Embryogenic Maize Research Articles

Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.

RBR3, a member of the retinoblastoma-related family from maize, is regulated by the RBR1/E2F pathway.

Retinoblastoma-related (RBR) proteins regulate cell division in higher eukaryotes by controlling the adenovirus E2 promoter binding factor (E2F)/dimerization partner (DP) family of transcription factors that regulate expression of many genes involved in cell-cycle progression. We identified a previously undescribed member of the maize RBR family, RBR3, which has the characteristic structure and binding activities of pocket proteins, where interaction depends on a LxCxE motif in the partner proteins and a critical cysteine within the B pocket domain. Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclin-dependent kinases. During endosperm development, RBR3 expression is restricted to the mitotic stage preceding the onset of endoreduplication. This finding suggests a role distinct from RBR1, which is constitutively expressed. Two sites in the RBR3 promoter bind to complexes containing maize E2F1 and DP proteins. Expression of wheat dwarf virus RepA protein, which blocks RBR1 activity and stimulates cell proliferation, dramatically up-regulates RBR3, but not RBR1, RNA in embryogenic maize calli. The results indicate that RBR3 expression is controlled by RBR1 through the activity of E2F/DP and that RBR3 is the maize equivalent of mammalian p107. Furthermore, maize and related grasses might have evolved a compensatory mechanism among distinct types of RBR proteins to ensure robust control of pocket protein activity.

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.

Molecular cloning and characterization of a maize transglutaminase complementary DNA

Two related complementary DNA clones, TGZ15 and TGZ21, encoding active maize transglutaminase (TGase) have been isolated for the first time in plants by molecular cloning (Patent Pending PCT/ES03/00247). Southern and northern blot analyses indicate that the two cDNAs probably corresponded to two different single-copy genes in the maize genome. Northern blot analyses revealed that the transcript is expressed preferentially in young leaves and differentiated embryogenic maize callus. This expression is dependent on light exposure time. TGase activity of the proteins encoded by clones TGZ15 and TGZ21 was detected in bacterial extracts overexpressing them, using two enzymatic assays. TGase activity was significantly higher than that of the empty-phagemid bacterial extracts. As in other TGases, this activity was inhibited by monodansyl cadaverine (MDC), GTP and the absence of exogenous Ca ++. Likewise, light-stimulated Ca ++-dependent TGase activity was detected in thylakoids and grana of maize chloroplast, which was inhibited by MDC, GTP, DIECA and Diuron.

Whisker-mediated transformation of embryogenic callus of maize.

The present study was designed to establish embryogenic callus as a target tissue for whisker-mediated transformation of maize (Zea mays L.). Silicon carbide whiskers were used to deliver the bar and uidA (GUS) genes into embryogenic maize callus. Samples of osmotically-treated Type II callus were vigorously agitated in the presence of whiskers and plasmid DNA using a standard laboratory vortex or a modified dental amalgamator. On average, three transgenic callus lines were obtained per 100 samples treated. Plants were regenerated from several GUS-expressing callus lines and DNA analyses confirmed stable integration and inheritance. As with other direct DNA delivery methods involving embryogenic maize callus, integration patterns of the inserted DNA appeared to be complex. Although currently less efficient than microparticle bombardment on a per target basis, whisker-mediated transformation of embryogenic callus represents a viable method for transgenic maize production.

Expression of murine adenosine deaminase (ADA) in transgenic maize.

A murine adenosine deaminase (ADA) gene, driven by the maize ubi-1 promoter and intron region, was transformed into embryogenic maize callus, along with a bar and gusA gene-containing plasmid, using microparticle bombardment. Selection in the presence of either the herbicide Basta or the adenosine analogue 2'-deoxyadenosine resulted in transgenic cultures that expressed GUS and accumulated a 41-kD protein that immunoprecipated with an ADA-specific polyclonal antibody. ADA enzyme activity was observed in extracts from transgenic callus as well as regenerated plants and progeny. Culltures expressing ADA grew in the presence of 200 mg/l 2'-deoxyadenosine, a concentration which completely inhibited the growth of non-transgenic cultures. ADA activity appeared to segregate in progency of regenerated plants as a single, dominant Mendelian trait. These results suggest that ADA, in combination with adenosine analogue selection, represents a potentially viable selectable marker system for transgenic maize production.

Completion of the sexual cycle and demonstration of genetic recombination in Ustilago maydis in vitro.

