Relationship of chlorophyll, seed moisture and ABA levels in the maturing Brassica napus seed and effect of a mild freezing stress

Chlorophyll (Chl) retention by mature seed of canola as the result of an early frost or other environmental factors (the "green seed problem") causes serious economic losses. The relationship of seed degreening to rate of moisture loss by seed and silique and the role of ABA in this process were investigated as a function of developmental age. During the normal predesiccation stage (28–45 days after pollination), seed of Brassica napus (cv. Westar) loses Chl rapidly but seed moisture slowly. After a mild freezing stress, there is a rapid loss of moisture from silique walls, followed by accelerated loss of seed moisture. Chl degradation ceases at 35–45% seed moisture. ABA levels in silique walls of frozen plants (determined by enzyme‐linked immunosorbant assay) increased after freezing, apparently in response to moisture loss. In contrast, ABA levels in the seed increased dramatically 1 day after freezing, then decreased to control levels. The influence of the rate of seed moisture loss on Chl degradation was investigated by fast and slow drying of isolated seed under controlled humidity conditions. Seed dried rapidly at 22% RH retained most of its Chl, whereas seed dried slowly at 86% RH lost Chl as fast or faster than seed on control (unfrozen) plants. In all treatments, Chl loss stopped at about 40% seed moisture.     

Freezing injury to ovules, pollen and seeds in winter rape

Winter rapeseed (Brassica napus, cv. Samouraï) flowersearly in spring and, under field conditions, short freezingperiods can occur. Unacclimatized plants were freeze-stressed(–3°C for 4 h) at different developmental stages ofbuds, open flowers and seeds. The dissection of pistils from stressed plants showed that freezingresults in shrivelled ovules. We assessed freezing injury onthe basis of per cent of shrivelled ovules: ovule sensitivitybegins early (8 d before anthesis) but increases up to anthesis.Crosspollination of stressed pistils with non-stressed pollenshowed that recording of freeze-injured ovules is a good methodfor early estimation of the effect of stress on seed yields. On the other hand, stress does not reduce the viability of pollen,except when it was applied at the binucleate pollen stage. Useof frozen pollen x nonstressed pistils has little effect onseed yields. Freeze injury on seeds was assessed by seed filling:seeds are very susceptible just after fertilization until 20d after fertilization (DAF). Freezing seems to inhibit seedfilling. A germination test of stressed seeds during their developmentindicated that embryo viability is not affected if the stressoccurs after 35 DAF. As the embryos develop, resistance to stressincreases. Key words: Brassica napus, rapeseed, freeze injury, pollen and ovule, seed filling     

Phenotypic plasticity and bet‐hedging in a heterocarpic winter annual/spring ephemeral cold desert species of Brassicaceae

Phenotypic plasticity can play an important role in colonization and survival of plants in an environmentally fluctuating habitat. The primary aim of this study was to determine the influence of level of abiotic (soil moisture and nutrient availability) and biotic (density and herbivory) factors on phenotypic plasticity in the number and proportional mass allocation to the heteromorphic dehiscent upper (high dispersal, low dormancy) and indehiscent lower (low dispersal, high dormancy) siliques of individuals of the cold desert winter annual/spring ephemeral Diptychocarpus strictus derived from different seed morphs. Plants produced from seeds sown in an experimental garden were subjected to different levels of soil moisture, nutrient supply, density and simulated herbivory. Mass allocated to vegetative and reproductive components was measured and number of upper and lower siliques counted. Except for number ratio of upper: lower siliques under nutrient supply, levels of the four treatments resulted in significant variation in total plant mass, reproductive mass, number of siliques (upper and lower), number and mass of each silique morph, individual seed mass, upper/lower silique ratio and mass allocation to each organ in an individual. In favorable environments, the upper/lower silique ratio was relatively high, while in unfavorable environments it was relatively low. The relative allocation to upper and lower siliques was significantly negatively correlated, suggesting that allocation to upper siliques in good growth conditions occurred at the expense of allocation to lower siliques. This appears to be the first report on the effect of herbivory on diaspore morph ratio in heterocarpic plants and one of only a very few on the effect of density on morph ratio in this group. In D. strictus, stressful vs nonstressful growth conditions caused a shift in the ratio of heteromorphic diaspores, which themselves are assumed to be bet‐hedging.     

