased 48 or 96 h after infection when the activities of other enzymes were rising. In flag leaves in the field this activity was differentiated only after infection with more a virulent strain. A tendency appeared in triticale seedlings for association of the resistance to the pathogen with lower enzymatic constitutive activities. This relationship became more evident in triticale infected by S. nodorum and may imply that although the investigated enzymes are certainly involved in general, non-specific defense mechanism, they do not decide on the resistance to pathogen at least in the early stages of infection and cooperate with other factors in the complex pathogen-plant interaction. One can also assume that the enzymatic activities are associated with severity of infection rather than resistance to pathogen.     

Production and characterization of alloplasmic lines of a triticale ‘Rosner’

Summary The transfer of cytoplasms of various Triticum and Aegilops species to a hexaploid triticale (Rosner) has been attempted using 30 alloplasmic lines and a euplasmic line of common wheat as cytoplasmic donors. The average rate of F₁ hybrid production (seed setting rateXgermination rate) following an ordinary method of crossing is only 0.09%, whereas this rate is increased to 3.1% by use of embryo culture. The first backcross of the F₁ plants with triticale pollen is again difficult, the hybrid production being 0.9%. Further backcrosses proceed smoothly in most cases. As a consequence, the following seven cytoplasms have been transferred to triticale: T. dicoccum, T. aestivum, Ae. squarrosa, Ae. cylindrica, Ae. juvenalis, Ae. ovata and Ae. speltoides. None of these alien cytoplasms causes more meiotic instability than does the triticale's own cytoplasm. Two cytoplasms of T. dicoccum and T. aestivum, both belonging to the B plasma type, have no effect upon any of triticale's characters. Two D type cytoplasms of Ae. squarrosa and Ae. cylindrica cause about 50% reduction of male fertility but exert no other remarkable effects. This fact suggests a partial functional compensation of the effect of a 1D chromosome upon interacting with D cytoplasm by a rye chromosome substituting for it in triticale. A D² cytoplasm of Ae. juvenalis causes earlier heading and complete male sterility, accompanied by some reduction of growth vigor. An M⁰ type cytoplasm of Ae. ovata and an S type cytoplasm of Ae. speltoides cause a great heading delay, complete male sterility, and severe reduction of vigor. From the viewpoint of triticale breeding, none of these cytoplasms appears superior to the triticale's own cytoplasm. However, from the viewpoint of genetics, the hexaploid triticale is an effective tester for differentiating the B, S, and D plasma types.Contribution No. 466 from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan     

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.     

Leaf optical properties during and after drought stress in triticale and maize genotypes differing in drought tolerance

The effect of a short (7 days) and prolonged (14 days) soil drought (D) on leaf optical properties (R reflectance, T transmittance and A absorbance) in PAR and NIR range of irradiation, and on changes in leaf water potential (ψ), leaf injury index (LI), leaf thickness (LT) and chlorophyll (a + b) content (Chl) was studied for maize and triticale genotypes differing in drought tolerance. Under control conditions (C) leaves of maize in comparison to triticale were better hydrated, were thicker and had higher content of chlorophyll (a + b). In non-stressed plants, small differences were observed in measurements of R, T and A. In the range from 500 to 600 nm, the differences between D-resistant and D-sensitive were observed only in transmittance (T) and in range from 700 to 1,100 nm in absorbance (A). In genotypes belonging to the group of D-sensitive T in PAR range and A in NIR range were two times higher than in D-resistant ones. However, in NIR range R for D-sensitive genotypes was lower than for D-resistant ones. The drought stress caused the decrease in ψ, Chl, LT and the increase in leaf injury index (LI). Soil drought applied within 14 days caused larger changes in these physiological characters in comparison to 7 days drought. The observed harmful influence of drought was more visible for maize than triticale. Moreover for genotypes belonging to D-sensitive ones, changes were larger than for D-resistant ones. Similar to changes in ψ, LT and LI drought stress caused changes in leaf optical properties parameters R, A and T. In the PAR range, the highest changes were observed in R, whereas changes in T and A, which were not considerable. Both in maize and triticale, increase in R was higher in plants subjected to 14 days drought than in plants exposed to drought for 7 days. In maize, increase in R was larger for D-sensitive genotype. For both species, changes in T and A of PAR range were small. In NIR range, an increase in R and A, and decrease in T were observed. After 7 days of recovery in plants subjected to shorter period of drought significant differences were still visible in most cases. The same was observed for ψ, LT, LI and Chl parameters. It shows that the period of 7 days rehydration is too short to remove the injuries caused by drought stress. This results indicate that measurements of R, T and A might be useful in practical application for the estimation of the drought tolerance level. Some limitations in the practical application for plant breeding may be caused by relatively high cost of necessary equipment.     

