Evaluation of in vitro selection regimes for a mitochondrial trait (methomyl resistance) in cms-T maize

Several factors affecting the success of selection in plant populations were examined for their relevance to in vitro selection. Three in vitro selection schemes and two growth assessment procedures were evaluated for effectiveness in selecting for a mitochondrial trait in maize: resistance to the insecticidal compound methomyl. Regenerable maize callus was derived from immature embryos of the three-way hybrid P39/IL766A2 x W182BN containing Texas male sterile cytoplasm (cms-T). Either low, gradually increasing, or high selection pressures were used to grow callus over a period of 3–5 months. There was no significant difference in recovery of resistant plants using these 3 methods. Growth of callus on medium containing methomyl was assessed by increase in fresh weight during the final month of selection or by increase in number of callus pieces over the course of selection. These quantitative measures of growth were unreliable indicators for gain in resistance within the callus population. A procedure for recovery of methomyl resistant and male-fertile cms-T plants is suggested.     

In vitro microspore selection in maize anther culture with oxidative-stress stimulators

Summary. In order to produce doubled-haploid maize plants tolerant of oxidative stress, in vitro microspore selection was carried out in anther culture with reactive oxygen species (ROS) progenitors such as paraquat, menadione, tert-butylhydroperoxide (t-BHP), and methionine combined with riboflavin. All the ROS progenitors reduced the anther induction, the formation of microspore-derived structures, and their regeneration potential. Abnormal cell divisions and progeny cell degradation could be observed during the development of microspores treated with ROS progenitors. Menadione and t-BHP influenced the microspore developmental pathway, as menadione induced the formation of embryoids, while t-BHP increased the proportion of calli in the microspore-derived structures. As the result of in vitro selection, 15, 10, 10, and 3 fertile doubled-haploid plants were obtained in cultures treated with paraquat, t-BHP, methionine combined with riboflavin, and menadione, respectively. Correspondence and reprints: Agricultural Research Institute, Hungarian Academy of Sciences, Brunszvik utca 2, 2462 Martonvásár, Hungary.     

Nuclear genotype affects mitochondrial genome organization of CMS-S maize

Summary A WF9 strain of maize with the RD subtype of the S male-sterile cytoplasm (CMS-S) was converted to the inbred M825 nuclear background by recurrent backcrossing. The organization of the mitochondrial genomes of the F₁ and succeeding backcross progenies was analyzed and compared with the progenitor RD-WF9 using probes derived from the S1 and S2 mitochondrial episomes, and probes containing the genes for cytochrome c oxidase subunit I (coxI), cytochrome c oxidase subunit II (coxII) and apocytochrome b (cob). Changes in mitochondrial DNA (mtDNA) organization were observed for S1-, S2-, and coxI-homologous sequences that involve loss of homologous restriction enzyme fragments present in the RD-WF9 progenitor. With the coxI probe, the loss of certain fragments was accompanied by the appearance of a fragment not detectable in the progenitor. The changes observed indicate the effect of the nuclear genome on the differential replication of specific mitochondrial subgenomic entities.     

Two-locus theory in recurrent selection for general combining ability in maize

Summary Two-locus theory for recurrent selection for general combining ability in maize was developed. The theory featured: (a) recombination of the selfed progeny of selected parents; and (b) linkage disequilibrium in the initial gametic array. The theory indicated: (a) that initial linkage disequilibrium exerts a permanent influence upon selection progress; (b) that interposition of one or more generations of random mating before each cycle reduces the permanent effect in ensuing cycles; and (c) that random mating done before initiation of selection is more efficient in removing the influence of linkage disequilibrium on selection progress than random mating done between subsequent cycles.     

Results of a diallel trial and a breeding experiment for in vitro aptitude in maize

Summary Some characteristics of in vitro culture of somatic tissues of maize were analysed by a diallel trial. Eight genetically different pure strains, chosen for their aptitudes, were used. The results show that there is considerable genetical variation for the characteristics of in vitro culture and that it should be possible to breed for aptitude to in vitro culture. The linear regression of hybrids on mid-parent reveals a significant heritability for such aptitude. Through selection we have improved plant regeneration after a long period of callus growth.     

In vitro pollination as a model for studying fertilization in maize (Zea mays L.)

Summary In vitro pollination was conducted using excised segments of maize female spikelets to determine the effects of age and silk length on fertilization efficiency and developmental pattern. Ovary development after 15 days resulted in: (1) normal kernels, (2) abnormal kernels and (3) enlarged ovaries; the percentages of each class varied with age. Evidence of double fertilization was observed in both normal and abnormal kernels. In vitro fertilization was traced using silk excision and autoradiography with ³²P-radiolabelled pollen and occurred between 4 and 7 h after the pollination of 4.5-cm-long silks. This study supports the validity of the in vitro pollination method for studying fertilization and emphasizes the importance of using a developmentally sensitive index (silk length) for establishing female developmental stage.     