Effects of Heat Stress during Seed Filling Stage on <i>Brassica napus</i> Seed Oil Accumulation and Chlorophyll Fluorescence Characteristics

As global temperature rise, the threat of heat stress to rapeseed production is becoming more obvious. Exploring the response characteristics of two important biological pathways, oil accumulation and photosynthesis, to heat stress during B. napus seed filling is helpful in the genetic improvement of heat-tolerant rapeseed. The effects of heat stress on seed oil accumulation and chlorophyll fluorescence characteristics of 29 B. napus germplasms with different oil content and environmental sensitivity, including 6 rapeseed varieties which exhibited environment-sensitive/insensitive and with high, medium or low oil content, were tested by whole plant heat stress or the in vitro silique culture system. Both assay exhibited similar trend on oil content of the rapeseed germplasms. The heat effect on the chlorophyll fluorescence kinetic parameters F_v/F_m, ETR and Y(II) were also consistent. Heat stress significantly decreased oil content, although there was abundant genetic variation on heat tolerance among the genotypes. Correlation analysis showed that the decrease rate of F_v/F_m of silique heat-stressed B. napus developing seed was positive correlative to the decrease rate of mature seed oil content of the whole plant heat-stressed rapeseed (R = 0.9214, P-value < 0.01). Overall, the results indicated that heat stress inhibited oil accumulation and photosynthesis in B. napus developing seed. The decrease rate of chlorophyll fluorescence parameter F_v/F_m of heat-stressed developing seed could be used as the index of heat tolerant rapeseed identification. Further, two heat insensitive rapeseed varieties with high oil content were identified.     

A CACTA‐like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed

Rapeseed (Brassica napus L.) is a model plant for polyploid crop research and the second‐leading source of vegetable oil worldwide. Silique length (SL) and seed weight are two important yield‐influencing traits in rapeseed. Using map‐based cloning, we isolated qSLWA9, which encodes a P450 monooxygenase (BnaA9.CYP78A9) and functions as a positive regulator of SL. The expression level of BnaA9.CYP78A9 in silique valves of the long‐silique variety is much higher than that in the regular‐silique variety, which results in elongated cells and a prolonged phase of silique elongation. Plants of the long‐silique variety and transgenic plants with high expression of BnaA9.CYP78A9 had a higher concentration of auxin in the developing silique; this induced a number of auxin‐related genes but no genes in well‐known auxin biosynthesis pathways, suggesting that BnaA9.CYP78A9 may influence auxin concentration by affecting auxin metabolism or an unknown auxin biosynthesis pathway. A 3.7‐kb CACTA‐like transposable element (TE) inserted in the 3.9‐kb upstream regulatory sequence of BnaA9.CYP78A9 elevates the expression level, suggesting that the CACTA‐like TE acts as an enhancer to stimulate high gene expression and silique elongation. Marker and sequence analysis revealed that the TE in B. napus had recently been introgressed from Brassica rapa by interspecific hybridization. The insertion of the TE is consistently associated with long siliques and large seeds in both B. napus and B. rapa collections. However, the frequency of the CACTA‐like TE in rapeseed varieties is still very low, suggesting that this allele has not been widely used in rapeseed breeding programs and would be invaluable for yield improvement in rapeseed breeding.     