Hybrid necrosis in triticale and the expression of necrosis genes in allopolyploids

Summary The occurrence in triticale of four different genes causing hybrid necrosis is described: Ne1 and Ne2 in the B genome of wheat and Ner1 and Ner2 in the rye genome. Hybrid necrosis develops due to dominant complementary interaction of two genes. This interaction in triticale, however, may take place not only between genes belonging to the same genome but also between genes of different genomes. In triticale, these genes can cause hybrid necrosis in four different combinations. The inheritance of the phenomenon in triticale is, therefore, more complicated than it is in wheat or rye. To avoid hybrid necrosis in triticale, attention should be paid that no necrosis genes are introduced into the primary triticale stocks from the wheat and rye parents. The expression of necrosis genes is influenced by the level of ploidy. Any additional genome — A, B, D, or R — may exert a suppressing effect on the expression of necrosis genes. Therefore, when identifying genotypes of triticale with regard to their necrosis genes, the level of ploidy has to be accounted for. Moreover, the present results illustrate that gene expression in polyploids is not only determined by interactions with other single genes but that it may also be modified by the total genotype of the respective individual.     

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.     

Protein inhibitors of microbial proteinases from wheat,rye and triticale

Specific protein inhibitors of microbial serine proteinases were isolated from wheat (Triticum aestivum L.), rye (Secale cereale L.) and triticale using affinity chromatography on subtilisin-Sepharose 4B. The wheat inhibitor had an isoelectric point (pI) at pH 7.2, while the rye inhibitor consisted of two forms with pI values of 6.8 and 7.1. In triticale, two components were present with pIs 7.2 and 6.8. All the inhibitors had M _r values of approx. 20 000. The isolated proteins were effective inhibitors of subtilisins Carlsberg and BPN, and of fungal proteinases (EC 3.4.21.14) from the genus Aspergillus, but they were completely inactive against trypsin (EC 3.4.21.4) chymotrypsin (EC 3.4.21.1) and pancreatic elastase (EC 3.4.21.36). The inhibitors formed complexes with subtilisin in a molar ratio of 1:1. The results of chemical modifications seem to indicate that the isolated inhibitors have methionine residues in their reactive sites.Abbreviation pI isoelectric point     

Anthocyanin expression in marker free transgenic wheat and triticale embryos

Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T₀ lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale. Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented embryos were only observed visually in mature T₁ seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic lines.     

Development of commercially valuable traits in hexaploid triticale lines with <Emphasis Type="Italic">Aegilops</Emphasis> introgressions as dependent on the genome composition

Introgressive hybridization is an efficient means to improve the genetic diversity of cultivated cereals, including triticale. To identify the triticale lines with Aegilops introgressions, genotyping was carried out with ten lines obtained by crossing hexaploid triticale with genome-substitution forms of the common wheat cultivar Aurora: Aurolata (AABBUU), Aurodes (AABBSS), and Aurotika (AABBTT). The genome composition of the triticale lines was studied by in situ hybridization, and recombination events involving Aegilops and/or common wheat chromosomes were assumed for nine out of the ten lines. Translocations involving rye chromosomes were not observed. Substitutions for rye chromosomes were detected in two lines resulting from crosses with Aurolata. Genomic in situ hybridization (GISH) with Ae. umbellulata DNA and molecular genetic analysis showed that chromosome 1R was substituted with Ae. umbellulata chromosome 1U in one of the lines and that 2R(2U) substitution took place in the other line. Fluorescence in situ hybridization (FISH) with the Spelt1 and pSc119.2 probes revealed a translocation from Ae. speltoides to the long arm of chromosome 1B in one of the two lines resulting from crosses with Aurodes and a translocation in the long arm of chromosome 7B in the other line. In addition, the pSc119.2 probe revealed chromosome 1B rearrangements in four lines resulting from crosses with Aurolata and in a line resulting from crosses with Aurotika. The lines were tested for main productivity parameters. A negative effect on all productivity parameters was demonstrated for Ae. umbellulata chromosome 2U. The overwinter survival in all of the lines was similar to or even higher than in the original triticale cultivars. A substantial increase in winter resistance as compared with the parental cultivar was observed for the line carrying the T7BS-7SL translocation. The line with the 1R(1U) chromosome substitution seemed promising for the baking properties of triticale.     

