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.     

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.     

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 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.     

PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis

One hundred maize zygotic embryos microdissected at the transition stage were used to construct a cDNA library after non-selective PCR (NS-PCR) amplification of whole cDNA populations. The library contains 2.3 × 10⁵ recombinants and two different calmodulin cDNAs were cloned using a heterologous probe from petunia. Calmodulin expression was confirmed throughout maize embryogenesis at the mRNA, amplified cDNA and protein levels. Sequence analysis suggests a maize origin for both clones and negligible nucleotide changes linked to PCR. This library is the first described for early plant embryos and represents a breakthrough to isolate genes involved in embryo differentiation.     

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.     

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.     

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.     

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.     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

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.     

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.     

Stability of deletion,insertion and point mutations at the bronze locus in maize

Summary Phenotypic revertants from several kinds of mutations, including deletions, have been detected by pollen analysis at the wx and Adh loci in maize. Mutations in these genes give phenotypic revertants with median frequencies of 0.7 and 0.5×10^–5, respectively. However, the nature of such revertants can only be analyzed following their recovery from conventional matings. In the current study large seed populations derived from crosses involving several bz (bronze) locus mutations in maize were examined for reversion to a Bz (purple) expression. Deletion, insertion and point mutations were included in the study. Principally, over 2 million gametes of the bz-R mutation, which is shown here to be associated with a 340 base pair deletion within the transcribed region of the gene, have been screened for reversion. No revertants from it or any of the other bz mutations have been recovered, even though a total of almost 5 million gametes from homoallelic crosses have been examined to date. Results from seed analysis are discussed in reference to those from pollen analysis in maize.     

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.     

Haplo-diploid gene expression and pollen selection for tolerance to acetochlor in maize

The objectives of this research were to determine if genes controlling the reaction to the herbicide acetochlor in maize (Zea mays L.) are active during both the haploid and the diploid phases of the life cycle and if pollen selection can be utilized for improving sporophytic resistance. Pollen of eight inbred lines, previously characterized through sporophytic analysis for the level of tolerance to acetochlor, showed a differential reaction to the herbicide forin vitro tube length; moreover, such pollen reactions proved to be significantly correlated (r =0.786^*,df=6) with those of the sporophytes producing the pollen. Pollen analysis of two inbred lines (i.e. Mo17, tolerant, and B79, susceptible) and their single cross showed that thein vitro pollen-tube length reaction of the hybrid was intermediate between those of two parents. An experiment on pollen selection was then performed by growing tassels of Mo17xB79 in the presence of the herbicide. Pollen obtained from treated tassels showed a greater tolerance to acetochlor, assessed asin vitro tube length reaction, than pollen obtained from control tassels. Moreover, the backcross [B79 (Mo17xB79)] sporophytic population obtained using pollen from the treated tassels was more tolerant (as indicated by the fresh weight of plants grown in the presence of the herbicide) than was the control backcross population. The two populations did not differ when grown without the herbicide. These findings indicate that genes controlling the reaction to acetochlor in maize have haplodiploid expression; consequently, pollen selection can be applied for improving plant tolerance.     

Plant recovery from maize somatic embryos subjected to controlled relative humidity dehydration

Summary Maize (Zea mays L.) embryogenic type-II calli were grown on medium containing 0,0.1 μM ABA or 60 g/liter sucrose or both before dehydration of solitary somatic embryos under three relative humidity regimes for up to 6 wk. Viability of dehydrated embryos after 2 wk rehydration was assessed by their ability to produce chlorophyll (greening), roots, coleoptiles, and/or leaves. Only embryos sequentially pretreated with ABA and high sucrose remained viable after 2 wk of dehydration at 70% RH. Up to 34% of the somatic embryos survived 2 wk dehydration at 70% RH, whereas embryos dehydrated at 50 or 90% RH exhibited reduced viability (8.7 and 0.8%, respectively). Approximately 15% of the embryos dehydrated at 70% RH developed into plants, whereas 0.9 and 0% of embryos dehydrated at 50 and 90% RH produced plants. Three percent of maize somatic embryos remained viable after 6 wk of dehydration at 70% RH, and 1.7% developed into plants. Embryo size influenced the ability of maize somatic embryos to survive dehydration. Only embryos greater than 5 mm survived 2 wk dehydration at 70% RH.