In vitro seed maturation in Brassica rapa L.: Relationship of silique atmosphere to storage reserve deposition

The Brassica rapa L. silique is a self-contained environment that maintains hypoxia around the developing seeds, and in which carbon dioxide accumulates to very high concentrations (>30,000 ppm). How the silique microenvironment modulates the composition and amount of storage reserves in the seeds is of interest because of the important agricultural role played by canola (B. rapa and Brassica napus) as an oilseed. Because of the small volume and dynamic nature of this microenvironment in Brassica, a standardized system was needed to study the environmental role played in storage reserve deposition. For this purpose we have developed a silique culture system that permits maturation of seed in vitro. Siliques excised from plants just 11 days after pollination complete the ripening of their seeds after 20 days of culture in light (200 μmol/m²/s) on MS medium containing 30 g/l sucrose, 0.25 mg/l BAP, and 0.025 mg/l NAA. Cytochemical localization and biochemical analyses revealed that storage reserves were affected by the in vitro maturation system. Although following a comparable ripening timeline to that occurring on the plant, and producing fully germinable seeds, in vitro maturation resulted in a 40% reduction in seed weight and the mature seeds contained decreased lipid, but increased protein, starch and soluble carbohydrates. To study the internal atmosphere surrounding the seeds, we developed a method to capture silique gases in helium with subsequent quantification of O₂ and CO₂ in the sample by gas chromatography. Analysis of the internal silique atmosphere showed that in vitro siliques provided seeds with a less oxygenated environment than they experience attached to the plant. Carbon dioxide concentrations remained high later into the maturation sequence in vitro than on the plant. When sampling gases from siliques attached to plants, we found multiple samples from the same plant resulted in higher variance than when only a single silique was sampled, suggesting that connection to the plant directly influences internal silique gases. Lower O₂ in the in vitro siliques was correlated with depressed lipid content in their mature seeds, supporting the conclusion that oxygen availability limits lipid accumulation. Previous studies showed how environmental factors influence Brassica embryos grown in tissue culture. These systems fail to preserve the component of metabolic regulation that is enforced by the silique wall tissues. Our in vitro maturation system provides a useful tool for specialized investigations since both the gaseous and hormonal environments can be readily manipulated.     

The amino acid permease AAP8 is important for early seed development in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The development of seeds depends on the import of carbohydrates and amino acids supplied by the maternal tissue via the phloem. Several amino acid transporters have been reported to be expressed during seed and silique development in Arabidopsis thaliana (L.) Heynh. Here we show that mutants lacking the high affinity amino acid permease 8 (At1g10010) display a severe seed phenotype. The overall number of seeds and the number of normally developed seed is reduced by ∼50% in siliques of the Ataap8 T-DNA insertion mutant. This result could be reproduced in plants where expression of AtAAP8 is targeted with an RNAi approach. The seed phenotype is correlated with a specifically altered amino acid composition of young siliques. Aspartic acid and glutamic acid are significantly reduced in young siliques of the mutants. In correlation with the fact that AAP8 is a high affinity transporter for acidic amino acids, translocation of ¹⁴C-labelled aspartate fed via the root system to seeds of the mutants is reduced. AAP8 plays a crucial role for the uptake of amino acids into the endosperm and supplying the developing embryo with amino acids during early embryogenesis.     

Sequence,expression divergence,and complementation of homologous <Emphasis Type="Italic">ALCATRAZ</Emphasis> loci in <Emphasis Type="Italic">Brassica napus</Emphasis>

The genomic era provides new perspectives in understanding polyploidy evolution, mostly on the genome-wide scale. In this paper, we show the sequence and expression divergence between the homologous ALCATRAZ (ALC) loci in Brassica napus, responsible for silique dehiscence. We cloned two homologous ALC loci, namely BnaC.ALC.a and BnaA.ALC.a in B. napus. Driven by the 35S promoter, both the loci complemented to the alc mutation of Arabidopsis thaliana, yet only the expression of BnaC.ALC.a was detectable in the siliques of B. napus. Sequence alignment indicated that BnaC.ALC.a and BolC.ALC.a, or BnaA.ALC.a and BraA.ALC.a, possess a high level of similarity. The understanding of the sequence and expression divergence among homologous loci of a gene is of due importance for an effective gene manipulation and TILLING (or ECOTILLING) analysis for the allelic DNA variation at a given locus. S. Hua and I. H. Shamsi contributed equally to this work.     

Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of<Emphasis Type="Italic"> Brassica napus</Emphasis> L.<Emphasis Type="Italic"> in planta</Emphasis>

Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0–10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [¹⁴C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [¹⁴C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [¹⁴C]acetate and [¹⁴C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.Abbreviations DAF Days after flowering - DAG Diacylglycerol - G3PAT Glycerol-3-phosphate acyltransferase - Glycerol-3P Glycerol-3-phosphate - PA Phosphatidic acid - PC Phosphatidylcholine - TAG Triacylglycerol,     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.Abbreviations ABA abscisic acid - bp base pairs - DAF days after flowering - HAI hours after the start of imbibition - kb kilobase (pairs)     

AtPER1 enhances primary seed dormancy and reduces seed germination by suppressing the ABA catabolism and GA biosynthesis in Arabidopsis seeds

Seed is vital to the conservation of germplasm and plant biodiversity. Seed dormancy is an adaptive trait in numerous seed‐plant species, enabling plants to survive under stressful conditions. Seed dormancy is mainly controlled by abscisic acid (ABA) and gibberellin (GA) and can be classified as primary and secondary seed dormancy. The primary seed dormancy is induced by maternal ABA. Here we found that AtPER1, a seed‐specific peroxiredoxin, is involved in enhancing primary seed dormancy. Two loss‐of‐function atper1 mutants, atper1‐1 and atper1‐2, displayed suppressed primary seed dormancy accompanied with reduced ABA and increased GA contents in seeds. Furthermore, atper1 mutant seeds were insensitive to abiotic stresses during seed germination. The expression of several ABA catabolism genes (CYP707A1, CYP707A2, and CYP707A3) and GA biosynthesis genes (GA20ox1, GA20ox3, and KAO3) in atper1 mutant seeds was increased compared to wild‐type seeds. The suppressed primary seed dormancy of atper1‐1 was completely reduced by deletion of CYP707A genes. Furthermore, loss‐of‐function of AtPER1 cannot enhance the seed germination ratio of aba2‐1 or ga1‐t, suggesting that AtPER1‐enhanced primary seed dormancy is dependent on ABA and GA. Additionally, the level of reactive oxygen species (ROS) in atper1 mutant seeds was significantly higher than that in wild‐type seeds. Taken together, our results demonstrate that AtPER1 eliminates ROS to suppress ABA catabolism and GA biosynthesis, and thus improves the primary seed dormancy and make the seeds less sensitive to adverse environmental conditions.     

Potential gene flow of two herbicide-tolerant transgenes from oilseed rape to wild B. juncea var. gracilis

Four successive reciprocal backcrosses between F₁ (obtained from wild Brassica juncea as maternal plants and transgenic glyphosate- or glufosinate-tolerant oilseed rape, B. napus, as paternal plants) or subsequent herbicide-tolerant backcross progenies and wild B. juncea were achieved by hand pollination to assess potential transgene flow. The third and forth reciprocal backcrosses produced a number of seeds per silique similar to that of self-pollinated wild B. juncea, except in plants with glufosinate-tolerant backcross progeny used as maternal plants and wild B. juncea as paternal plants, which produced fewer seeds per silique than did self-pollinated wild B. juncea. Germination percentages of reciprocal backcross progenies were high and equivalent to those of wild B. juncea. The herbicide-tolerant first reciprocal backcross progenies produced fewer siliques per plant than did wild B. juncea, but the herbicide-tolerant second or third reciprocal backcross progenies did not differ from the wild B. juncea in siliques per plant. The herbicide-tolerant second and third reciprocal backcross progenies produced an amount of seeds per silique similar to that of wild B. juncea except for with the glufosinate-tolerant first and second backcross progeny used as maternal plants and wild B. juncea as paternal plants. In the presence of herbicide selection pressure, inheritance of the glyphosate-tolerant transgene was stable across the second and third backcross generation, whereas the glufosinate-tolerant transgene was maintained, despite a lack of stabilized introgression. The occurrence of fertile, transgenic weed-like plants after only three crosses (F₁, first backcross, second backcross) suggests a potential rapid spread of transgenes from oilseed rape into its wild relative wild B. juncea. Transgene flow from glyphosate-tolerant oilseed rape might be easier than that from glufosinate-tolerant oilseed rape to wild B. juncea. The original insertion site of the transgene could affect introgression.     