Development and assessment of DArT markers in triticale

Triticale (X Triticosecale Wittm.) is a hybrid derived by crossing wheat (Triticum sp.) and rye (Secale sp.). Till date, only a limited number of simple sequence repeat (SSRs) markers have been used in triticale molecular analyses and there is a need to identify dedicated high-throughput molecular markers to better exploit this crop. The objective of this study was to develop and evaluate diversity arrays technology (DArT) markers in triticale. DArT marker technology offers a high level of multiplexing. Development of new markers from triticale accessions was combined with mining the large collection of previously developed markers in rye and wheat. Three genotyping arrays were used to analyze a collection of 144 triticale accessions. The polymorphism level ranged from 8.6 to 23.8% for wheat and rye DArT markers, respectively. Among the polymorphic markers, rye markers were the most abundant (3,109) followed by wheat (2,214) and triticale (719). The mean polymorphism information content values were 0.34 for rye DArT markers and 0.37 for those from triticale and wheat. High correlation was observed between similarity matrices derived from rye, triticale, wheat and combined marker sets, as well as for the cophenetic values matrices. Cluster analysis revealed genetic relationships among the accessions consistent with the agronomic and pedigree information available. The newly developed triticale DArT markers as well as those originated from rye and wheat provide high quality markers that can be used for diversity analyses and might be exploited in a range of molecular breeding and genomics applications in triticale.     

Creating new forms of 4x, 6x and 8x primary triticale associating both complete R and D genomes

Summary Strains of Aegilops squarrosa L. and Ae. ventricosa L. were pollinated either by Secale cereale L. or tetraploid triticale. Using in vitro culture of immature F1 embryos, the four corresponding hybrids were obtained. Successful doubling occurred following colchicine treatment, leading to the creation of new amphidiploid structures (C1 plants). These correspond to primary triticale forms involving, at three different levels of ploidy, both R and D full complements. The various combinations were compared for their response at successive steps of the process. Crosses involving Ae. squarrosa present a higher fruit setting than those with Ae. ventricosa, which in contrast yield colchicine treated-plants with better grain fertility. Experimental data on the cytological behaviour and fertility of colchicine-treated as well as amphidiploid plants are presented. The importance of this material in triticale breeding is discussed.     

The Responses of Pro‐ and Antioxidative Systems to Cold‐hardening and Pathogenesis Differ in Triticale (x Triticosecale Wittm.) Seedlings Susceptible or Resistant to Pink Snow Mould (Microdochium nivale Fr., Samuels & Hallett)

Two winter triticale (x Triticosecale Wittm.) cultivars, Magnat (susceptible to pink snow mould) and Hewo (relatively resistant), were used in a model system to test the effect of prehardening and different cold‐hardening regimes on pro‐ and antioxidative activity in seedling leaves. The concentration of hydrogen peroxide and the activity of total superoxide dismutase, catalase, peroxidase and ascorbic peroxidase were analysed spectrophotometrically. As there has been no previous analysis of the pro/antioxidative reaction of cereals to Microdochium nivale infection has been undertaken to‐date, this is the first in the series describing our results. We confirmed that both exposure to abiotic stress of low temperature and subsequent low light intensity, as well as biotic stress of M. nivale infection, change the pro‐ and antioxidative activity in model plants. Genotypes differed substantially in their hydrogen peroxide content: susceptible cv. Magnat generally showed higher levels during all the experiments. This result can lead to the conclusion that cv. Magnat is also more susceptible to low temperature and low light intensity than cv. Hewo. Simultaneous measurements of antioxidative activity indicated that the increased activity of catalases and peroxidases and the consequent lower H₂O₂ level are correlated with a higher resistance to low temperature, low light intensity and pink snow mould in triticale seedlings. The higher H₂O₂ level observed in the susceptible line is likely to be derived from the imbalance of reactive oxygen species production and consumption in this genotype under stress conditions.     

