Maize Tissue Cultures Research Articles

Maize tissue culture, transformation, and genome editing

Maize tissue culture, transformation, and genome editing

Contribution of the Zea mays insulin-like growth factor (ZmIGF) to the embryogenic competence of maize tissue cultures

Establishment of somatic embryogenic cultures is highly influenced by the plant genotype and the explant type. In maize, immature embryos generate embryogenic callus (E), whereas quiescent embryos produce non-embryogenic callus (NE). E callus shows active growth and high capacity of plant regeneration, while NE callus shows slow growth and no regeneration capacity. Active growth is needed for the establishment of embryogenic cultures; therefore, lack of this characteristic pose a handicap for plant regeneration from NE callus. To correct the slow growth on NE callus, the Zea mays insulin-like growth factor (ZmIGF), a peptide that promotes growth by activating the target of rapamycin (TOR) pathway, was used as media supplement. Additionally, since the TOR pathway is connected to the auxin pathway, ZmIGF participation in cell differentiation was considered. To this end, this research explores ZmIGF effect, beyond growth and proliferation on a Mexican maize landrace, which has shown high somatic embryogenic capacity. Expression levels of reported genes involved in the embryogenesis and differentiation processes were evaluated in maize E, NE, and NE-ZmIGF (NE-Zm) growth-activated calluses. A tendency to upregulate messenger RNA (mRNA) expression was observed for genes encoding transcription factors and auxin transport. Some genes related to epigenetic control showed downregulation. Global DNA methylation and chromatin modifications results suggest an epigenetic activation state on E callus and a repression status on NE callus. ZmIGF induced modifications at DNA methylation and chromatin over NE callus, which changed its original repression state to an active one. Overall, results suggest that the expression of genes related to auxin signaling, mainly transport and efflux carriers, are essential for accomplishing plant regeneration through somatic embryogenesis (SE).

Reproduction of corn lesion nematode on maize and white clover callus tissue cultures

Maize (Zea mays L.) and 'white clover' (Trifolium repens L.) are the important crops throughout the world. Both sustain damage from many species of nematodes; however additional information is needed to develop reliable techniques for studying the interactions between these plants and the nematodes that parasitize them.An experiment was conducted to study the reproduction of the corn lesion nematode, Pratylenchus zeae Graham on callus of five maize inbred lines and three white clover cultivars for incubation periods of 30, 45, and 60 days.In maize test, highly significant differences were observed in nematode production overtime, while reproduction of P. zeae did not diffar significantly among five inbred lines.The analysis showed non significant differences in nematode number per gram of callus either among three white clover cultivars or among periodes of incubation. The maize inbreds and white clover cultivars did not exhibit a wide range in levels of resistance of the corn lesion nematode.

Study Progress on Tissue Culture of Maize Mature Embryo

Abstract It has been paid more and more attention on maize tissue culture as it is a basic work in maize genetic transformation, especially huge breakthrough has been made in maize tissue culture utilizing mature embryos as explants in the recent years. This paper reviewed the study progress on maize tissue culture and plant regeneration utilizing mature embryos as explants from callus induction, subculture, plant regeneration and browning reduction and so on.

TCUP: A Novel hAT Transposon Active in Maize Tissue Culture

Transposable elements (TEs) are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced TEs that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP), which is transcribed at high levels in long-term maize black Mexican sweet (BMS) tissue culture and is transcribed in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected novel TCUP insertions in two long-term tissue-cultured cell lines of the genotype Hi-II A × B and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

Tissue culture-induced genomic alteration in maize (Zea mays) inbred lines and F1 hybrids

Genomic alteration is a common phenomenon associated with plant tissue culture, which often encompasses genetic changes and epigenetic modifications (e.g. cytosine methylation). Here, we studied genomic alteration in maize by assessing calli and regenerated plants derived from three inbred lines (M17, J7 and JC) and two pairs of reciprocal F1 hybrids (pair I: M17/J7 and J7/M17 and pair II: M17/JC and JC/M17). By employing two molecular markers, the amplified fragment length polymorphism and methylation-sensitive amplified polymorphism, we found that both types of genomic alterations occurred in calli and regenerated plants of all the studied maize inbred lines and F1 hybrids, but the extent and pattern of changes varied substantially across the genotypes. Among the three inbred lines, M17 showed markedly higher frequencies of both genetic (from 2.1% to 3.8%) and methylation alterations (from 6.5% to 9.9%, by adding up the various patterns) than the other two lines which showed similar frequencies for both types of alterations (genetic: 0.5–1.8%, methylation: 2.1–3.7%). Of the two F1 hybrid pairs, while pair I showed genetic variation frequencies similar to that of the inbred parent with lower changing frequency and pair II was intermediate of those of the parents, both pairs showed frequencies of methylation alteration more or less intermediate of those of their inbred parental lines. Parent-of-origin effects in both genetic and methylation changes were detected in only one of the hybrid pairs (primarily pair II) for a given changing pattern. Statistical testing confirmed the genotypic difference in both genetic and methylation (hypomethylation) alterations among the regenerants. Taken together, it could be concluded that the frequency and pattern of both genetic and cytosine methylation alterations in maize tissue culture were largely genetic context-dependent traits, but stochasticity also played an important part. F1 hybrids were not significantly more stable than their inbred parental lines under tissue culture conditions.

