Inheritance of nuclear DNA content in leaf epidermal cells of Zea mays L.

 The nuclear DNA content (ploidy level) of maize leaf-epidermal cells was investigated by Feulgen cytophotometry in two lines, Illinois High Protein (IHP) and Illinois Low Protein (ILP), their reciprocal hybrids, and their F₂s. Epidermal cells have a 2C, 4C or 8C nuclear DNA content. The mean DNA content per nucleus in IHP was significantly higher than in ILP; the mean DNA content per nucleus in hybrids was intermediate between the parental lines, and the same DNA content was measured in reciprocal crosses. In F₂s the same mean DNA content as in F₁s was observed but with larger variability than in the F₁, possibly indicating genetic segregation. It is inferred that the ploidy level in the leaf epidermis is inherited, and incomplete dominance occurs in hybrids. The same behaviour in the different genotypes was observed for epidermal cell-surface area, except that an increase of mean surface area occurred in the F₁, probably due to heterotic effects. The difference in the accumulation of 4C and 8C nuclei in leaf epidermis parallels that reported between two genotypes for the endosperm tissue: to the greater chromosome endoreduplication found in the endosperm there were correspondingly higher frequencies of 4C and 8C nuclei in the leaf epidermis, indicating a higher general tendency to chromosome endoreduplication in IHP than in ILP. It is suggested that the accumulation of 4C nuclei (G₂-block) in the leaf epidermis may be regarded as the initial step of chromosome endoreduplication, the two phenomena being related to the control of the sequence DNA synthesis-mitosis, possibly involving the same genes in both endosperm and leaf. However, the inheritance of DNA content per nucleus in epidermal tissue seems to be different from that observed in endosperm tissue of the same genotypes, suggesting that differences may occur in the regulation of the activity of these genes. Received: 19 November 1996 / Accepted: 29 November 1996     

Revisiting the hetero-fertilization phenomenon in maize

Development of a seed DNA-based genotyping system for marker-assisted selection (MAS) has provided a novel opportunity for understanding aberrant reproductive phenomena such as hetero-fertilization (HF) by observing the mismatch of endosperm and leaf genotypes in monocot species. In contrast to conventional approaches using specific morphological markers, this approach can be used for any population derived from diverse parental genotypes. A large-scale experiment was implemented using seven F(2) populations and four three-way cross populations, each with 534 to 1024 individuals. The frequency of HF within these populations ranged from 0.14% to 3.12%, with an average of 1.46%. The highest frequency of HF in both types of population was contributed by the pollen gametes. Using three-way crosses allowed, for the first time, detection of the HF contributed by maternal gametes, albeit at very low frequency (0.14%-0.65%). Four HF events identified from each of two F(2) populations were tested and confirmed using 1032 single nucleotide polymorphic markers. This analysis indicated that only 50% of polymorphic markers can detect a known HF event, and thus the real HF frequency can be inferred by doubling the estimate obtained from using only one polymorphic marker. As expected, 99% of the HF events can be detected by using seven independent markers in combination. Although seed DNA-based analysis may wrongly predict plant genotypes due to the mismatch of endosperm and leaf DNA caused by HF, the relatively low HF frequencies revealed with diverse germplasm in this study indicates that the effect on the accuracy of MAS is limited. In addition, comparative endosperm and leaf DNA analysis of specific genetic stocks could be useful for revealing the relationships among various aberrant fertilization phenomena including haploidy and apomixis.     