The heterobasidiomycetes responsible for plant smuts obligatorily require their hosts for the completion of the sexual cycle. Accordingly, the sexual cycle of these fungi could so far be studied only by infecting host plants. We have now induced Ustilago maydis, the causative agent of corn smut, to traverse the whole life cycle by growing mixtures of mating-compatible strains of the fungus on a porous membrane placed on top of embryogenic cell cultures of its host Zea mays. Under these conditions, mating, karyogamy and meiosis take place, and the fungus induces differentiation of the plant cells. These results suggest that embryogenic maize cells produce diffusible compounds needed for completion of the sexual cycle of U. maydis, as the plant does for the pathogen during infection.

Intron-mediated enhancement of gene expression in maize (Zea mays L.) and bluegrass (Poa pratensis L.)

We report a strength comparison of a large variety of monocot and dicot intron-containing fragments inserted in the 5' untranslated leader, between the CaMV 35S promoter and the uidA gene (coding for the ß-glucuronidase: GUS). Relative strengths of the intron-containing fragments were evaluated by comparing transient GUS expression after particle bombardment in embryogenic maize and bluegrass suspension cultures. Our results confirm a dramatic dependence on the presence of an intron for chimeric gene expression in both species. On average, the maize first intron of ubi1 provided the highest enhancement of gene expression in maize and bluegrass (71- and 26-fold enhancement, respectively). Half of the introns tested affected gene expression differently in bluegrass and maize. This suggests that the intron-mediated enhancement of gene expression generally obtained with maize may not be fully applicable to all monocots. We also report enhancement of gene expression (92-fold) in a monocot species by a dicot intron (chsA intron).

Polyamine and free amino acid variations in NaCL-treated embryogenic maize callus from sensitive and resistant cultivars

Summary The effect of different NaCl concentrations on maize embryogenic calluses derived from immature embryo cultures of a) the salt-sensitive inbred line W64Ao2, b) the resistant hybrid Arizona 8601 and c) the 0.4% NaCl pre-adapted W64Ao2 (WpA) calluses was studied. The effect of salt stress on growth as well as on polyamine (putrescine, spermidine and spermine) and amino acid contents of the treated calluses was determined. Enzymatic activities of the polyamine biosynthetic enzymes arginine and ornithine decarboxylase were also analyzed. A significant decrease in the growth of calluses in relation to increased salt concentrations and to the tolerance of the callus was observed. Embryogenic Arizona calluses showed the lowest growth inhibition and W64Ao2 calluses the highest inhibition after 60 days of culture in saline medium. WpA calluses showed an intermediate response. At high-salt concentrations (1.2–2.0%), calluses showed a significant increase in total polyamine content, especially caused by a rise in putrescine. This increase was proportionally higher for Arizona and WpA than for W64 calluses. Whereas the spermidine content of Arizona calluses was augmented with salt, a spermidine decrease was observed for W64 from 0.8 % NaCl. Arginine decarboxylase activity increased from 1.2 to 2.0 % NaCl for Arizona calluses, while in W64 calluses this increase was lower than in Arizona up to 2.0 %. With respect to free amino acid contents, the levels of free proline, which represent 50 % of the total free amino acid content, decreased with increasing salt in the medium. The highest amino acid increases were observed for arginine, alanine, glutamine, glutamic acid and y-aminobutyric acid. Arginine decarboxylase activity showed significant increments in relation to salt stress, which may be related to putrescine and some amino acid variations. Relationships between all the analyzed parameters and the polyamine synthesis and degradation processes under stress conditions are discussed.

Production of fertile transgenic maize plants by silicon carbide whisker‐mediated transformation

SummaryA simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos‐resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.

Transformation and inheritance of a hygromycin phosphotransferase gene in maize plants.

Embryogenic maize (Zea mays L.) callus cultures were transformed by microprojectile bombardment with a chimeric hygromycin phosphotransferase (HPT) gene and three transformed lines were obtained by selecting for hygromycin resistance. All lines contained one or a few copies of the intact HPT coding sequence. Fertile, transgenic plants were regenerated and the transmission of the chimeric gene was demonstrated through two complete generations. One line inherited the gene in the manner expected for a single, dominant locus, whereas two did not.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.

Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus

Protoplasts isolated from embryogenic callus cultures derived from immature embryos ofZea mays L. are suitable for analysis of transient gene expression using electroporation-mediated DNA transfer. Expression of introduced genes is comparable to the levels obtained with protoplasts from Black Mexican Sweet suspension cultures. Two different promoters, that directing synthesis of the 35S RNA of cauliflower mosaic virus and the maizeAdh1 promoter were placed in front of the luciferase reporter gene to assess protoplast gene expression and the impact of an intron on expression level.