In vitro pollination fertilization of maize: influence of explant factors on kernel development

The influence of donor plant genotype, ear maturity, explant size, and the ratio of ovule-to-cob tissue on kernel development from in vitro pollinated ovules was examined. All genotypes evaluated in this study were capable of in vitro pollination/fertilization, however, significant differences were observed for the responses measured. Genotype means for complete kernel formation ranged from 1.5% to 25.4% with B73 exhibiting the highest response. Averaged over all genotypes, ear maturity effects were not significant, however, the genotype x ear maturity mean square was significant for swelling percentage. Explant size had a profound effect on in vitro kernel development. Averaged over all genotypes and ear maturities, 30-ovule explants resulted in more than twice as many ovules classified as complete kernels when compared to 10-ovule explants. Ovule-to-cob tissue ratio was also found to have highly significant effects on all three variables measured. An ovule-to-cob tissue ratio of 4:24 resulted in the highest percentages of swelling, embryos with incomplete embryos, and complete kernels.     

Micromanipulation and in vitro fertilization with single pollen grains of maize

Summary The manipulation of single pollen grains of maize was studied. The effects of delivering substances both locally to the grain wall, tube or tip by a microcapillary and directly into the pollen grain by microinjection, and single grain pollination were investigated. Germination was induced by adding small amounts of water locally to the grains with either a microcapillary or with a waterdelivering emulsion without any other ingredients in the medium. The grains were overlayered by mineral or silicone oil so that tube growth proceeded without the grains bursting. There was no apparent penetration of high-molecular-weight substances (FITC-dextran, ethidium bromide labelled DNA) into the living grain either before or after pollination. Neither could the penetration of these substances be detected in both dry, viable and hydrated grains, tubes and tube tips, with or without treatment with Triton X-100 and dimethyl sulfoxide. By microinjection, however, the delivery of high-molecular-weight substances into grains was possible. Such injected grains successfully pollinated stigmas of cultured ear segments. Pollination with pore-injected grains was most efficient (mean 26%). No difference in fertilization rates between mass pollination (mean 41%) and single grain pollination (mean 39%) could be found. A mean fertilization rate of 29% could be obtained after microinjection. Seedlings developed 3 weeks after being pollinated by means of the in vitro pollination and fertilization method.     

Analysis of pollen-tube growth in cultured maize silks

Summary In vitro pollen-tube growth in maize was studied using an in vitro pollination system. In the cut-silk method, ovaries with silks were placed on medium in vitro, whereafter the silk was cut and the upper part of the silk was pollinated. Pollen tubes were not able to bridge the space between the two silk parts. Even when silk parts were tightly connected, pollen tubes still were not able to pass the cut ends and reach the lower silk part. Pollen-tube growth rates and the direction of tube growth were not influenced by the presence or absence of an ovary. Prepollination did not have any influence on pollen-tube growth rate. Measurements of pollen-tube growth rate also showed that there was no population effect, i.e. growth rate was not stimulated by pollination with an excess of pollen grains. We found that the direction in which maize pollen grew was determined only by the positioning of the silk hairs.     

In vitro fertilization of single,isolated gametes of maize mediated by electrofusion

Summary Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released from pollen grains after rupture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cellulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs of gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.     

In vitro selection of barley and wheat for resistance against Helminthosporium sativum

Summary Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive.     

Maturation of maize pollen in vitro

Summary Maturation of maize pollen was obtained in male reproductive structures cultured in vitro. Immature tassels containing microspores at the mid-uninucleate to late-binucleate stage of development were excised and spikelets, anthers, and/or isolated microspores were cultured on a medium capable of supporting pollen maturation. Microspore mitosis, culminating in the production of starch-filled, trinucleate pollen capable of germination, was observed after 7–15 days, depending on the genotype and stage at which the cultures were initiated. Up to 100%, 70%, and 20% of the cultured spikelets, anthers, and isolated microspores, respectively, produced mature pollen, which germinated, however, at different frequencies (i.e., spikelets, 50–70%; anthers, 5–10%; microspores, <1%). Mature kernels were produced following fertilization with pollen from cultured spikelets and anthers. These procedures provide methods for the in vitro manipulation of a significant phase of the maize life cycle.     

