Light induces gene expression to enhance the synthesis of storage reserves in Brassica napus L. embryos.

KEY MESSAGE: In this manuscript, we disclosed the influence of light on the accumulation of storage reserves in B. napus embryos.1.Light induced the gene expression in the developing embryos of B. napus.2.Light promoted the starch synthesis in chloroplasts of B. napus embryos.3.Light enhanced the metabolic activity of storage reserve synthesis in B. napus embryos. Light influences the accumulation of storage reserves in embryos, but the molecular mechanism was not fully understood. Here, we monitored the effects of light on reserve biosynthesis in Brassica napus by comparing embryos from siliques grown in normal light conditions to those that were shaded or masked (i.e., darkened completely). Masked embryos developed more slowly, weighed less, and contained fewer proteins and lipids than control embryos. They also had fewer and smaller oil bodies than control embryos and lacked chloroplasts, where starch grains are usually synthesized. The levels of most amino acids, carbohydrates, and fatty acids were higher in masked embryos than in control or shaded embryos, whereas the levels of these metabolites in the masked endosperms were lower than those in control and shaded endosperm. Transcriptome analysis indicated that genes involved in photosynthesis (42 genes), amino acid biosynthesis (51 genes), lipid metabolism (61 genes), and sugar transport (13 genes) were significantly repressed in masked embryos. Our results suggest that light contributes to reserve accumulation in embryos by inducing the expression of metabolic genes, thereby enhancing the biosynthesis of storage reserves.

CHR721, interacting with OsRPA1a, is essential for both male and female reproductive development in rice.

KEY MESSAGE: CHR721 functions as a chromatin remodeler and interacts with a known single-stranded binding protein, OsRPA1a, to regulate both male and female reproductive development in rice. Reproductive development and fertility are important for seed production in rice. Here, we identified a sterile rice mutant, chr721, that exhibited defects in both male and female reproductive development. Approximately 5% of the observed defects in chr721, such as asynchronous dyad division, occurred during anaphase II of meiosis. During the mitotic stage, approximately 80% of uninucleate microspores failed to develop into tricellular pollen, leading to abnormal development. In addition, defects in megaspore development were detected after functional megaspore formation. CHR721, which encodes a nuclear protein belonging to the SNF2 subfamily SMARCAL1, was identified by map-based cloning. CHR721 was expressed in various tissues, especially in spikelets. CHR721 was found to interact with replication protein A (OsRPA1a), which is involved in DNA repair. The expressions of genes involved in DNA repair and cell-cycle checkpoints were consistently upregulated in chr721. Although numerous genes involved in male and female development have been identified, the mode of participation of chromatin-remodeling factors in reproductive development is still not well understood. Our results suggest that CHR721, a novel gene cloned from rice, plays a vital role in both male and female reproductive development.

Genome size analysis of field grown and somatic embryo regenerated plants in Allium sativum L.

In the present study, an efficient in vitro propagation protocol has been developed from clove explants of Allium sativum L., one of the oldest vegetable and medicinal plant used worldwide. Garlic is propagated vegetatively as cross-fertilization is strictly precluded due to sterile flowers. Due to a low rate of multiplication, limited genetic improvement possibility and increased germplasm degradation, plant tissue culture becomes an efficient and preferred tool for quality and rapid propagation of garlic. Here, the clove explants were cultured on Murashige and Skoog basal medium amended with different concentrations of Plant Growth Regulators (PGRs) namely 2,4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino purine (BAP), and 1-naphthalene acetic acid (NAA). Within 2 weeks of inoculation, white compact callus was formed, maximum callus induction frequency (85.99%) was on 1.5 mg l-1 2, 4-D added MS medium. Induced callus transformed into an embryogenic callus on 2, 4-D and BAP amended MS medium with highest embryogenic frequency (77.7%) was noted on 0.25 mg l-1 2, 4-D and 1.0 mg l-1 BAP added medium. Embryogenic callus differentiated into progressive stages of somatic embryos starting from globular, scutellar, and finally to coleoptilar stage of the embryo. Histological and scanning electron microscopic study of embryogenic callus was conducted, showing different stages of embryos, their origin and development, re-confirming somatic embryogenesis incidence in A. sativum. Green and mature somatic embryos were germinated and converted into plantlets on 0.5 mg l-1 BAP amended MS medium. The in vitro regenerated plants were cultured separately in IBA and NAA supplemented media for root induction. The MS medium amended with 1.0 mg l-1 IBA proved to be the best PGR treatment in inducing roots. The rooted plants were acclimatized and transferred ex vitro with about 87% survival rate. Cytological and flow cytometric analyses were performed to assess the genetic stability of in vitro regenerated plants. Cytological studies of in vitro regenerated plants showed 2n = 16 chromosome number and did not reveal any numerical variation in chromosomes. Flow cytometry was employed to measure the 2C DNA content of somatic embryo regenerated A. sativum plants and compared with in vivo grown garlic. The histogram peaks of relative 2C DNA content of in vitro regenerated plantlets were similar to the corresponding 2C DNA peak of in vivo grown plants. Flow cytometric 2C DNA content of embryo regenerated and field-grown A. sativum plants were the same, i.e., 33.45 pg and 33.56 pg, respectively, confirming genetic similarity. In conclusion, the present cytological and flow cytometric study suggest that the in vitro culture conditions are quite safe, did not encourage genetic alterations, and regenerants were "true to type."

