Influence of Water Stress on the Chemical Composition of Seeds of Two Lupins (Lupinus albus and Lupinus mutabilis)

We have performed chemical proximate analysis and determined the carbohydrate profile of mature seeds of Lupinus albus, cv. Rio Maior and Lupinus mutabilis, cv. Potosi plants, either well watered (ww) or subjected to a water-stress period (imposed from the 15th to 35th day after anthesis). Protein content from lupin seeds was 450 and 320 g kg⁻¹ of seed on dry weight basis, for ww plants, respectively, of L. albus and L. mutabilis. Water stress did not affect protein content. The oil content of ww plants was about 120 g kg⁻¹ of seed dry weight for L. albus and 180 g kg⁻¹ of seed dry weight for L. mutabilis. Water stress reduced those values of half. The sugar content was about 90 g kg⁻¹ for L. albus and 50 g kg⁻¹ of seed dry weight for L. mutabilis, in ww plants. Water stress led to an increase in sugar content to 200 and 130 g kg⁻¹ of seed dry weight, respectively, for L. albus and L. mutabilis. The α-galactosides amounted to 70 % of the total sugars (raffinose being 30 % and stachyose, 40 %) and sucrose was about 20 % of the total sugars. Water stress increased total carbohydrates and increased the sucrose/α-galactoside ratio, although reducing raffinose content and increasing sucrose content.     

Intraspecific Variation in Mean Endosperm Cell Cycle Time in Vicia faba (L.) and Interspecific Hybridization with Vicia narbonensis (L.)

The rate of early embryo-sac development was studied in seven faba bean (Vicia faba L.) cultivars grown in a controlled environment. The embryo-sac stage was determined from the number of endosperm nuclei per embryo-sac and the number of embryo cells during the first 12 days after pollination. Differences in early embryo-sac development were observed between the genotypes studied. In addition, five different V. faba×V. narbonensis crosses and reciprocals were made using genotypes with small differences in mean endosperm cell cycle time and genotypes with large differences. The percentage of success (pod set) in V. faba×V. narbonensis crosses ranged between 9 % and 59 % while in the reciprocals it ranged from 12 % up to 30 %. However, cytological studies showed that the high percentage of success (pod set) observed in the cross A-107 × A-202 was not associated with a higher percentage of interspecific hybrid embryos. The results indicate that genotypes of the two species with smaller differences in mean endosperm cell cycle time result neither in a higher percentage pod set nor in bigger hybrid embryos.     

Effects of Temperature on Pollen Tube Growth and Fruit Set in Reciprocal Crosses Between Cucumis sativus and C. metuliferus

Interspecific hybridization between Cucumis sativus and C. metuliferus may be used to introduce disease resistance into the cultivated cucumber. Hybrid plant development, however, is restricted by strong barriers to crossing. The present study was undertaken to examine temperature effects on pollen tube growth and fruit set in reciprocal crosses between C. sativus and C. metuliferus. When crossing C. sativus×C. metuliferus, at 20°C, pollen tube growth was arrested in the stylar region of the pistil and no fruits containing embryos were found. However, at 23° and 26°C, C. metuliferus pollen tubes penetrated into the C. sativus ovules. The enhanced pollen tube penetration was correlated with improved embryo development. Although pre-fertilization barriers were overcome, post-fertilization barriers caused abortion of hybrid embryo development at the globular stage. No effects of higher temperatures were found in the cross C. metuliferus×C. sativus; Pollen tubes were generally arrested in the stylar region of the pistil.     

Analysis of endosperm development of allotriploid × diploid/tetraploid crosses in <Emphasis Type="Italic">Lilium</Emphasis>