Primary dormancy in tomato (Lycopersicon esculentum cv. Moneymaker): studies with the sitiens mutant

Intact wild-type tomato (Lycopersicon esculentum cv. Moneymaker)seeds do not complete germination to the same percentage orat the same speed as intact ABA-deficient sitiens (sit^w) mutantseeds when seeds of both genotypes are imbibed on polyethyleneglycol (PEG) solutions of –0.3 to –1.5 MPa osmoticpotential. However, if the thicker testas of wild-type seedsare removed (stripped) from the micropyle without damaging theendosperm, both the percentage and speed of germination at lowexternal water potential are similar to that of sit^w mutantseeds. Removing the micropylar end of the testa from sit^w seedsdid not enhance either the speed or percentage of germinationon PEG solution. Despite similar germination percentage and speed between strippedwild-type seeds and either stripped or intact sit^w seeds underosmotic stress, some differences in seed metabolism are evidentbetween genotypes. The activity of endo-ß-mannanasewas greater in the endosperm of sit^w mutant seeds compared tothe endosperm of wild-type seeds when seeds were exposed toosmotic stress. Although     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

Two-generation marker-aided backcrossing for rapid conversion of normal maize lines to quality protein maize (QPM)

The low nutritive value of maize endosperm protein is genetically corrected in quality protein maize (QPM), which contains the opaque2 gene along with numerous modifiers for kernel hardness. We report here a two generation marker-based backcross breeding program for incorporation of the opaque2 gene along with phenotypic selection for kernel modification in the background of an early maturing normal maize inbred line, V25. Using the flanking marker distances from opaque2 gene in the cross V25×CML176, optimum population size for the BC₂ generation was computed in such a way that at least one double recombinant could be obtained. Whole genome background selection in the BC₂ generation identified three plants with 93 to 96% recurrent parent genome content. The three BC₂F₂ families derived from marker identified BC₂ individuals were subjected to foreground selection and phenotypic selection for kernel modification. The tryptophan concentration in endosperm protein was significantly enhanced in all the three classes of kernel modification viz., less than 25%, 25–50% and more than 50% opaqueness. BC₂F₃ lines developed from the hard endosperm kernels were evaluated for desirable agronomic and biochemical traits in replicated trials and the best line was chosen to represent the QPM version of V25, with tryptophan concentration of 0.85% in protein. The integrated breeding strategy reported here can be applied to reduce genetic drag as well as the time involved in a conventional line conversion program, and would prove valuable in rapid development of specialty corn germplasm.     

Phytotoxic effects of fumonisin B1 on maize seedling growth

Fumonisin B₁ toxin is produced by the fungusFusarium moniliforme Sheldon, which is systemic to maize (Zea mays L.) and maize seeds. The effects of zero to 100 parts per million fumonisin B₁ on the germination process of maize seeds was determined. The presence of fumonisin had no effect on percent seed germination, but fumonisin inhibited radicle elongation by up to 75% after 48 hours of imbibition. An analysis of amylase secretion in the maize endosperm indicated that fumonisins inhibited amylase production in the germinating seed. Isoelectric focusing of endosperm extracts indicated that secretion of the low pI class of amylases was affected more that other amylase isozymes. The results suggested that the presence of high levels of fumonisin in maize seed may have deleterious effects on seedling emergence.     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Dormancy in Dioscorea: Rapid Germination of Detached Embryos from Dormant Seeds of D. tokoro

The intact dormant seeds of Dioscorea tokoro germinate slowlyif at all between 11-23°C; for full and rapid germinationthey require prior chilling treatment [Okagami and Kawai (1982)Bot. Mag. Tokyo 95: 155]. The germination abilities of zygoticembryos detached from dormant seeds of this species were studiedunder various nutritional and temperature regimes. For germinationof embryos, the minimum nutritional components in Murashigeand Skoog's (1962) medium that were required were sucrose andNO^–₃ or SO^2–₄. As the source of carbohydrate forgermination of detached embryos, sucrose, mannose and maltosewere effective; glucose and fructose were less effective; andrhamnose was entirely unable to support germination. Embryos detached from dormant seeds, incubated with the sucroseplus KNO₃, germinated more rapidly with increasing temperatureup to 35°C. However, application of sucrose and KNO₃ didnot induce germination of intact seeds above 26°C. Therefore,it is very possible that the endosperm exerts an inhibitoryfunction on germination at such high temperatures. When seeds were incubated after a cut was made over a smallpart of the edge of the endosperm in which the radicle of theembryo is encased, germination occurred rapidly but the increasein germination percentage was slight. This result suggests thatthe endosperm suppots part of the germination inhibition bymeans of a mechanical barrier or its impermeability to wateror gases. Physiological features of the endosperm alone or interactionsbetween the embryo and endosperm may contribute significantlyto the characteristics of dormancy of intact seeds of this species. (Received May 30, 1988; Accepted January 11, 1989)     

