Assessment of polyembryony in lemon: rescue and in vitro culture of immature embryos

The 27 lemon cultivars analysed could be considered slightly or moderately polyembryonic, with 25 to 43% of seeds being polyembryonic and from 1.3 to 1.6 embryos per seed. On this basis, it is necessary to rescue zygotic embryos at an immature stage. Rescue and in vitro embryo development have been studied in two Citrus limon polyembryonic cultivars. Sucrose (50 and 70 g/l) was combined with Murashige and Skoog and Gamborg’s B5 media and tested for optimal growth response. An important effect of genotype was observed: embryos from cultivar ‘Eureka’ had greater survival, germination percentage, and radical development. While the sucrose concentration in the medium did not have an effect on germination, the medium affected the embryo survival and root development of the seedlings, Gamborg’s B5 medium giving the best results. The ability to form plants in vitro was affected by an increase of embryo developmental stage. The germination and seedling height were greater with embryos of seeds collected 135–150 days after anthesis.     

几个芒果品种的胚性及多胚苗遗传分析

通过目测,筛选出芒果多胚种子,观察其多胚形态;将多胚种子培育成多胚苗,观察其生长发育状况;取多胚苗和母树叶片进行体细胞染色体数目鉴定及同工酶的分析.结果表明,多胚类群的品种中有单胚现象出现,土芒、吕宋、象牙单胚出现的频率分别为1.04%、3.85%和5.44%;土芒和象牙胚数出现的范围分别为2-6和2-8.多胚种子子叶形态和胚轴的着生位置各异.种子多胚的萌发率、成活率及多胚苗的生长状况均与多胚种子的胚数、子叶发育大小等因素相关.多胚苗体细胞染色体数目是2n=40,未发现染色体的数目变异.POD同工酶分析表明,同一种子的多胚苗间酶谱上存在着差异.     

Polyembryony and apomixis in Eriotheca pubescens (Malvaceae - Bombacoideae)

Apomixis and adventitious polyembryony have been reported for several species of Bombacoideae, including Eriotheca pubescens, a tree species of the Neotropical savanna (Cerrado) areas in Brazil. However, the origin of polyembryonic seeds and their importance for the reproduction of the species remained to be shown. Here, we analyzed the early embryology of this species to establish the apomictic origin of extranumerary embryos. We also observed the geographic distribution of polyembryony in E. pubescens, and tested if apomixis was related to the source of pollen (self or cross) and population density. Moreover, we tested if polyembryonic apomictic embryos would develop normally into seedlings. In the observed seed primordia, after a relatively long quiescent period, the zygote developed into a sexual embryo concurrently with adventitious apomictic embryos which developed from nucellus cells. Adventitious embryos develop faster than sexual ones and are morphologically similar, so that 44 days after anthesis it was virtually impossible to distinguish and trace the fate of the sexual embryo. Polyembryony is widely distributed in populations some 400 km distant, and only one strictly monoembryonic individual was observed during the study. The number of embryos per seed varied between fruits and individuals but was significantly higher in seeds from cross-pollinations than from selfs, although fruit and seed set after crosses were much lower than after selfs. Embryo development into seedlings depended on their weight at germination, but polyembryonic seeds germinated and produced up to seven seedlings per seed in greenhouse conditions. Adventitious embryony and apomictic seedlings would explain the mostly clonal populations suggested by molecular studies.     

几种具无融合生殖特性的植物多胚和多苗现象的观察

报道6种具无融合生殖特性的植物种子的胚数和萌发实生苗数的观察结果。金桔(Fortunellamargarita (Lour.)Swingle)、蜜桔(Citrus unshiu Marcoritch)和花椒(Zanthoxylum bungeanum Maxim.)具珠心胚,含多胚种子频率分别为97.50％、100％和45.00％;多胚种子的胚数范围分别为2～49,3～54和2～6。草地早熟禾(Poa pratensis L.)、滨草(Elymus rectisetus)和湖北海棠(Malus hupehensis(pampon.)Rehd.)具非减数配子体无融合生殖特性,含多胚种子频率依次为34.25％、8.11％和37.50％;前两种的多胚种子中胚数范围为2～3,后者为2～15。蜜桔、草地早熟禾和湖北海棠种子萌发多苗的频率分别为22.00％、6.14％和2.22％。描述了多胚种子中胚的形态、位置和分布。对6种植物含单胚种子的胚的来源进行了分析。初步结论:1.具无融合生殖现象的植物种子含多胚和萌发多苗的特性可作为寻找具无融合生殖特性的植物的形态学指标;2.对其筛选的材料进行大孢子发生、胚囊形成和早期胚胎发育的研究,以期阐明多胚来源和生殖类型。     