Lilium are Fritillaria-type plants. Triploid lilies, regardless of their male sterility, can be used as female parents to cross with appropriate males, in contrast to other triploid Polygonum-type plants, which are usually seedless. Up to now, however, little attention has been paid to the reason. Here we made allotriploid × diploid/tetraploid crosses in Lilium. Endosperm and progenies of LAA × AA/AAAA crosses were analyzed for ploidy level and genome composition. The endosperm of some LAA × AA/AAAA ovules developed well but there was little or no development of LAA × LL/OO endosperm. The endosperm genome composition of LAA × AA, LAA × AAAA, LAA × LL, LAA × OO are theoretically derived as 5A + 2L, 6A + 2L, 4A + 3L, 4A + 2L + O, respectively. Genomic in situ hybridization showed that the progenies of LAA × AA/AAAA were aneuploid. Based on the experimental results and analyses, our hypothesis is that five same genomes of endosperms are essential for its development in triploid × diploid/tetraploid crosses of Lilium. The hypothesis can explain the success or failure of 3x × 2x/4x crosses in Lilium and is of importance for lily breeders who need to know the likelihood of success when producing new cultivars of this economically important horticultural crop.     

Identification of barriers to interspecific crosses in the genus Trifolium

Summary A study of pre- and post-fertilisation barriers after interspecific crosses of diploid and tetraploid Trifolium pratense L. and wild species T. alpestre L., T. medium L. and T. sarosiense Hazsl. was aimed at finding of a promising cross combination for obtaining hybrids. The growth of pollen tubes was arrested in interspecific crosses mainly when T. pratense was at a diploid level. To investigate the post-fertilisation barriers in detail, the hybrid embryo viability was traced by two clearing treatments of immature seeds: (1) using chloral hydrate (which proved to be most appropriate); and (2) a mixture of benzyl benzoate and dibutly phthalate. In interspecific combinations T. pratense (4×) × either T. alpestre or T. sarosiense, enlargement of immature seeds occurred, but no hybrid embryo was traced. Of the wild species used as a male parent for crosses, T. medium was the only exception from the point of view of fertilisation. Globular, heart and the early torpedo stages of hybrid embryos were observed 7 days after pollination (DAP) but only when T. pratense was at a tetraploid level. When T. pratense (2×, 4×) was used as a male parent for interspecific crosses with T. alpestre, T. medium and T. sarosiense, strong defects in various stages of embryogenesis were observed, particularly wrinkled and narrowing embryo sacs caused by an expansion of endothelial cells. We conclude with the following finding: (1) to make crosses only in one direction with T. pratense as a female parent and T. medium as a male; (2) to use tetraploid plants of T. pratense; (3) and to excise hybrid embryos at an early torpedo stage, about 7 DAP.     

Interspecific hybridization between Vicia faba (L.) and Vicia narbonensis (L.): Early pod growth and embryo-sac development

Summary Fertilized embryo-sac development and pod growth was studied in one Vicia faba cultivar, one Vicia narbonensis population and their reciprocal crosses. The initial development of endosperm and embryo was at least four days faster in V. narbonensis than in V. faba. Pods and ovules developed also faster in V. narbonensis than in V. faba. The growth rate of the hybrid pods followed the growth rate of the mother species, but was slower than that of the pods from selfed flowers. In the cross V. narbonensis × V. faba the ovules stopped growing 9 days after pollination, while in the reciprocal cross they stopped growing 15 days after pollination. Hybrid embryo-sacs from V. faba × V. narbonensis were aborted before they reached the stage of 256 endosperm nuclei or 200 embryo cells. Selfed V. faba embryo-sacs reached this stage in less than 9 days after pollination. In the reciprocal cross the embryo-sacs were aborted before they reached the stage of 128 endosperm nuclei or 80 embryo cells. Selfed V. narbonensis embryo-sacs reached this stage at the 4th day after pollination. Given that at these stages the embryo has less than 200 cells it was concluded that an in-ovule embryo culture technique should be developed to obtain hybrid plants.     

The crossing of common wheat (Triticum aestivum L.) with cultivated rye (Secale cereale L.). II. Fertilization and early post-fertilization developments

Summary Fertilization and early seed development was studied in the variety Chinese Spring of common wheat (Triticum aestivum L.) after pollination with rye (Secale cereale L.) and selfing, and in the common wheat variety Hope after selfing. In all three combinations the first pollen tube reached the micropyle in about 40 min after pollination. When pollinated with rye the migration of the sperm nuclei to the egg cell and the polar nuclei was delayed by about an hour. In the subsequent development until 72 hours after pollination the average cellular and nuclear doubling times of embryo and endosperm were about 16 and 8 hours for the interspecific combination. 18 and 9 hours for Chinese Spring selfed and 20 and 12 hours for Hope selfed.     