Down‐regulation of BnDA1, whose gene locus is associated with the seeds weight,improves the seeds weight and organ size in Brassica napus

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down‐regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down‐regulated in B. napus by over expressed of AtDA1^R358K, which is a functional deficiency of DA1 with an arginine‐to‐lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.     

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H⁺-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.     

The zygotic embryogenesis and ripening of Brassica rapa seeds provokes important alterations in the levels of free and conjugated abscisic acid and polyamines

Prior to the embryogenic study of Brassica rapa (turnip tops), the PA (polyamine) changes during flower development were determined. Whereas the flower was closed, no notable changes were found. However, pollination strongly stimulated the synthesis of the F (free) and ASC (acid soluble conjugated) forms, the pistil and not the petals being the main responsible. During embryogenesis, major alterations appeared in the synthesis of free and conjugated PA and ABA. Seeds, and not the silique wall, contributed most to the total content of the different F-PA (free polyamines) in the silique, this contribution involving all the PA studied. During seed desiccation, F-Put remained almost undetectable whereas F-Spd and F-Spm were very high. This is also applicable to ASC-Spd and ASC-Spm, this not being noted in the silique wall. During the first 5 stages of silique embryogenesis, the wall had higher levels of AIC-PA (acid insoluble conjugated polyamines) than in the seeds, primarily due to AIC-Spd and AIC-Spm. By contrast, during the middle and final phases AIC-PA was found more in the seeds than in the silique wall. The free and conjugated ABA showed the presence of 3 and 2 peaks in the seeds and the silique wall, respectively. The first corresponded to the onset of development, the second appeared when the highest moisture content was reached, and the third coincided with the triggering of carotenoid synthesis when the seed and the silique wall had lost more than 70–75% of their moisture. Finally, it is shown that the dry seeds of turnip tops were highly heterogeneous, not only in their germinative capacity, but also in the levels of free and conjugated ABA, which increased inversely to the germinative capacity.     

Population-based resequencing analysis of improved wheat germplasm at wheat leaf rust resistance locus Lr21

Little is known about the genetic control of heterosis in the complex polyploid crop species oilseed rape (Brassica napus L.). In this study, two large doubled-haploid (DH) mapping populations and two corresponding sets of backcrossed test hybrids (THs) were analysed in controlled greenhouse experiments and extensive field trials for seedling biomass and yield performance traits, respectively. Genetic maps from the two populations, aligned with the help of common simple sequence repeat markers, were used to localise and compare quantitative trait loci (QTL) related to the expression of heterosis for seedling developmental traits, plant height at flowering, thousand seed mass, seeds per silique, siliques per unit area and seed yield. QTL were mapped using data from the respective DH populations, their corresponding TH populations and from mid-parent heterosis (MPH) data, allowing additive and dominance effects along with digenic epistatic interactions to be estimated. A number of genome regions containing numerous heterosis-related QTL involved in different traits and at different developmental stages were identified at corresponding map positions in the two populations. The co-localisation of per se QTL from the DH population datasets with heterosis-related QTL from the MPH data could indicate regulatory loci that may also contribute to fixed heterosis in the highly duplicated B. napus genome. Given the key role of epistatic interactions in the expression of heterosis in oilseed rape, these QTL hotspots might harbour genes involved in regulation of heterosis (including fixed heterosis) for different traits throughout the plant life cycle, including a significant overall influence on heterosis for seed yield.