Genetic and Cytogenetic Variation in Plants Regenerated from Organogenic and Friable, Embryogenic Tissue Cultures of Maize

Recent advances in maize(Zea mays L.) tissue culture methods permit reproducible establishment of rapid‐growing, friable, embryogenic (Type‐II) cultures from some genotypes. Although Type‐II cultures have replaced the more common Type‐I (relatively compact, organogenic) cultures for many applications, little information is available concerning somaclonal variation in plants regenerated from Type‐II cultures. This study was conducted to determine the relative frequency of genetic and cytogenetic variants in plants regenerated from Type‐I compared to Type‐II maize tissue cultures. Type‐I and Type‐II cultures were initiated from immature embryos of the inbred line A188, A188‐BC6 lines containing various genetic markers, and segregating progenies of an A188/B73 genetic background. Plants were regenerated 16 and 36 wk after culture initiation and were analyzed for pollen sterility, cytological abnormalities, and phenotypic variants in progeny generations. After 16 wk in culture, 33.3 and 20.8% of the regenerants from Type‐II and Type‐I cultures, respectively, possessed at least one abnormality. These values increased to 37.2 (Type‐II) and 24.2% (Type‐I) after 36 wk in culture. As expected, the frequency of chimeric plants was lower from Type‐II than from Type‐I cultures: however, culture age had a larger effect on the frequency of chimeric plants than culture type. Although reduced compared to Type‐I cultures, the frequency of chimerism in regenerants from Type‐II cultures was still quite high, ranging from 9.4 to 50.0% of the variant regenerants. The results demonstrate that genetic and cytogenetic abnormalities are frequently recovered in plants regenerated from both organogenic and friable, embryogenic maize tissue cultures.

Plant Regeneration from Protoplasts Isolated from Embryogenic Maize Cell Cultures

Genetic engineering of maize via protoplast technology has been limited due to lack of plant regeneration from maize protoplasts. We have developed a system of protoplast culture that results in high plating efficiency from embryogenic protoplasts and can be followed by plant regeneration. Maize protoplasts were grown on filters directly over a feeder layer of nurse cells in liquid medium. Initial condition and subsequent growth of feeder cells were critical in obtaining a plating efficiency of 10% from protoplasts. Protoplasts were digested from embryogenic cell suspension cultures, and recovered callus retained the morphogenic potential of initial donor cultures. The system has been successfully used with protoplasts of two maize inbreds, one of which is an important commercial line.

Establishment and characterization of long-term embryogenic maize callus and cell suspension cultures

Friable callus (type 2) was selected from three genotypes (A188, hybrid A188 × B73, and hybrid B73 × A188) of Zea mays L. The three genotypes of type 2 callus doubled in fresh weight after 1 week, and growth was better on N6 than on Murashige-Skoog (MS) medium. Type 2 callus of hybrid B73 × A188 was maintained in culture longer than A188 type 2 callus, and it regenerated higher numbers of plants than the other two genotypes. Type 2 callus of the hybrid B73 × A188 was used to establish cell suspensions. Suspension cells initially grew better on N6 than on MS medium, but after several months of subculture, cells in either N6 or MS medium grew at similar rates. Suspension cells were in mid-log phase by 5–7 days and in stationary phase by about 10 days depending on inoculum density. Growth rate was optimal when cells were transferred at mid-low phase and dry weight of the suspension cells increased at least 10-fold during a 10-day period. Suspension cells from 9-month-old cultures plated on solid medium regenerated plants at an efficiency similar to that of the friable type 2 callus but with more phenotypic abnormalities. Thus, cell suspensions derived from type 2 B73 × A188 callus, in culture for over 1 year, were capable of regenerating plants when 9-months old.

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 μm in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10(3)-10(7) protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10(6) protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1-0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.

Establishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline

Friable, embryogenic maize (Zea mays L.), inbred line A188, callus was established and maintained for more than one year without apparent loss of friability or embryogenic potential. Embryoid development was abundant in these cultures and plants were easily regenerated. Frequencies of friable-callus initiation and somatic-embryoid formation increased linearly with addition to N6 medium (C.C. Chu et al. 1975, Sci. Sin. [Peking] 18, 659-668) of up to 25 mM L-proline. Proline additions up to 9 mM to MS medium (inorganic elements of T. Murashige and F. Skoog 1962, Physiol. Plant. 15, 473-497, plus 0.5 mg 1(-1) thiamine hydrochloride and 150 mg 1(-1) L-asparagine monohydrate) did not stimulate embryoid formation. A major part of the difference between MS and N6 media could be attributed to their respective inorganic nitrogen components. L-Glutamine was not a satisfactory substitute for L-proline. Of 111 regenerated plants grown to maturity from three independent friable, embryogenic cell lines ranging in age from three to seven months, only four plants were abnormal based on morphology and pollen sterility. Seed was produced by 77% of the regenerated plants.