The overexpression of rice ACYL-CoA-BINDING PROTEIN2 increases grain size and bran oil content in transgenic rice.

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while ß-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822, and -766/-762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value.

Microstructural and histochemical characteristics of Lycium barbarum L. fruits used in folk herbal medicine and as functional food.

Lycium barbarum L. fruits, referred to as functional food, have long been used in traditional and folk herbal medicine due to their therapeutic properties. The fruit microstructure was analysed using light, scanning and transmission electron microscopes. The distribution of bioactive compounds in drupe tissues was assessed with histochemical and fluorescence assays. The analysis of the microstructure has shown that the fruit is covered by a skin with an amorphous cuticle and a layer of amorphous epicuticular waxes on the surface. The skin is composed of a single-layered epidermis with thickened walls and one layer of hypodermis with slightly thickened periclinal walls. The pericarp cells contain different types of chromoplasts, which most often contained exhibited reticulotubules/fibrils of carotenoid pigments and phytoferritine deposits. The results of the histochemical assays demonstrated that the secondary metabolites with high phytotherapeutic importance were located in all layers of the pericarp and seeds and, specifically, in the drupe exocarp and endocarp. The phytochemicals were represented by polysaccharides (LBP), lipid compounds (carotenoids, essential oils, sesquiterpenes, steroids), polyphenols (tannins and flavonoids), and alkaloids. This study, which is the first report of the microstructure and localisation of bioactive compounds in wolfberries, is a valuable complement of phytochemical analyses and can be helpful for enhancement of the therapeutic effect of the fruit as well as preliminary assessment of the medicinal potential in the search for new pharmaceuticals. Detailed anatomical studies are crucial for exploration of determinants of fruit quality and useful for identification of diagnostic taxonomic traits.

Recovery of Oil Palm (Elaeis guineensis Jacq.) Somatic Embryos Cryostored For 20 Years.

　　BACKGROUND: A cryopreservation protocol has been established for oil palm somatic embryos (SEs), the efficiency of which must be evaluated, both in terms of regeneration and of long-term storage capacity, before its large-scale routine use. OBJECTIVE: To test the survival and recovery of 29 clones of oil palm somatic embryos cryostored for 20 years. MATERIALS AND METHODS: Clumps of SEs were pregrown for 7 days on medium containing 0.75 M sucrose, dehydrated in air-tight containers containing silica gel to moisture contents between 19-35% fresh weight, and then immersed directly in liquid nitrogen and stored in cryotanks for 20 years. RESULTS: Survival of SEs cryopreserved and rewarmed immediately displayed an average value of 19.1% for the 29 clones tested while survival of SEs rewarmed after 20 years of cryostorage was significantly higher, with an average of 33.2% for the 28 surviving clones. Out of these 28 surviving clones, three were lost due to contamination or regrowth decline, six produced only shoots and the rest proliferated. CONCLUSION: It is possible to cryostore oil palm SEs for extended periods and to regenerate proliferating cultures and plantlets from the cryopreserved material. The cryopreservation protocol established can thus be efficiently used to store oil palm germplasm and to manage large-scale production in industrial laboratories.

