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.     

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.     

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 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.     

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.     

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 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.     

In vitro culture of immature tassels of an inbred field variety of Zea mays,cv. Oh43

The medium and conditions which permit in vitro culture of immature tassels of Zea mays cv. Oh43 are reported. Final fresh weight, total number of spikelets and the number of normal spikelets were enhanced by optimal concentrations of sucrose (0.3 M), kinetin (10^–7 M) and casein hydrolysate (30 mg l^–1) when added to the Murashige and Skoog salts, White's vitamins and glycine and inositol. A single cultured tassel produces up to 200 normal spikelets which contain anthers bearing germinable pollen.     

In vitro fertilization and seed development inTrifolium

Summary Pistils ofTrifolium repens L. andT. ambiguum Bieb. were cultured on an agar-based modified Murashige-Skoog medium. Pistils with and without accessory floral parts were removed from flowers of selected clones ofT. repens, hand-pollinated under aseptic conditions, and planted on the medium. Pistils cultured without accessory floral parts showed no evidence of fertilization after 2 weeks. However, 52% of thoseT. repens pistils cultured with calyx lobes and pedicels contained ovules with maturing embryos 12 days after in vitro cross-pollination. Pistils fromT. ambiguum intraspecific cross-pollinations could not be cultured successfully under the same conditions; however, addition of various combinations of auxin, cytokinin, and gibberellic acid enhanced embryo growth. Fertilization and partial embryo development occurred in interspecific crosses betweenT. ambiguum andT. repens orT. hybridum only whenT. ambiguum was used as the pistillate parent. These results indicate that embryological development under in vitro conditions closely parallels in situ development although growth regulator requirements may vary among species. This work is Technical Contribution 1785 from the South Carolina Agricultural Experiment Station and was supported by SCAES-USDA Cooperative State Research Agreement No. 616-15-65.     

From in vitro fertilization to early embryogenesis in maize

Summary The development of in vitro fertilization methods in plants, the characterization of developmental mutants, and the adaptation of molecular biology techniques to construct cDNA libraries from minute samples, all represent important recent technical break-throughs. They allow the study of fertilization and early embryogenesis at a molecular level and considerable improvement in the under-standing of higher plant reproduction can be predicted over the next few years. Important biological questions, such as polyspermy, gamete fusion physiology, asymmetrical cell division, embryo axis formation, can now be addressed experimentally in maize, which appears as a major study model in this area.Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

In vitro selection for methomyl resistance in CMS-T maize

Summary Many plants resistant to methomyl (Lannate), an insecticide which selectively damages maize with the Texas (T) type of cytoplasmic male sterility (CMS-T), were obtained by in vitro selection and also without selection. The selection procedure used 0.6–0.7mM methomyl and callus from CMS-T versions of several field and sweet corn genotypes (W182BN, Wf9, P39, MDM1, SW1 and hybrids of SW1, IL766A1, IL766A2, and 442 with W182BN-N). Addition of 1 mM methomyl to the regeneration medium greatly reduced recovery of methomyl-sensitive escapes. Resistance was linked with reversion to male fertility and maternally inherited. Most progeny of resistant plants exhibited stable maternally inherited resistance for two generations in field tests. First-generation progeny of seven culture-derived plants segregated for resistance and sensitivity; this suggests that ears of these seven regenerants were cytoplasmically chimeral. Resistance to methomyl was associated with resistance to T toxin from Helminthosporium maydis race T and with changes in mitochondrial physiology. Prolonged culture (14–16 months versus 6–8 months) increased the frequency of resistance among both selected and non-selected regenerants. Little or no resistance was found among regenerants from certain genotypes. Selection with methomyl may be useful for production of improved sweet corn lines and as a source of mitochondrial mutants. This system is also convenient for studies of the effects of nuclear background and of culture and selection systems on the generation of cytoplasmic mutants.     

Effects of red and blue light on the composition and morphology of maize kernels grown in vitro

Growth and development of plants are known to be affected by exposure to red and blue light. Mechanisms by which light quality influences gene expression in maize (Zea mays L.) embryos have not been explored. Maize kernels can be cultured in vitro allowing experimental manipulation of environmental factors during seed development. We used the in vitro kernel culture system to investigate the response of developing maize seeds, which normally develop without exposure to light, to controlled light quality. Kernels grown under red light accumulated more dry weight than those grown in darkness, whereas kernels grown under blue light accumulated less. Reciprocal color shift experiments showed that light quality during the first week in culture had more influence on kernel weight than during the subsequent three weeks in culture. Soluble sugars were higher in both light treatments than in darkness. Blue-grown kernels had higher amino acid and lower lipid levels than red-or dark-grown kernels. Embryo morphology was markedly affected by red light, under which the upper shoot axis was longer than under blue light or in darkness. Embryo morphology was influenced by light quality during the later stages of development rather than the first week. We suggest, based on these results, that gene expression in the embryo and endosperm of developing maize seeds is sensitive to light quality, and the mechanism and time dependence of this effect warrant further study. In vitro maize kernel culture affords a convenient system for such light quality experiments.     