AFLP markers closely linked to a major gene essential for nucellar embryony (apomixis<Emphasis Type="Italic">)</Emphasis> in <Emphasis Type="Italic">Citrus maxima</Emphasis> × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Some citrus varieties express a form of apomixis termed nucellar embryony in which the adventive embryos develop from nucellus tissue surrounding the embryo sac. This trait results in many seeds containing multiple embryos (polyembryony). Inheritance of the frequency of polyembryony was studied in 88 progeny from a cross of Citrus maxima (monoembryonic) × Poncirus trifoliata (polyembryonic). The frequency of polyembryonic seed produced by each progeny was determined by scoring 100–500 seeds for the number of seedlings to emerge from each seed. Two groups of eight individuals from each extreme of the population were chosen for bulked segregant analysis with amplified fragment length polymorphism markers amplified with 256 primer combinations. Candidate markers identified in the bulks as linked to the trait were tested on the 32 individuals used to create the bulks and then on the remaining plants in the population. Five candidate markers tightly linked to polyembryony in P. trifoliata were identified. Specific marker alleles were present in nearly all progeny that produced polyembryonic seed, and alternate alleles were present in nearly all progeny that produced only monoembryonic seed. The region defined by these markers very likely contains a gene that is essential for the production of polyembryonic seeds by apomixis, but also shows segregation distortion. The proportion of polyembryonic seeds varied widely among the hybrid progeny, probably due to other genes. Scoring 119 progeny of a P. trifoliata selfed population for the closely linked markers and the proportion of polyembryonic seeds confirmed close linkage between these markers and polyembryony.     

Polyploidy and polyembryony in Anemopaegma (Bignonieae, Bignoniaceae)

Polyploidy is a key process in plant evolution, with the asexual formation of embryos representing a way through which polyploids can escape sterility. The association between polyploidy and polyembryony is known to occur in Bignoniaceae. In this study, we investigate polyembryony in four polyploid species of Anemopaegma: A. acutifolium, A. arvense, A. glaucum and A. scabriusculum as well as in one diploid species, A. album. Polyembryony was observed only in polyploid species. We used seed dissection and germination tests to compare the number of polyembryonic seeds. We tested how the pollen source influences the number of polyembryonic seeds and the number of embryos per seed and tested the correlation between the number of viable seeds per fruit and mean number of embryos per seed. The number of polyembryonic seeds observed by seed dissection was higher than the number of polyembryonic seeds determined by the germination test, with the number of embryos produced per seed being higher than the number of seedlings. The dissection of seeds of A. glaucum indicated that a higher number of polyembryonic seeds and a higher number of embryos were present in seeds from cross-pollination than in seeds from self-pollination. On the other hand, germination tests indicated that a higher number of polyembryonic seeds were present in fruits from self-pollination than from cross-pollination. The mean number of embryos per seed was not influenced by the number of viable seeds per fruit in fruits from open pollination. These results indicate a positive relationship between polyembryony and polyploidy in Anemopaegma.     

Polyembryony increases embryo and seedling mortality but also enhances seed individual survival in Handroanthus species (Bignoniaceae)

Polyembryony seems to be advantageous to mother plants in detriment of their siblings which face competition since the beginning of seed development. This competition may limit the turnover of embryos into seedlings and their survival ability. We analysed polyembryony frequency and embryo to seedling turnover in three Handroanthus species with sporophytic apomixis. We tested if the embryo number per seed affected seed and embryo morphometry, seedling survival ability and seed individual survival (i.e. survival of at least one seedling per seed). The number of embryos per seed was compared with seedling number at different developmental stages. All 14 populations showed high frequencies of polyembryonic seeds (21–91%). As the number of embryos per seed increased (up to eight embryos/seed), there was a reduction of mean embryo mass, area, seedling length, individual seedling survival ability, and embryo to seedling turnover. There was also an increase in embryo morphological anomalies. However, enhanced seed individual survival was also observed. Thus, the high frequency of polyembryonic seeds and the increase in seed individual survival support the idea that polyembryony represents an alternative reproductive mechanism which can favours these species.     