Study on reproductive barriers in 4x–2x crosses in Dianthus caryophyllus L

Reproductive barriers often exist in 4x–2x crosses in Dianthus caryophyllus L. and seriously lead to low seed set and breeding efficiency. In this study, we inspected pre-fertilization and post-fertilization barriers in crosses between ‘butterfly’ (2n = 4x = 60) and the fertile breeding line ‘NH10’ (2n = 2x = 30) and ‘NH14’ (2n = 2x = 30) using fluorescence microscopy and paraffin section. The growth of the pollen tubes up to the ovary was observed in crosses of ‘butterfly’ × ‘NH10’ and ‘butterfly’ × ‘NH14’. However, abnormalities of pollen tube including coiled and spiky and callose deposition in incompatible tubes has also been detected. In the cross ‘butterfly’ and ‘NH10’, the number of pollen tubes at the base of the style reached the highest value, 67.2 ± 10.8 at 48 h after pollination, but the number of ovules with micropylar penetration was low, 19 ± 4.5. Similarly, in the cross between ‘butterfly’ and ‘NH14’, pollen tubes at the base of the style at 48 h after pollination was 51 ± 6.2 and the ovules with penetrated micropyles per ovary at 48 h after pollination was 11 ± 2.4. It indicated the pollen tubes difficultly penetrated into the ovules in 4x–2x crosses. In addition, it was observed that non-fused of egg and sperm or non-fused polar nucleus resulted in endosperm without embryos, and embryos without endosperm. Embryos aborted at different developmental stages (prembryo to cotyledon embryo, main globular embryo), which might be related to a deficient endosperm development in reciprocal crosses. The results suggested that pre-fertilization and post-fertilization barriers were detected in these combinations. Due to the post-fertilization barriers in 4x–2x crosses, the embryo rescue might be contributed to obtain hybrid F1 plants.     

Influence of Telomeric Heterochromatin Loss on Growth and Development of the Early Embryo and Endosperm in Triticale

The influence of telomeric heterochromatin on early embryo and endosperm development and the production of aberrant endosperm nuclei were investigated using eight triticale (×Tritocmecale Wittmack) lines homozygous for the presence or absence ol blocks of telomeric heterochromatin from rye chromosome arms 1RL, 2RS, (SRS, and 7RL. The loss of heterochromatin from two chromosomes (6R, 7R) produced a significantly lower percentage of aberrant endosperm nuclei than did the loss from one chromosome (6R). Deletion of a third block had a significant additional effect in only one of four lines. The differences between lines for rales of embryo and endosperm development were significant even within a karyotypc, presumably because of genetic variation. The onset of cellularization of the endosperm tended to obscure the detection of aberrancy, and since it evidently occurred at different times in different lines, may have been largely responsible for the apparent reduction in aberrant nuclei in lines with less heterochromatin.     

Early Embryo-Sac Development and Intraspecific Variation in the Mean Endosperm Cell Cycle Time in Vicia narbonensis (L.)

The early endosperm development in seven Vicia narbonensis populations was studied under a 23/ 16 ° C day/night temperature regime. Double fertilization occurred in less than 24 h after pollination. Endosperm divisions were synchronous until the 8th division of the endosperm nuclei. Aberrant endosperm nuclei were first observed alter the 8th division and their number increased after the 10th division. Endosperm nuclei continued to divide until the 12th day after pollination. During the following days a decrease in the number of endosperm nuclei was observed together with a substantial increase in the number of aberrants. Endosperm nuclei finally disintegrated. Neither cellularization nor starch deposition was observed in the endosperm. A positive correlation (r 0.69) was recorded between early pod length and the number of endosperm nuclei found in the ovules within each pod. In the populations studied the number of endosperm nuclei increased at a steady but different rate. This was followed by embryo development as well. It was concluded that intraspecific variation in the mean endosperm cell cycle time exists in Vicia narbonensis and that populations A 174 and A 202 should be more compatible to Vicia faba ev. ‘Polycarpe’ than population A 201.     