Tillage and N fertilization effects on maize root growth and root:shoot ratio

Two methods for estimating the size of the maize (Zea mays l.) root system from soil cores taken in the field were compared. The spatially weighed block method of estimation accounted for variation in root density by using 18 samples per plant which varied in distance from plant and soil depth. This method was compared to an estimation which averaged all of the 18 samples together. Both methods gave surprisingly similar estimates for total root growth. Increased root growth in the surface soil layers, due to tillage and N fertilization, did not impact on the estimation of total root growth. Total root length remained unchanged or increased with N fertilization, while root weight remained the same or decreased. Root mass per length decreased with N fertilization. The estimated size of the root system was used to calculate root:shoot weight ratios. The largest root:shoot ratio was found in the vegetative stage and decreased throughout the rest of the season. In this field experiment, the estimated size of the root system at 8 weeks after planting was not significantly different from the size at silking or harvest. Nitrogen fertilization significantly decreased the root:shoot weight ratio. However, tillage did not significantly change the ratio.     

New lights in early steps of in vitro fertilization in plants

Studies using in vitro fertilization systems in animals and lower plants have led to a better understanding of the initial steps of fertilization and their underlying mechanisms. These mechanisms remain to be elucidated in flowering plants. Recent progress related to the development of in vitro fertilization systems using maize as a plant model is presented in this review. Their potential for leading to a better understanding of the process of gametic recognition and fusion and of the early events triggering egg activation and zygote formation are also discussed.     

In vitro fertilization and preimplantation embryo development of African green monkeys (Cercopithecus aethiops)

Ovaries of five adult female African green monkeys were stimulated by repeated administrations of equine chorionic gonadotrophin (eCG), followed by a single administration of human chorionic gonadotrophin (hCG). Oocytes were collected from enlarged follicles 28 h after hCG administration and incubated in vitro for 288 h. Oocytes that had extruded the first polar body were inseminated with spermatozoa that had been incubated for 4 to 6 h in medium with caffeine and dibutyryl cyclic AMP. Of these oocytes, 66% were fertilized and the incidence of polyspermy was 37%. Eighty-two percent of the fertilized eggs cleaved, with some developing into expanded blastocysts. Am. J. Primatol. 43:43–50, 1997. © 1997 Wiley-Liss, Inc.     

Development of maize caryopses resulting from in-vitro pollination

Intact maize (Zea mays L.) ovaries were excised from unpollinated ears (pistillate inflorescences of field-grown plants and placed on defined, agar-based media in Petri dishes. Application of pollen to the end of silks (styles) positioned outside the Petri dish resulted in fertilization of 46% of the ovaries. The extent of subsequent kernel (caryopsis) development varied. After 40 days some kernels had only embryo development while others had embryo and variable endosperm development. About 5% of the initial ovaries developed into normal kernels; 60% of the kernels with some endosperm germinated under laboratory conditions, and 70% of the embryos excised from the embryo-only kernels germinated on culture media.Contribution from the Department of Agromy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA. Paper no. 9641 scientific journal series, Minnesota Agricultural Experiment Station     

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.     

A preliminary report on the establishment of pregnancies in an in vitro fertilization (IVF) programme at the University of British Columbia (UBC)

Between July 1982 and November 1983, two pregnancies were established using in vitro fertilization and embryo transfer (IVF and ET) procedures with three different schedules to induce follicular maturation. All women were cycling normally and had inoperable or absent fallopian tubes. Of 83 oocytes aspirated from 24 patients (31 cycles), 75% were considered mature and 25% immature by the morphological characteristics of the oocytes and cumulus cells. Oocytes were preincubated for 6–24 hours, and after insemination, 60% cleaved to the two-to-four-cell stage. The superovulation induction schedule employing hMG administered according to the individually adjusted treatment scheme established two pregnancies. This schedule was considered the superior regimen, as it gave the highest proportion of mature oocytes (89%) which cleaved (78%). The pregnancy-attaining follicle showed a high progesterone:estradiol-17β ratio (P₄/E₂) in its microenvironment of aspirated follicular fluid, culture media of granulosa cells, and oocyte-cumulus complex. Our observations indicate a high P₄/E₂ ratio in the pregnancy-attaining follicle, and thereby reflect a further parameter in influencing maturation of the oocytes most likely to implant.     

Predictive variables of in vitro fertilization and pre-implantation embryo development in the mouse

The present study aims to analyze the cause-effect relationships among several in-vitro fertilization and pre-implantation embryo development variables in the mouse. Superovulation of hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of pregnant mare's serum gonadotropin at the estrus stage of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Ovulated cumulus-enclosed oocytes were inseminated with sperm from hybrid males of 12-16 weeks of age. The multiple linear regression analyses performed indicated that (a) total number of ovulated oocytes is a good predictor of both fertilization frequency and total number of cells in day-5 blastocysts; (b) fertilization frequency predicts percentage of day-5 blastocysts; (c) total number of cells in day-5 blastocysts is predicted by percentage of day-5 blastocysts; and (d) total number of cells in day-5 blastocysts predicts percentage of apoptotic cells, number of inner cell mass (ICM) and trophectoderm (TE) cells, and ICM/TE ratio in day-5 blastocysts. Mitotic index in day-5 blastocysts was positively correlated with total number of ovulated oocytes, percentage of ovulated cumulus-enclosed oocytes, fertilization frequency, percentage of day-5 blastocysts and total number of cells in day-5 blastocysts. On the contrary, it was negatively correlated with percentage of apoptotic cells in day-5 blastocysts.