Identification of opaque-2 genotypes in segregating populations of Quality Protein Maize by analysis of restriction fragment length polymorphisms

Quality Protein Maize (QPM) is a name given to genetically modified opaque-2 maize with hard endosperm. The opaque-2 mutation conditions a reduction in the amount of zein seed storage protein; zeins are deficient in the essential amino acids lysine and tryptophan, and mutant seed have a higher nutritional value. To utilize the potential of opaque-2 maize, elite inbreds can be converted to o2/o2 forms and subsequently to hard endosperm opaque-2. Since opaque-2 is recessive and endosperm specific, conventional backcross procedures to convert elite inbreds to opaque-2 forms are inefficient. To alleviate this problem, a marker-assisted selection procedure was developed for the Texas A&M University Quality Protein Maize breeding program. Hybridization of an O2 cDNA probe to blots of DNA from plants carrying O2 and o2 alleles showed that restriction fragment length polymorphisms (RFLPs) exist between the W64A o2 allele and O2 alleles of Mo17 and TX5855 inbred lines. To identify the opaque2 genotypes in segregating populations, an RFLP marker assay combining the O2 cDNA probe and HindIII-digestion of genomic DNA was developed. The effectiveness of the O2 RFLP marker assay was tested under field conditions using F₂ and backcross populations of several hard endosperm opaque-2 lines. A comparison of the genotypes identified by RFLP analysis with the seed phenotypes of the next generation indicated that this procedure is accurate and can be used for identifying O2/O2, O2/o2, and o2/o2 genotypes of individual juvenile plants in breeding populations.     

Application of embryo rescue after interspecific crosses in the genus Rhododendron

Interspecific hybridization between evergreen pot azalea (Tsutsusi) cultivars and genotypes of other Rhododendron subgenera or sections (Rhododendron, Hymenanthes, Pentanthera, Vireya) is significantly hampered by many prezygotic and postzygotic barriers. The objective of our work was to overcome spontaneous abortion and lack of endosperm formation and to increase germination rates by establishing an embryo rescue protocol. The optimal germination medium for immature Rhododendron seeds was a basal medium supplemented with 145 μM GA₃. This medium induced germination of fertilized ovules after several sexual combinations of subgenera. Its use was clearly more efficient than in vivo sowing. The direction of the cross significantly influenced the occurrence of abortion, germination and albinism. The obtained seedlings were multiplied on Woody Plant Medium + 4.5 μM 2iP, and rooted afterwards. Finally, about 9% of the germinated ovules resulted in vigorously growing seedlings that were successfully acclimatized.     

Inducing salt tolerance in maize (Zea mays L.) through seed priming with chloride salts: Growth and ion transport at early growth stages

The study was conducted to determine whether salt tolerance could be induced in maize at germination stage by soaking of seeds for 8 h in distilled water or in 200 meq·L⁻¹ of NaCl, KCl, CaCl₂·2H₂O. Both primed and un-primed seeds were subjected for 14 days to 0, 100 or 200 mol·m⁻³ NaCl under controlled conditions. Although all priming agents were effective in alleviating adverse effects of salt stress on maize at germination stage, CaCl₂·2H₂O proved to be more effective since the seeds primed with this salt had significantly higher final germination, rate of germination and fresh and dry weights of plumules and radicles than those treated with other salts or distilled water. Concentration of Na⁺, K⁺ and Ca²⁺ increased significantly in all parts of germinating seeds of maize seeds primed with NaCl, KCl, or CaCl₂·2H₂O, respectively. In addition, seeds primed with CaCl₂·2H₂O were the highest in Cl⁻ accumulation in all parts of the germinating seeds, followed by seeds treated with NaCl and KCl. Most of the Ca²⁺ was retained in seeds and mesocotyl, because of which, transport of this ion to plumules and radicles was low.     

Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25°C, but not at 15°C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15°C and 25°C.

The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25°C than at 15°C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15°C and 25°C after the puncture force was reduced to between 0.3 and 0.4 newtons.

Application of gibberellic acid₄₊₇ (100 microliters per liter) resulted in earlier germination at 15°C and 25°C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O₂ concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid₄₊₇, but only at 25°C. At 15°C, high O₂ concentrations slowed germination and endosperm strength decline.

     

Increased hexokinase levels in endosperm of mutant maize

Hexokinase activity was measured in endosperms of shrunken-2 (sh₂) and starchy maize. Initial increases in hexokinase were observed for developing endosperms of both genotypes, and the enzyme declined in both as the seeds matured. A higher level of hexokinase was observed in developing sh₂ than in starchy endosperm. This difference persisted throughout maturation and occurred also in germinating seeds. Soluble hexokinase activity per endosperm continued to increase in sh₂ for about 8 days (22–30 days after pollination) after the enzyme in starchy endosperm had attained maximum activity and begun to decline. Hexokinase was predominantly soluble in both genotypes so the differences observed are not due to altered distribution of enzyme between particulate and soluble fractions.     

Comparison of soluble starch synthases and branching enzymes from leaves and kernels of normal andamylose-extender maize

Soluble starch synthases (SS) and branching enzymes (BE) from 20-day-old maize leaves and 22-day-old seeds of normal and amylose-extender (ae) were purified by DEAE-cellulose chromatography. Elution profiles of leaf extracts showed one major SS and two BE fractions from both genotypes. The SS fractions from normal and ae leaf extracts were capable of citrate-stimulated starch synthesis and had different reaction rates with various primers. The two BE fractions from normal leaf extracts differed significantly from each other but not when compared to the same BE from ae. Comparison of BE fractions from ae and normal leaves showed no differences based on chromatographic, kinetic, and immunological properties. Comparison of the leaf enzymes with endosperm enzymes showed major differences. Leaf extracts did not contain SSII or BEIIb observed in endosperm extracts. Developing ae endosperm lacks BEIIb activity and ae is the structural gene for BEIIb. The tissue specific expression of BEIIb in the endosperm provides the basis for explaining the tissue-specific expression of ae. We propose that as BEIIb is expressed in the endosperm, but not leaves, allelic substitution at the ae locus modifies only endosperm starch synthesis.     

Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.)

Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided by the addition of P₂₄ medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P₂₄ medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency, but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred to the greenhouse, where they have been successfully established in substrate.     

In vitro screening for resistance against Septoria nodorum blotch in wheat

This study was carried out to develop an in vitro test for the identification of genotypes resistant to Septoria nodorum blotch. The basis for this project was a previous study in which a crude extract of S. nodorum was used as a selective agent (Keller et al. 1994). It was possible to distinguish resistant and susceptible cultivars in an in vitro test with zygotic embryos. In our project we wanted to test whether this in vitro test can also be used to detect resistant and susceptible genotypes in early segregating populations. Specific crosses between eight winter wheat lines showing contrasting resistance reaction for S. nodorum blotch on leaves and ears were made. The resistance level of both leaf and ear was evaluated after artificial inoculation in the field for the parental lines, the F₁ progenies, as well as for segregating F₃ and F₄ populations. In addition, this plant material was tested in vitro using methods similar to those described by Keller et al. (1994), i.e. culturing immature zygotic embryos and mature seeds on selective media. A good agreement between in vitro screening and field resistance on the ear was found for the parental lines, the F₁ and F₄ generation but not for the F₃ generations. This leads to the conclusion that the in vitro screening might be integrated into wheat breeding programs. Populations showing a high susceptibility to the pathogen metabolites in vitro could be discarded. Another promising implementation for wheat breeding would be the screening of advanced breeding material or candidate partners in a crossing program for resistance on the ear. However, the in vitro screening is not precise enough to select single plants in early segregating populations. Received: 18 January 1999 / Accepted: 30 April 1999     