Reduction of generation time in Eucalyptus globulus

The plant growth retardant paclobutrazol was applied by foliar spray to seedlings of Eucalyptus globulus (Labill.) to determine its effects on reproductive activity. It was shown to induce flowering in vegetatively juvenile seedlings and increase reproductive activity in vegetatively adult material. The former case represented a 50% (three year) reduction in generation time for commercial seedlings. Paclobutrazol treatment did not appear to have a negative effect on any aspect of reproductive development, germination of new seed, or early seedling growth, while having a distinct positive effect on reproductive output. This suggests that paclobutrazol may be an effective tool enabling tree breeders to reduce generation time and increase reproductive output in E. globulus. Foliar spraying during autumn was shown to be an effective means and timing of paclobutrazol application, with results evident in the following flowering season. Considerable persistence of paclobutrazol effects was noted in the second season after application. Pot size and growth temperatures also play roles in determining the extent of new reproductive activity each season in E. globulus seedlings.     

Genetic analysis of a polyembryonic mutant in rye

We have obtained one plant regenerated from rye tissue culture which showed a high percentage of polyembryonic seeds in its progeny. The mutation inducing the development of extra embryos is also influencing erroneous cell division, mitosis and meiosis. The genetic analysis indicated that the aptitude for polyembryonic seed formation is a heritable trait controlled by a dominant gene. However, for expression of the phenotype the female parent should have a specific cytoplasm.     

Fungal endophytes in seeds and needles of Pinus monticola

Using a sequence-based approach, we investigated the transmission of diverse fungal endophytes in seed and needles of Pinus monticola, western white pine. We isolated 2003 fungal endophytes from 750 surface-sterilized needles. In contrast, only 16 endophytic isolates were obtained from 800 surface-sterilized seeds. The ITS region was sequenced from a representative selection of these endophytes. Isolates were then assigned to the most closely related taxa in GenBank. Although 95 % of the endophytes in needles from mature trees belonged to the Rhytismataceae, 82 unique ITS sequences were obtained from at least 21 genera and 10 different orders of fungi. Significantly, none of the endophytes in seed were rhytismataceous (χ² = 180; P < 0.001). Similarly, needles of greenhouse seedlings yielded only non-rhytismataceous isolates, whereas seedlings of the same age that had naturally regenerated near older white pines in roadless areas were colonized by rhytismataceous endophytes almost to the same extent as in mature trees. Only one of 17 rhytismataceous isolates were able to grow on a medium containing only 0.17 % nitrogen, whereas 25 of 31 non-rhytismataceous endophytes grew. Rhytismataceous endophytes are dominant in needles of P. monticola, but they appear to be absent in seed, and unlikely colonists of nitrogen-limiting host tissues such as the apoplast.     

MEGAGAMETOPHYTE ORGANIZATION IN A POLYEMBRYONIC LINE OF LINUM USITATISSIMUM

A haploid-diploid twin-producing polyembryonic line “RA91” of Linum usitatissimum L., flax, known to produce twin embryos with a frequency in excess of 32% was employed to determine the cytological factors relating to the production of haploid embryos using light and transmission electron microscopy. Potentially polyembryonic flax megagametophytes contain at maturity either a conventional 3-celled egg apparatus (36% of megagametophytes), or contain supernumerary eggs forming either a 4- (50%) or 5-celled (14%) egg apparatus. In megagametophytes with supernumerary eggs, one egg cell appears to occupy the conventional position, whereas additional supernumerary eggs surmount the synergids and are smaller cells presumably derived from division of the true egg. Although supernumerary eggs appear similar in ultrastructure, they differ in location, size, and unlike typical egg cells, appear to have a complete, contiguous cell wall adjacent to the synergid. Such supernumerary cells are postulated as the origin of such haploid embryos, with the diploid member formed by conventional fertilization events with the micropylar egg.     

咖啡多胚现象与多胚苗形态发育的研究

通过目测筛选出咖啡多胚豆(种子),对其进行离体胚培养获得幼小植株,观察分析了多胚及其幼苗的形态发育特征。结果表明,不同倍性咖啡种质均存在多胚现象,不同材料的双胚率不同(变幅在1.06‰ ~ 4.46‰）,其中四倍体材料普遍比二倍体的高。多胚豆中胚的形态和着生位置各异。多胚再生植株生长发育特点各不相同,一般大胚再生植株生长发育正常;小胚植株长势弱,出现畸形植株及生长过程中夭亡等现象。同一咖啡豆中远离正常胚位的额外胚可能是由胚囊外的体细胞发育而形成的不定胚。     