Maize tissue culture plant regeneration ability can be improved by polyethylene glycol treatment

Type II maize callus of the HiII genotype can be separated into regenerable and non-regenerable types based on the visible morphology of the callus. When the non-regenerable morphotype of callus initiated 6 mo or a yr earlier was treated with from 2% to 20% polyethylene glycol (PEG; 3,350 molecular weight) for three subculture periods of 21 d each, the morphotype changed to regenerable, and the callus did become highly regenerable. The PEG treatments did not improve the plant regeneration ability of the regenerable morphotype or of an old culture initiated 4 yr earlier.

Metabolic effects of glyphosate change the capacity of maize culture to regenerate plants

Since the presence of glyphosate in maize tissue cultures of proprietary line L2 was very detrimental to plant regeneration, we determined metabolic changes associated with the glyphosate effects on plant regeneration in maize cultures. The polar fraction composition and soluble and cell-wall-bound phenolics were analyzed in the regenerable (R) and non-regenerable (NR) calluses of maize line L2. The tissues with high regeneration capacity had low sugar and 4-aminobutyric acid (GABA) concentrations and increased concentrations of most amino acids, polyamines and indole-3-butenol in the soluble polar fraction and higher ferulic acid/coumaric acid and ferulic acid/diferulic acid ratios and higher levels of the predominant G (guaiacyl) units in the cell wall fraction compared with NR calluses as was found before with H99 and HiII maize R and NR tissues, indicating an association of these metabolites with the capacity of maize cultured tissue to regenerate plants. We also found that di-coumaroyl spermidine and coumaroyl-feruoyl spermidine are present in the soluble fraction of L2 R tissues and are practically absent in NR tissues. However, we did not see such differences in HiII and H99 samples, which indicate that these are genotypic features not related to the capacity to regenerate plants in maize tissue cultures. Glyphosate treatment caused the accumulation of shikimic and quinic acids (not detected in untreated samples) in R and NR calluses (with higher levels found in R tissues) and also decreased the FA/diFA ratio in cell wall phenolics, polyamine and amino acid levels, and increased sugar concentrations in the R L2 tissues, indicating a metabolic shift of R callus to NR tissues.

The Stimulatory Effect of the Antibiotic Cefotaxime on Plant Regeneration in Maize Tissue Culture

In order to elucidate the effects of the antibiotic cefotaxime on callus growth and morphogenesis, we incubated embryogenic maize calli (Zea mays L.) of A188 and R91 lines and of their F1 hybrid with 50–500 mg/l cefotaxime throughout several subcultures. Cefotaxime did not affect the induction frequency and growth of the embryogenic callus but enhanced its morphogenesis. In both tested lines and a hybrid, the highest increase in the number of regenerated plants was observed at the antibiotic concentration of 150 mg/l. The degree of morphogenesis stimulation and the range of cefotaxime concentrations effective in stimulation of plant regeneration depended on the properties of calli obtained from tested genotypes.

Histone Hyperacetylation in Maize in Response to Treatment with HC-Toxin or Infection by the Filamentous Fungus Cochliobolus carbonum.

HC-toxin, the host-selective toxin produced by the filamentous fungus Cochliobolus carbonum, inhibits maize (Zea mays L.) histone deacetylases (HDs) in vitro. Here we show that HDs are also inhibited by HC-toxin in vivo, as demonstrated by the accumulation of hyperacetylated forms of the core (nucleosomal) histones H3.1, H3.2, H3.3, and H4 in both maize embryos and tissue cultures. Hyperacetylation of H4 and all isoforms of H3 in tissue cultures of inbred Pr (genotype hm/hm) occurred at 10 ng/mL (23 nM). The effect was host-selective; acetylation of histones in the near isogenic inbred Pr1 (genotype Hm/Hm) did not occur in tissue cultures or embryos treated with 0.2 [mu]g/mL or 10 [mu]g/mL HC-toxin, respectively. Hyperacetylation of histone H4 in embryos of Pr1 began to occur at 50 [mu]g/mL. HC-toxin, and 200 [mu]g/mL HC-toxin caused equal hyperacetylation in Pr and Pr1 embryos. Hyperacetylated core histones, especially of the isoforms of histone H3, accumulated in leaves of inbred Pr, but not Pr1, after infection by toxin-producing strains of C. carbonum. Accumulation of hyperacetylated histones began at 24 h after inoculation, before the development of visible disease symptoms. Hyperacetylation of H2A or H2B histones were not detected in any of the studies. The results are consistent with HD being a primary site of action of HC-toxin.