Genotypic variation between maize (Zea mays L.) single cross hybrids in response to drought stress

Effects of soil drought on growth and productivity of 16 single cross maize hybrids were investigated under field and greenhouse experiments. The Drought Susceptibility Index (DSI) was evaluated in a three year field experiment by the determination of grain loss in conditions of two soil moisture levels (drought and irrigated) and in a pot experiment by the effects of periodical soil drought on seedling dry matter. In the greenhouse experiment response to drought in maize genotypes was also evaluated by root to shoot dry mater ratio, transpiration productivity index, indexes of kernel germination and index of leaf injury by drought and heat temperature. The obtained values of DSI enabled the ranking of the tested genotypes with respect to their drought tolerance. The values of DSI obtained in the field experiment allow to divide the examined genotypes into three, and in the greenhouse experiment into two groups of drought susceptibility. The correlation coefficients between the DSI of maize hybrids in the field and the greenhouse experiments was high and statistically significant, being equal to 0.876. The ranking of hybrids drought tolerance, identified on the basis of field experiments was generally in agreement with the ranking established on the basis of the greenhouse experiment. In the greenhouse experiment statistically significant coefficients of correlation with DSI values in hybrids were obtained for the ratio of dry matter of overground parts to dry matter of roots, both for control and drought treatments, whereas in the estimation of the transpiration productivity coefficient and total dry matter the correlation coefficients were not statistically significant. In this study several laboratory tests were carried out for the drought tolerance of plants (kernel germination, leaf injury) on 4 drought resistant and 4 drought sensitive maize hybrids. Statistically significant correlation coefficients between DSI and the examined parameter of grain germination and leaf injury were obtained for the determination of promptness index (PI), seedling survival index (SS) and leaf injuries indexes (IDS, ITS) as a result of exposure to 14 days of soil drought, osmotic drought −0.9 MPa and exposure to high temperature 45 ° or 50 °C. The results of laboratory tests show that in maize the genetic variation in the degree of drought tolerance is better manifested under severe conditions of water deficit in the soil.     

Phytochrome-induced Germination, Endosperm Softening and Embryo Growth Potential in Datura ferox Seeds: Sensitivity to Low Water Potential and Time to Escape to FR Reversal

In Datura ferox seeds, the far-red absorbing form of phytochrome(Pfr) induces endosperm softening, larger embryo growthpotential,and germination. We investigated the effect of exposing theseeds to a range of water potentials in the presence of Pfronits induction of these responses. In addition, the escape timeto far-red-light (FR) reversal of the three responses wasdetermined. Low water potential inhibited Pfr action on endosperm softeningand germination in a similar way. In both cases, a 50% reductionin the response to a saturating red-light (R) irradiation wasobserved at a water potential of c. —0·5 MPa andtherewas very good correlation between the percentage numberof seeds with softened endosperm at 45 h after R and germinationat 72 h after R (R2=0·95). In contrast, the effect ofdecreasing the external water potential on Pfr induction ofa larger embryogrowth potential was more complex. Moderate decreasesin water potential (—0·3 to —0·5 MPa)enhanced Pfr action and thegrowth potential of the embryos waslarger (20—25%) than the water controls; water potentialsbelow —0·7 MPa inhibited the Pfr stimulus. The escape time to FR reversal of the R effect was shorter forthe increase in embryo growth potential than for endospermsoftening.Twenty-four h after R, the embryo response had escaped in morethan 80% of the population whereas endospermsoftening and germinationwere susceptible to FR inhibition in 100% of the seeds. These results indicate that in D. ferox seeds the increase inembryo growth potential is not sufficient for germination andthatendosperm softening is a necessary condition. Key words: Germination, dormancy, phytochrome, endosperm softening, water potential