Compatibility relations between the edible carrot Daucus carota and D. pusillus, a related wild species from the Argentinian Pampas

To establish the feasibility of hybridization between the wild carrot species Daucus pusillus Michx. (2n = 2x = 22; 2n = 2x = 22 and 20), collected in the pampas grasslands of Argentina, and the edible carrot, Daucus carota L. (2n = 2x = 18), controlled pollinations were attempted on the plant. Due to the difficulties encountered, flowers of 12 accesions and three commercial cultivars were excised from individual plants and pollinated in Petri dishes following an incomplete diallel design. After processing, the pollinated pistils (four to six per genotypic combination) were observed under a microscope with UV light. Pollen tubes reaching the ovaries and/or the ovules (compatible relation) were observed in six out of nine D. pusillus × D. pusillus and seven out of 18 D. pusillus × D. carota genotypic combinations. In the eight D. carota × D. pusillus genotypic combinations, only ungerminated pollen, pollen not adhered to the stigmas or pollen tubes overlapping the stylar tissue were observed. Additional flowers were pollinated in a sample of compatible genotypic combinations and the pollinated pistils were in vitro culture to study embryo and endosperm development. Eight out of nine pistils from the intraspecific and nine out of 13 from the interspecific crosses enlarged to form apparently normal schizocarps. Histological analyses revealed normal development of embryo and endosperm. The breeding barriers between the two species are incomplete, making feasible the obtainment of interspecific hybrids by conventional techniques.     

Genetic effects of embryo and endosperm for four malting quality traits of barley

Seed of seven cultivars of two-rowed barley (Hordeum Vulgare L.) and F2 seed from a half-diallel set of crosses among the cultivars were malted in two years to obtain data on diastatic power (DP), alpha-amylase activity (αAA), beta- amylase activity (βAA) and malt nitrogen (N) content. Embryo and endosperm genetic effects on the traits were studied by using a genetic model including genotype × environment interaction for malting quality characters. Variation of the four malting quality traits was affected by gentic effects and environmental interaction. Performance of DP and βAA was mainly controlled not only by endosperm dominance effects but also by embryo genotype × environment interaction and endosperm dominance × environment interaction. Variation of αAA and malt N content was controlled by both embryo and endosperm genetic effects, but the embryo dominance and endosperm additive effects contributed a major part to the total genetic effects. Significant interaction variances (embryo additive × environment and dominance × environment and endosperm dominance × environment) were also observed for αAA and malt N content. Diastatic power was related positively to βAA. Malt N content was associated positively with DP, largely because of the relationship between malt N and βAA. No obvious phenotype association between DP and αAA was found. General narrow-sense heritabilities of αAA and malt N content were 26.1% and 27.8%, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient hybridization between Lycopersicon esculentum and L. peruvianum via 'embryo rescue' and in vitro propagation

For the transfer of valuable traits from wild species into the cultivated tomato, excised globular-stage embryos 13 and 15 days after pollination (DAP) were cultured in vitro. Plants were regenerated from interspecific crosses of Lycopersicon esculentum cv. ‘Early pink’×L. peruvianum PI270435, and backcrosses of L. esculentum‘Giban (JF) No. 1’× (‘Early pink’× PI270435). Somatic embryos and single cotyledons emerged on hypocotyl sections of the embryos. Five to nine plantlets per embryo were obtained by clonal propagation. The hybrid nature of the plants is confirmed by comparing hybrids and parents in their ability to regenerate shoots from leaf segments in vitro, by comparing plant morphology and characteristics and by chromosome number. This study describes an efficient ‘embryo rescue’ method, as well as somatic embryogenesis by clonal propagation. A novel and simple method for the characterization of the interspecific hybrids is also reported.     

AFLP, ISSR and RAPD markers reveal high levels of genetic diversity among Lupinus spp.