Production of normal,germinable and viable pollen from in vitro-cultured maize tassels

Summary Immature tassel meristems (1.0–1.5 cm long) of Zea mays L. inbred, Oh43, and single cross hybrid, Se60, cultured on a nutrient liquid medium underwent extensive development through to maturity and produced normal, mature, trinucleate pollen grains. The grains germinated on nutrient agar and on receptive silks and also produced viable kernels. No differences were observed between in vitro-produced pollen and in vivo pollen (pollen from greenhouse-grown plants) in characteristics such as pollen size, in vitro and in situ germination, and pollen tube growth in vitro. The kernels produced with in vitro pollen grew into mature plants (in vitro plants) which were similar to in vivo plants (plants produced with in vivo pollen), with no significant differences for all the morphological characteristics measured, and no phenotypic and cytological abnormalities. Gel electrophoresis of polypeptides revealed no major differences between in vitro and in vivo seedlings. This demonstration of fertilization and production of normal, uniform plants with pollen from cultured tassels has significant potential in basic and applied research studies.     

Breeding for resistance to downy mildews and stalk rots in maize

Summary The present review includes information on distribution, symptoms, inoculation techniques, disease rating, sources of resistance, genetics of resistance, breeding approaches for resistance, and the present status of resistance breeding with respect to Sclerophthora and Peronosclerospora downy mildews and Erwinia, Cephalosporium and Fusarium stalk rots. Some suggestions highlighting research gaps pertinent to future breeding strategies are mentioned.Publication No. 2993, Experiment Station, GB Pant University of Agriculture and Technology, Pantnagar, India     

Sporophytic response to pollen selection for Alachlor tolerance in maize

In order to assess the efficiency of male gametophytic selection (MGS) for crop improvement, pollen selection for tolerance to herbicide was applied in maize. The experiment was designed to test the parallel reactivity to Alachlor of pollen and plants grown in controlled conditions or in the field, the response to pollen selection in the sporophytic progeny, the response to a second cycle of MGS, and the transmission of the selected trait to the following generations. The results demonstrated that pollen assay can be used to predict Alachlor tolerance under field conditions and to monitor the response to selection. A positive response to selection applied to pollen in the sporophytic progeny was obtained in diverse genetic backgrounds, indicating that the technique can be generally included in standard breeding programs; the analysis of the data produced in a second selection cycle indicated that the selected trait is maintained in the next generation.     

A new source of cytoplasmic male sterility in maize induced by the nuclear gene,iojap

Summary Cytoplasmic male sterility (cms) was found in plants derived from the F₂ progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.     

Genome-wide analysis of maize cytoplasmic male sterility-S based on QTL mapping

Three types of sterile cytoplasm in cytoplasmic-male-sterility (CMS) maize, T, C and S, can be identified according to their fertility-restoration and mitochondrial DNA RFLP patterns. CMS-S, which is the least stable among the three types of CMS, is controlled by sterile cytoplasm interactions with certain nuclear-encoded factors. We constructed a high-resolution map of loci associated with male-restoration of CMS-S in BC1 populations of maize. The map covers 1730.29 cM, including 32 RFLP, 51 SSR 62 RAPD and 21 AFLP markers. Genome-wide QTL analysis detected 6 QTLs with significant effects on male fertility as assessed by their starch-filled pollen ratios. Four QTLs out of six were located between the SSR markers MSbnlg1633-Mrasg20, MSbnlg1662-Msume1126, MSume1230-MSumc1525, and RAPD marker MraopQ07-2-MraopK06-2 on chromosome 2. Two other minor loci were mapped between MraopK16-1-Mraopi4-1, on chromosome 9, and between Msuncbnlg1139-MraopR10-2, on chromosome 6. The Rf3 nuclear restoring gene was precisely located on the chromosome 2, 2.29 cM to the left of umc1525 and 8.9 cM to the right of umc1230. The results provide important markers for marker-assisted selection of stable CMS-S maize.     

In vitro development of young maize ears

The effects of sucrose, cytokinin and auxin on in vitro growth and development of immature maize ears (female inflorescences) isolated from the first and second nodes of a prolific and a nonprolific hybrid were evaluated. The explants were cultured on an agar-solidified Murashige and Skoog medium and tested at four sucrose concentrations, five cytokinin (6-benzylamino purine and zeatin) concentrations, and six zeatin/indole-3-acetic acid (IAA) combinations. Sucrose at 60 g l^–1 resulted in the best ear growth and development in both hybrids and both node positions. Ear lengths from the first and second nodes of the prolific hybrid were further increased by 100 and 62% respectively, by the addition of zeatin (1 mg l^–1) + IAA (1.5 mg l^–1), whereas the ears of the nonprolific hybrid increased by 45 and 33%, respectively. Ear development was also increased by these treatments (114, 166, 57 and 57%, respectively), compared with the control without hormones. When growing in vivo, the second ears of the prolific hybrid were able to reach anthesis (stigma exposition) while in the nonprolific they could not develop beyond the style elongation stage.     

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.