Arabidopsis LORELEI, a Maternally Expressed Imprinted Gene, Promotes Early Seed Development.

In flowering plants, the female gametophyte controls pollen tube reception immediately before fertilization and regulates seed development immediately after fertilization, although the controlling mechanisms remain poorly understood. Previously, we showed that LORELEI (LRE), which encodes a putative glycosylphosphatidylinositol-anchored membrane protein, is critical for pollen tube reception by the female gametophyte before fertilization and the initiation of seed development after fertilization. Here, we show that LRE is expressed in the synergid, egg, and central cells of the female gametophyte and in the zygote and proliferating endosperm of the Arabidopsis (Arabidopsis thaliana) seed. Interestingly, LRE expression in the developing seeds was primarily from the matrigenic LRE allele, indicating that LRE expression is imprinted. However, LRE was biallelically expressed in 8-d-old seedlings, indicating that the patrigenic allele does not remain silenced throughout the sporophytic generation. Regulation of imprinted LRE expression is likely novel, as LRE was not expressed in pollen or pollen tubes of mutants defective for MET1, DDM1, RNA-dependent DNA methylation, or MSI-dependent histone methylation. Additionally, the patrigenic LRE allele inherited from these mutants was not expressed in seeds. Surprisingly, and contrary to the predictions of the parental conflict hypothesis, LRE promotes growth in seeds, as loss of the matrigenic but not the patrigenic LRE allele caused delayed initiation of seed development. Our results showed that LRE is a rare imprinted gene that functions immediately after double fertilization and supported the model that a passage through the female gametophyte establishes monoalleleic expression of LRE in seeds and controls early seed development.

A mutational approach for the detection of genetic factors affecting seed size in maize.

KEY MESSAGE: Genes influencing seed size. The designation emp (empty pericarp) refers to a group of defective kernel mutants that exhibit a drastic reduction in endosperm tissue production. They allow the isolation of genes controlling seed development and affecting seed size. Nine independently isolated emp mutants have been analyzed in this study and in all cases longitudinal sections of mature seeds revealed the absence of morphogenesis in the embryo proper, an observation that correlates with their failure to germinate. Complementation tests with the nine emp mutants, crossed inter se in all pairwise combinations, identified complementing and non-complementing pairs in the F1 progenies. Data were then validated in the F2/F3 generations. Mutant chromosomal location was also established. Overall our study has identified two novel emp genes and a novel allele at the previously identified emp4 gene. The introgression of single emp mutants in a different genetic background revealed the existence of a cryptic genetic variation (CGV) recognizable as a variable increase in the endosperm tissue. The unmasking of CGV by introducing single mutants in different genetic backgrounds is the result of the interaction of the emp mutants with a suppressor that has no obvious phenotype of its own and is present in the genetic background of the inbred lines into which the emp mutants were transferred. On the basis of these results, emp mutants could be used as tools for the detection of genetic factors that enhance the amount of endosperm tissue in the maize kernel and which could thus become valuable targets to exploit in future breeding programs.

Immunolocalization of pectic polysaccharides during abscission in pea seeds (Pisum sativum L.) and in abscission less def pea mutant seeds.

BACKGROUND: In pea seeds (Pisum sativum L.), the presence of the Def locus determines abscission event between its funicle and the seed coat. Cell wall remodeling is a necessary condition for abscission of pea seed. The changes in cell wall components in wild type (WT) pea seed with Def loci showing seed abscission and in abscission less def mutant peas were studied to identify the factors determining abscission and non-abscission event. METHODS: Changes in pectic polysaccharides components were investigated in WT and def mutant pea seeds using immunolabeling techniques. Pectic monoclonal antibodies (1 â†’ 4)-ß-D-galactan (LM5), (1 â†’ 5)-α-L-arabinan(LM6), partially de-methyl esterified homogalacturonan (HG) (JIM5) and methyl esterified HG (JIM7) were used for this study. RESULTS: Prior to abscission zone (AZ) development, galactan and arabinan reduced in the predestined AZ of the pea seed and disappeared during the abscission process. The AZ cells had partially de-methyl esterified HG while other areas had highly methyl esterified HG. A strong JIM5 labeling in the def mutant may be related to cell wall rigidity in the mature def mutants. In addition, the appearance of pectic epitopes in two F3 populations resulting from cross between WT and def mutant parents was studied. As a result, we identified that homozygous dominant lines (Def/Def) showing abscission and homozygous recessive lines (def/def) showing non-abscission had similar immunolabeling pattern to their parents. However, the heterogeneous lines (Def/def) showed various immunolabeling pattern and the segregation pattern of the Def locus. CONCLUSIONS: Through the study of the complexity and variability of pectins in plant cell walls as well as understanding the segregation patterns of the Def locus using immunolabeling techniques, we conclude that cell wall remodeling occurs in the abscission process and de-methyl esterification may play a role in the non-abscission event in def mutant. Overall, this study contributes new insights into understanding the structural and architectural organization of the cell walls during abscission.