Dominant and Recessive Mutations Conferring Resistance to S-2-aminoethyl-L-cysteine in Maize

Summary Maize tissue culture was use to select plants resistant to the lysine analogue, S-2-aminoethyl-L-cYsteine (AEC). Embryogenic calli of the maize ({itZea mays} L.) inbred me Cat-100-1 were mutagenize with NaN3 and selected in medium containing 0.25 mM AEC. Several plants were regenerated, transferred to soil and grown to maturity. Two plants, AEC-1 an AEC-5, 0 63 regenerated plants, when self-pollinated, segregate resistant and sensitive pants. The resistance of the AEC-1 plant proved to be determined by a single dominant gene while the resistance of AEC-5 was determined by a single recessive gene after the analysis of R 1 progenies obtained from AEC-1 and AEC-5 regenerated plants. Soluble amino acids from the endosperm of both mutants were extracted an quantified by high performance liquid chromatography. Both resistant mutants presented an increase soluble lysine concentration when compare with sensitive ones.

Lipid Metabolism in Maize Tissue-Culture

Lipid metabolism in maize ( Zea mays L.) tissue culture was studied using 7 strains or inbred lines. The lipid content from the embryogenic callus derived from these lines followed the same trend as the lipid content found in the kernels, but there was no consistent lipid pattern observed for the leaf or root callus from these same lines relative to the tissue that it originated from. Also, embryogenic calli were used to select cell lines resistant to ceruleni an inhibitor of fatty-acid synthesis. While it was hypothesized that the cerulenin-resistant calli might give rise to an overproducing-oil line, the selected resistant calli did not exhibit an increase in lipid content relative to the control calli.

DNA methylation and tissue culture-induced variation in plants

Plant cells growing in an artificial culture environment make numerous genetic mistakes. These alterations are manifested as increased frequencies of single-gene mutations, chromosome breakages, transposable element activations, quantitative trait variations, and modifications of normal DNA methylation patterns. Evidence is presented that indicates a high frequency of DNA hypomethylation as the result of the tissue culture process. Fifteen percent of the methylation changes appear to have been homozygous in the original regenerated plants. A hypothesis is advanced that relates DNA methylation to the variety of genetic alterations found among maize tissue culture regenerants and their progenies. The epigenetic nature of DNA methylation raises questions concerning the stability of tissue culture-induced changes in self-pollinations and crosses.

Catalase isozymes are useful markers of differentiation in maize tissue cultures

To examine the temporal pattern of catalase expression during callus induction, cultures obtained from immature embryos and leaves of several maize inbred lines at several times after explanting were evaluated. The results indicate that irrespective of the primary explant used, CAT-1 is the predominant isozyme in established callus. Catalase expression during the egeneration phase in the absence of auxin was assayed in the cultures. CAT-2 was found to be the isoform specific to this developmental time. The results indicate that different catalase isozymes are expressed at tissue culture level and callus initiation and subsequent plant regeneration can be easily monitored by means of catalase isozymes.

Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family.

We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.

Segregation of transgenes in maize

Progeny recovered from backcrossed transgenic maize tissue culture regenerants (R0) were analyzed to determine the segregation, expression, and stability of the introduced genes. Transgenic A188 x B73 R0 plants (regenerated from embryogenic suspension culture cells transformed by microprojectile bombardment; see [9]) were pollinated with nontransformed B73 pollen. Inheritance of a selectable marker gene, bar, and a nonselectable marker gene, uidA, was analyzed in progeny (R1) representing four independent transformation events. Activity of the bar gene product, phosphinothricin acetyltransferase (PAT), was assessed in plants comprising the four R1 populations. The number of R1 plants containing PAT activity per total number of R1 plants recovered for each population was 2/7, 19/34, 3/14 and 73/73. Molecular analysis confirmed the segregation of bar in three R1 populations and the lack of segregation in one R1 population. Cosegregation analysis indicated genetic linkage of bar and uidA in all four R1 populations. Analysis of numerous R2 plants derived from crossing transformed R1 plants with nontransformed inbreds revealed 1:1 segregation of PAT activity in three of four lines, including the line that failed to segregate in the R1 generation. Integrated copies of bar in one line appeared to be unstable or poorly transmitted.

Dominant mutations causing alterations in acetyl-coenzyme A carboxylase confer tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides in maize.