The frequency of polyembryonic seedlings and polyploids from ms1 soybean

Summary Seed from homozygous recessivems ₁ genetic male-sterile soybean (Glycine max (L.) Merr.) plants was studied for frequencies of polyembryonic seedlings and different levels of polyploidy among abnormal seedlings from six different source populations: Amesms ₁ (Ams), North Carolinams ₁ (NCms), Tonicams ₁ (Tms), Urbanams ₁ (Ums), and F₄ generation seed obtained from crosses ofms ₁ to two chromosome interchange lines (Ams x Clark T/T and Ums x KS-172-11-3). Frequencies of polyembryony observed in Tms, Ums, Ams, NCms, F₄ seed from Ams x Clark T/T, and F₄ seed from Ums x KS-172-11-3 were 3.6%, 2.4%, 3.1%, 2.5%, 2.2% and 0.1%, respectively. Frequencies of abnormal seedlings from these six sources varied from 1.7% (Ums X KS-172-11-3) to 16.8% (Ams X Clark T/T). Frequencies of polyploids among the abnormal seedlings ranged from 6.8% in Ums x Ks-172-11-3 to 66.7% in Tms. On average, the frequency of polyploid individuals from monoembryonic seedlings was 1.22%. Chromosome number of these seedlings varied from 20 to 200. Variation of the frequencies of polyembryonic seedlings and polyploid progeny among abnormal seedlings suggested that the mechanism(s) controlling the characters of polyembryony and formation of polyploids was associated with thems ₁ gene and was affected by other gene(s) or environmental factors.Joint contribution: Agricultural Research Service, US Department of Agriculture, and Journal Paper No. J-11255 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA. Project 2471     

Polyembryony in Citrus. Accumulation of seed storage proteins in seeds and in embryos cultured in vitro.

Citrus exhibits polyembryonic seed development, an apomictic process in which many maternally derived embryos arise from the nucellus surrounding the developing zygotic embryo. Citrus seed storage proteins were used as markers to compare embryogenesis in developing seeds and somatic embryogenesis in vitro. The salt-soluble, globulin protein fraction (designated citrin) was purified from Citrus sinensis cv Valencia seeds. Citrins separated into two subunits averaging 22 and 33 kD under denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cDNA clone was isolated representing a citrin gene expressed in seeds when the majority of embryos were at the early globular stage of embryo development. The predicted protein sequence was most related to the globulin seed storage proteins of pumpkin and cotton. Accumulation of 33-kD polypeptides was first detected in polyembryonic Valencia seeds when the majority of embryos were at the globular stage of development. Somatic Citrus embryos cultured in vivo were observed to initiate 33-kD polypeptide accumulation later in embryo development but accumulated these peptides at only 10 to 20% of the level observed in polyembryonic seeds. Therefore, factors within the seed environment must influence the higher quantitative levels of citrin accumulation in nucellar embryos developing in vivo, even though nucellar embryos, like somatic embryos, are not derived from fertilization events.     

In vitro embryo culture of Scirpus acutus Muhl.

An in vitro embryo culture protocol was developed for Scirpus acutus Muhl. A maximum of 85.6% of germination was obtained when isolated embryos were cultured in vitro, a result similar to those reported in the literature with traditional dormancy breaking treatments. In vitro seedling development was optimal in half-strength Murashige and Skoog (1962) liquid medium. An average of 3–4 shoots were produced from the initial seedlings. Clusters of plantlets were successfully acclimatized and transferred to soil. These results corroborate the findings of previous studies stating that seed dormancy in Scirpus is caused by the seed/fruit coats. In vitro embryo culture thus allows for the production of Scirpus acutus Muhl. seedlings that can be transferred to natural or artificial wetlands.     

Non-target effects of neonicotinoid seed treatments; mortality of coccinellid larvae related to zoophytophagy

Third and fourth instar Harmonia axyridis Pallas larvae feed directly on corn seedlings; therefore they may be at risk if the seedlings are chemically treated. Neonicotinoids are broad-spectrum systemic insecticides that are frequently applied to corn seeds prior to planting to protect seedlings from early-season root and leaf-feeding. In this study, H. axyridis larvae were exposed for 360 min to corn seedlings that had been grown from seeds treated with either thiamethoxam or clothianidin. Neurotoxic symptoms (trembling, paralysis, and loss of coordination) were observed in 72% of the H. axyridis larvae; recovery following neurotoxic symptoms rarely occurred (7%). Starvation of larvae for 48 h prior to seedling exposure may increase tissue consumption; however, there was no difference in the number of starved or satiated larvae that displayed neurotoxic symptoms and died. Neurotoxic symptoms and larval mortality were significantly more likely to occur if larvae were exposed to seedlings grown from seeds treated with neonicotinoids compared to the control. In addition, clothianidin resulted in significantly greater larval mortality (80%) compared to thiamethoxam (53%). Symptoms and mortality were rarely observed if seedlings were not treated with a neonicotinoid seed treatment (3% and 3%, respectively). Coccinellids are abundant in agroecosystems in which neonicotinoid seed treatments are likely to be applied. Therefore, the use of neonicotinoids may have negative effects on these non-target species if early-season leaf-feeding occurs in the field.     