The Lupinus genus includes a number of important crop species. The use of defined nucleotide sequences for the analysis of genetic diversity among these species has revealed modest levels of diversity. The aim of this study was to access AFLP, ISSR and RAPD markers to evaluate the genetic diversity among L. albus, L. angustifolius, L. cosentinii, L. hispanicus, L. luteus, L. mutabilis, L. pilosus and L. polyphyllus. Unexpectedly, low levels of genetic similarity were found (ranging from 0.205 to 0.432), regardless of the type of molecular marker used. Nevertheless, these techniques consistently showed a greater genetic similarity between L. pilosus and L. cosentinii, L. mutabilis and L. polyphyllus and among L. luteus, L. hispanicus and L. angustifolius, clearly separating the Old World from the New World species. Such low genetic similarity among Lupinus spp. is most unlikely to be due to differences in coding sequences but could be the result of a long diverging process concerning non‐coding regions, which would represent a very important proportion of these genomes.     

Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis

Intergeneric sexual hybridizations were conducted between two genera of Rutaceae, with Satsuma mandarin (Citrus unshiu Marc) and Red tangerine (C. reticulata Blanco) as maternal parents, and Poncirus trifoliata (L.) Raf as the paternal parent, in an effort to generate hybrid populations for both molecular mapping and rootstock breeding. Embryo rescue is important for citrus sexual breeding because polyembryony can interfere with hybrid embryo recovery. Immature embryos of 80, 85, 90 days after pollination (DAP) from the Satsuma mandarin (S) × trifoliate orange (P) cross, 80 and 85 DAP from the Red tangerine (R) × trifoliate orange (P) cross, were cultured on MG1.0 medium consisting of MT basal medium supplemented with 1.0 mg l⁻¹ GA₃ and 4% sucrose. The results showed that 80 DAP was the optimal time for embryo rescue of the tested crosses, as evidenced by embryos at this stage exhibiting high germination rates, 37.3% for S × P and 51.3% for R × P. Among the eight tested media, MT medium supplemented with 0.5 mg l⁻¹ GA₃ was the best one for Satsuma mandarin, and MT plus 1.0 mg l⁻¹ GA₃ for Red tangerine. A total of 85 plants were obtained from S × P, and 340 from R × P. Out of them, 44 progenies from S × P and 111 from R × P were sufficiently confirmed to be hybrids by morphological characterization and SSR analysis. In addition, two hybrid callus lines were obtained from S × P and R × P respectively.     

In vitro rescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis

Embryo development in vivo has been studied in four Citrus aurantium L. polyembryonic genotypes. Seeds were collected 65, 85, 105, 125 and 220 days after pollination (DAP). None of the immature seeds harvested 65 and 85 DAP contained visible embryos. A single embryo at a more advanced developmental stage was observed in the central position at the micropylar apex of the embryo sac in about 74% of seeds harvested at 105 DAP, while at 125 and 220 DAP the majority of seeds had two or more embryos at the same developmental stage crowded together. Restriction fragment length polymorphism (RFLP) analysis of lowand high-copy-number nuclear DNA was used to distinguish zygotic from nucellar seedlings. Analysis of plantlets derived from in vitro culture of the bigger embryos, located in the central position at the micropylar apex of the embryo sac of seeds harvested at 105 DAP, established that the frequencies of zygotic embryos ranged from 82 to 88%. Media for immature embryo germination in vitro were based on the nutrients and vitamins of Murashige and Skoog (MS) and Murashige and Tucker (MT) media supplemented with various concentrations of sucrose and growth regulators. A total of 76% of globular stage embryos (<0.3mm) germinated on MT medium containing 150 mM sucrose and 14.4 μM gibberellic acid. Heart stage embryos (0.3-0.8 mm) germinated at 95% on MT medium supplemented with 150 mM sucrose and 2.9 μM gibberellic acid. The addition of 500 mg/l malt extract to MS medium increased the germination of early cotyledon stage (0.8-2.0mm) embryos to 98%. The optimum sucrose concentration for embryo rescue was 150 mM for the three embryo developmental stages. The ability to form plants in vitro strongly increased with increasing embryo developmental stage.     