Changes of leaf water potential and gas exchange during and after drought in triticale and maize genotypes differing in drought tolerance

Influence of drought (D) on changes of leaf water potential (Ψ) and parameters of gas exchange in D-resistant and D-sensitive genotypes of triticale and maize was compared. Soil D (from −0.01 to −2.45 MPa) was simulated by mannitol solutions. At −0.013 MPa significant differences in Ψ, net photosynthetic rate (P _N), transpiration rate (E), stomatal conductance (g _s), and internal CO₂ concentration (C _i) of D-resistant and D-sensitive triticale and maize genotypes were not found. Together with the increase in concentration of the mannitol solution the impact of D on E and g _s for D-sensitive genotypes (CHD-12, Ankora) became lower than for the D-resistant ones (CHD-247, Tina). Inversely, impact of D on Ψ was higher in D-sensitive than D-resistant genotypes. From 1 to 3 d of D, a higher decrease in P _N was observed in D-resistant genotypes than in the D-sensitive ones. Under prolonged D (5–14 d) and simultaneous more severe D the decrease in P _N was lower in D-resistant than in D-sensitive genotypes. Changes in Ψ, P _N, E, and g _s caused by D in genotypes differing in the drought susceptibility were similar for triticale and maize. Compared to control plants, increase of C _i was different for triticale and maize genotypes. Hence one of the physiological reasons of different susceptibility to D between sensitive and resistant genotypes is more efficient protection of tissue water status in resistant genotypes reflected in higher decrease in g _s and limiting E compared to the sensitive ones. Other reason, observed in D-resistant genotypes during the recovery from D-stress, was more efficient removal of detrimental effects of D.     

Changes in specific area and energy of root surface of cereal plants in Al-solution cultures. Water vapor adsorption studies

Surface areas and energetic properties of the shooting stage roots of rye (Secale L.), triticale (Triticale), barley (Hordeum L.) and four wheat (Triticum L.) varieties were estimated from experimental water vapor adsorption data. Roots stressed during 10 days at pH 4 with aluminium concentrations ranging from 0 to 40 mg dm^–3 were studied. Roots grown continuously at pH 7 were taken as controls. The surface properties of the roots grown at pH 4 without Al addition were apparently the same as those of the control roots. With the increase of the concentration of the aluminium treatment the surface area of the roots increased for all of the plants, beginning at 5 mg Al dm^–3 for barley, at 10 mg Al dm^–3for wheat and triticale, and at 40 mg Al dm^–3 for rye. The average water vapor adsorption energy of the root surface decreased in general with the increase of Al stress concentration for all plants but triticale, for which this increased. The sensitive cereal varieties seem to have greater amount of high energy adsorption centers (more polar surface) than the resistant ones (lower surface polarity), however more data is needed to justify this hypothesis. For Al-sensitive roots, fraction of high energy adsorption sites decreased and fraction of low energy sites increased under the Al stress. Smaller changes in adsorption energy sites were noted for roots of Al-resistant plants.     

Effect of copper on shoot and root regeneration in wheat,triticale, rape and tobacco tissue cultures

CuSO₄ (0.1–100 M) significantly enhanced shoot regeneration from calli of wheat and triticale and of tobacco leaf disc cultures. In cultures of wheat and triticale, CuSO₄ also stimulated root formation. When equal concentrations of CuSO₄ were applied in different media, it was found that the components of the basal media had only modifying effects. CuSO₄ pretreatment promoted plant survival when regenerated wheat plants were transferred directly to potting soil. In contrast with CuSO₄, AgNO₃, which also stimulated shoot regeneration, inhibited rooting in wheat and triticale. In Brassica napus callus cultures, AgNO₃ strongly increased morphogenesis, whereas CuSO₄ had no significant effect.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid     

Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat₂, M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat₂ (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat₂-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat₂-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat₂ can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.