Fungicide seed treatments increase growth of perennial ryegrass

Field, laboratory and glasshouse experiments were carried out to measure effects of seed treatments with captan or thiram on growth of perennial ryegrass (Lolium perenne L.). Field-sown captan- or thiram-treated seed gave twice as many seedlings as untreated seed. Spaced plants growing from fungicide-treated seed produced almost 6 times more dry matter 16 weeks after sowing than those from untreated seed. This effect, though diminishing with time, was still apparent more than a year after sowing. Fungicides in sterile agar growth medium were phytotoxic to seedlings at concentrations of 10μg/ml and greater. Seedlings grown from treated seeds sown from 5 to 15 mm away from developing colonies of the virulent seedling pathogenFusarium oxysporum Schlecht. were more than 4 times larger than those grown from untreated seeds. Captan-treated seed sown into pots containing field soil produced more and larger seedlings than untreated seed. Methyl bromide fumigation of the same soil also increased both number and size of seedlings. Fungicidal, rather than direct chemical effects, at early stages of seedling growth, account for increased growth of plants from fungicide-treated seed.     

Redundancy modulation of nuclear DNA sequences in Dasypyrum villosum

In order to assess fluid domains in the genome of Dasypyrum villosum, Feulgen/DNA cytophotometric determinations and molecular and cytological DNA-DNA hybridization experiments were carried out in resting embryos and developing seedlings from yellow and brown caryopses belonging to different populations. The cytophotometric data showed that the basic amount of nuclear DNA is, on average, 12% higher in 2-day-old seedlings from yellow caryopses as compared to those from brown caryopses. It increases in each individual during seed germination, to a higher extent in seedlings from yellow caryopses than in those from brown caryopses. DNA content also differs up to 13% between plants within a caryopsis-colour group and up to 40% between populations. Dot-blot hybridization of a 396-bp D. villosum-specific DNA repeat to genomic DNA extracted from embryos in dry seeds, or from seedlings belonging to single progenies of plants from different populations, confirmed the cytophotometric results. The redundancy in the genome of sequences hybridizing to the 396-bp element differs significantly both between populations and between plant progenies within a population. During seed germination these sequences are the more amplified the less they are redundant in the genome of resting embryos, and amplification occurs to a significantly-greater extent in seedlings from yellow caryopses than in those from brown caryopses. ³H-labelled 396-bp sequences hybridize at or near the telomeres of most chromsome pairs though only to the shorter of the two subtelocentric pairs. The hybridization level is higher in seedlings from yellow caryopses that in those from brown caryopses, and a linear correlation exists between the number of silver grains counted over the labelled regions of each chromosome pair in the two groups of seedlings. Possible control mechanisms of the observed changes in the nuclear genome, and the role of these changes in developmental pregulation and environmental adaptation, are discussed.     

Embryo culture and taxane production inTaxus spp

Summary We have previously shown (Flores and Sgrignoli, 1991) that immature embryos ofTaxus brevifolia andT. X media are capable of precocious germination and can grow into seedlings in vitro. The cultural and environmental parameters for embryo germination and conversion into seedlings have been optimized and extended toT. baccata andT. cuspidata. A 14-h photoperiod improved embryo germination and growth into seedlings. A pregermination cold treatment of the seeds had a positive effect on both the onset and percentage of germination. Embryos from cold-treated seeds germinated earlier and at a higher frequency than those from control seeds. Boron was necessary for embryo germination, and levels of this micronutrient were established for optimal growth and germination ofT. brevifolia andT. X media cv. Hicksii embryos. Gupta and Durzan’s medium was superior to White’s for embryo germination and root formation. Naphthaleneacetic acid stimulated root formation in embryo-derived seedlings. We also found that immature embryos could be induced to form callus with embryogenic potential. Taxol and related taxanes were detected in embryo- derived seedlings.