Genetical control of alkaloid production in Lupinus mutabilis and the effect of a mutant allele Mutal isolated following chemical mutagenesis

Summary A new mutant allele, described here as mutal, which reduces the alkaloid content in dry matter of lupinus mutabilis has been identified following seed treatment with ethyl methane sulphanate. The allele, when homozygous, reduces the alkaloid content from levels of > 2.0 per cent found in seed dry matter of existing populations to 0.2–0.3 per cent and produces plants with vegetative and seed tissues that are organoleptically sweet. Component alkaloids in plants homozygous for mutal differ in respect of the percentage composition of sparteine and of lupanine, as well as possibly of oxa-sparteine and 4-hydroxylupanine, but none is eliminated in genotypes homozygous for the mutant allele. Alkaloid concentration, in so far as not under the control of mutal, has low heritability but lines were isolated following two generations of successive selection which possessed reduced alkaloid levels. These were interpreted to have arisen as a result of recombination of alleles affecting reduction in alkaloid level at several loci. the mutant, mutal, has been established in a pure breeding line which represents a crucial additional step in the evolution of Lupinus mutabilis towards full status as a crop plant.     

Production of interspecific hybrids between <Emphasis Type="Italic">Lilium longiflorum</Emphasis> and <Emphasis Type="Italic">L. lophophorum</Emphasis> var. <Emphasis Type="Italic">linearifolium</Emphasis> via ovule culture at early stage

Interspecific hybridization was carried out between Lilium longiflorum and L. lophophorum var. linearifolium by using the cut style method of pollination, as a contrast, intraspecific hybridization between L. longiflorum ‘Gelria’ and L. longiflorum was also made, but no mature seeds and offspring were obtained from the two combinations under in vivo condition. Ovules excised from each carpel 5–35 days after pollination (DAP) were cultured on B5 or half-strength B5 medium containing sucrose at different concentrations in vitro. In L. longiflorum × L. lophophorum var. linearifolium, only 1.17% of ovules excised at 10 DAP developed into seedlings, and in L. longiflorum ‘Gelria’ × L. longiflorum, only 0.99% of ovules excised at 25 DAP developed into seedlings; none of the ovules excised at other different DAP in the two cross combinations produced any seedlings. The results showed that interspecific hybridization had a more serious post-fertilization barrier than the intraspecific hybridization, and that a lower concentration (3%) of sucrose led to better embryo development and higher percentage of seedlings in ovule cultures. All hybrid seedlings obtained were successfully transplanted to soil and grew normally. The progenies investigated were identified as true hybrids based on inter-simple sequence repeat (ISSR) analysis.     

The effect of ovule age on ovary-slice culture and ovule culture in intraspecific and interspecific crosses with Tulipa gesneriana L.

The efficiency of two embryo rescue techniques, direct ovule culture and ovary-slice culture followed by ovule culture, is studied in crosses with Tulipa gesneriana as maternal genotype. The germination percentages increased, in most cases, significantly with increasing ages of te ovary-slices and ovules at the start of the cultures. The low number of embryos recovered at early culture dates is caused by a higher rate of embryo abortion and by retarded embryo development. The germination percentages for ovary-slice culture followed by ovule culture was mostly comparable to direct ovule culture. Unique hybrids have been obtained from the crosses T. gesneriana × T. agenensis and T. gesneriana × T. praestans. This revised version was published online in July 2006 with corrections to the Cover Date.     

An embryological study of a Pisum sativum x Vicia faba cross

Summary Flowers of Pisum sativum plants grown in the field and greenhouse were self-pollinated and pollinated with Vicia faba. Germination of pollen and pollen tube growth were slower in the Pisum x Vicia cross. About 2% fertile ovules in the field and 8% in the greenhouse were obtained from the Pisum x Vicia cross, compared to 26% and 48% for Pisum x Pisum. Development of the Pisum x Vicia embryo was normal in form, though much slower than the Pisum x Pisum embryo, and about the sixth day it began to collapse. The greatest difference between the two types of crosses was observed in the endosperm. While the Pisum x Pisum endosperm nuclei divided normally and endosperm developed throughout the ovule cavity and around the embryo, the Pisum x Vicia endosperm nuclei formed only scattered masses of densely stained nuclei which were not always in close proximity to the hybrid embryo. It was concluded that faillure of some ovules to develop following Pisum sativum x Vicia faba cross pollination was the result of slow germination of pollen and slow growth of the Vicia pollen tubes, with subsequent lack of fertilization. Collapse of fertilized ovules was associated with abnormal and limited development of the hybrid endosperm, possibly leading to lack of nourishment and eventual collapse of the hybrid embryo.