A partially dominant mutation exhibiting increased tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides was isolated by exposing susceptible maize (Zea mays) tissue cultures to increasingly inhibitory concentrations of sethoxydim (a cyclohexanedione). The selected tissue culture (S2) was greater than 40-fold more tolerant to sethoxydim and 20-fold more tolerant to haloxyfop (an aryloxyphenoxypropionate) than the nonselected wild-type tissue culture. Regenerated S2 plants were heterozygous for the mutant allele and exhibited a high-level, but not complete, tolerance to both herbicides. Homozygous mutant families derived by self-pollinating the regenerated S2 plants exhibited no injury after treatment with 0.8 kg of sethoxydim per ha, which was greater than 16-fold the rate lethal to wild-type plants. Acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) is the target enzyme of cyclohexanedione and aryloxyphenoxypropionate herbicides. ACCase activities of the nonselected wild-type and homozygous mutant seedlings were similar in the absence of herbicide. ACCase activity from homozygous tolerant plants required greater than 100-fold more sethoxydim and 16-fold more haloxyfop for 50% inhibition than ACCase from wild-type plants. These results indicate that tolerance to sethoxydim and haloxyfop is controlled by a partially dominant nuclear mutation encoding a herbicide-insensitive alteration in maize ACCase.

Selection and Characterization of Sethoxydim- Tolerant Maize Tissue Cultures

;Black Mexican Sweet' (BMS) maize (Zea mays L.) tissue cultures were selected for tolerance to sethoxydim. Sethoxydim, a cyclohexanedione, and haloxyfop, an aryloxyphenoxypropionate, exert herbicidal activity on most monocots including maize by inhibiting acetyl-coenzyme A carboxylase (ACCase). Selected line B10S grew on medium containing 10 micromolar sethoxydim. Lines B50S and B100S were subsequent selections from B10S that grew on medium containing 50 and 100 micromolar sethoxydim, respectively. Growth rates of BMS, B10S, B50S, and B100S were similar in the absence of herbicide. Herbicide concentrations reducing growth by 50% were 0.6, 4.5, 35, and 26 micromolar sethoxydim and 0.06, 0.5, 5.4, and 1.8 micromolar haloxyfop for BMS, B10S, B50S, and B100S, respectively. Sethoxydim and haloxyfop concentrations that inhibited ACCase by 50% were similar for BMS, B10S, B50S, and B100S. However, ACCase activities were 6.01, 10.7, 16.1, and 11.4 nmol HCO(3) (-) incorporated per milligram of protein per minute in extracts of BMS, B10S, B50S, and B100S, respectively, suggesting that increased wild-type ACCase activity conferred herbicide tolerance. Incorporation of [(14)C]acetate into the nonpolar lipid fraction was higher for B50S than for BMS in the absence of sethoxydim providing further evidence for an increase in ACCase activity in the selected line. In the presence of 5 micromolar sethoxydim, [(14)C]acetate incorporation by B50S was similar to that for untreated BMS. The levels of a biotin-containing polypeptide (about 220,000 molecular weight), presumably the ACCase subunit, were increased in the tissue cultures that exhibited elevated ACCase activity indicating overproduction of the ACCase enzyme.

Tissue culture isolation of a second mutant locus for increased threonine accumulation in maize

Regenerable maize (Zea mays L.) tissue cultures were selected for ability to grow in the presence of inhibitory (1.0-1.5 mM) concentrations of L-lysine plus L-threonine. Testcross kernels from one regenerated plant (LT20) segregated for wild-type and high free threonine concentration in a 1∶1 ratio consistent with a single dominant gene for high free threonine. Free threonine concentrations (nmol/mg dry weight) increased an average of 29-fold in bulked F2 kernel samples from heterozygous mutant plants, and the total (free plus protein-bound) threonine concentration increased 68%. Increases in protein-bound methionine, lysine and glycine concentrations were also noted, suggesting a possible effect of the mutation on protein concentration and composition. Allelism tests with a previously selected mutant line, Ltr (*)19, showed that two unlinked, codominant genes conditioned the high free threonine phenotype. Based on a separate study of aspartate kinase feedback inhibition characteristics in the two mutant lines, we propose that the mutant alleles [gene and allele designations are according to guidelines for maize genetic nomenclature (Burnham et al. 1975)] be designated Ask-LT19 and Ask2-LT20 for the Ltr (*)19 and LT20 mutants, respectively.

Enhancement of production and regeneration of embryogenic type II callus in Zea mays L. by AgNO3

In order to evaluate the impact of ethylene in maize tissue culture, silver nitrate has been used as an inhibitor of ethylene action. Type II callus initiation rate was improved when immature embryos were cultured on a modified Murashige & Skoog medium containing various concentrations of silver nitrate (5, 10, 20 mgl-1). Regeneration ability of calli initiated and maintained in presence of silver nitrate was enhanced. No modification of callus growth rate neither of ethylene production has been detected.