[Acceleration of Embryonic Development of Pinus sibirica Trees with a One-Year Reproductive Cycle].

The study of the formation of embryonic structures in Pinus sibirica forms with a one-year reproductive cycle showed that the acceleration of the embryonic process manifested itself as a reduction of the coenocytic stage of the female gametophyte development (1.5 months instead of 1 year). The egg was not fertilized because of the asynchronous maturation of male and female gametophytes. Seeds without embryos were formed. We assumed that the acceleration of the reproductive process in Pinus sibirica was caused by a mutation in the female generative organs.

Water Stress and Foliar Boron Application Altered Cell Wall Boron and Seed Nutrition in Near-Isogenic Cotton Lines Expressing Fuzzy and Fuzzless Seed Phenotypes.

Our previous research, conducted under well-watered conditions without fertilizer application, showed that fuzziness cottonseed trait resulted in cottonseed nutrition differences between fuzzy (F) and fuzzless (N) cottonseed. Under water stress conditions, B mobility is further limited, inhibiting B movement within the plant, affecting seed nutrition (quality). Therefore, we hypothesized that both foliar B and water stress can affect B mobility, altering cottonseed protein, oil, and mineral nutrition. The objective of the current research was to evaluate the effects of the fuzziness seed trait on boron (B) and seed nutrition under water stress and foliar B application using near-isogenic cotton lines (NILs) grown in a repeated greenhouse experiment. Plants were grown under-well watered conditions (The soil water potential was kept between -15 to -20 kPa, considered field capacity) and water stress conditions (soil water potential between -100 and -150 kPa, stressed conditions). Foliar B was applied at a rate of 1.8 kg B ha(-1) as H3BO3. Under well-watered conditions without B the concentrations of seed oil in N lines were higher than in F lines, and seed K and N levels were lower in N lines than in F lines. Concentrations of K, N, and B in leaves were higher in N lines than in F lines, opposing the trend in seeds. Water-stress resulted in higher seed protein concentrations, and the contribution of cell wall (structural) B to the total B exceeded 90%, supporting the structural role of B in plants. Foliar B application under well-watered conditions resulted in higher seed protein, oil, C, N, and B in only some lines. This research showed that cottonseed nutrition differences can occur due to seed fuzziness trait, and water stress and foliar B application can alter cottonseed nutrition.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

Starting to gel: how Arabidopsis seed coat epidermal cells produce specialized secondary cell walls.

For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls.

COBRA-LIKE2, a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE family, plays a role in cellulose deposition in arabidopsis seed coat mucilage secretory cells.

Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation.

The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development.

The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development.

[In situ investigation to three dimensional structures of Chinese medicines seeds].

This paper is aimed to microscopic identification of traditional Chinese medicines (TCMs) using an in situ imaging method. In this study, two kinds of Zingiberaceae seeds, Amomi Rotundus Fructus and Alpiniae Katsumadai Semen, were investigated by synchrotron radiation in-line X-ray phase-contrast computed tomography (IXPCT) imaging method. The results showed that the microstructures of these Zingiberaceae seeds could be clearly obtained from the virtual slices information in different observing angles. It proves that IXPCT is an effective imaging method, which can provide the imaging information for the microscopic identification of the intact TCMs in situ and non-destructively.

Distribution of potato spindle tuber viroid in reproductive organs of petunia during its developmental stages.

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.