Effects of SPAD Decline Value of Mid-season Hybrid Rice Leaves after Full-heading Stage on Productivity of Rice Fertilized with Nitrogen

[Objective] The aim was to research relationship between SPAD decline index after full-heading stage （SDIFHS） and productivity of rice. fertilized with nitro- gen in order to provide theoretical and practical references for selection and breed- ing of rice varieties. [Method] From 2008 to 2010, 18 mid-season hybrid rice vari- eties were researched every year to explore relationship＇ between SDIFHS and pro- ductivity of rice fertilized with nitrogen. [Result] The productivity of rice fertilized with nitrogen was of extremely significant positive corretation with SDIFHS, because the higher SPAD decline index is, the higher LAI decline index and the transformation ratio of dry matter to spikes in overground plant would be. [Conclusion] The re- search established a new method to predict productivity of rice fertilized with nitro- gen based on SPAD decline index.

Effects of Varying Temperature and Constant Temperature on Hatching Result of Fertilized Peacock Eggs

In order to investigate the effect of temperature on hatching result of fertilized peacock eggs, they were hatched under conditions of varying temperature and constant temperature respectively. The result shows that the hatching rate in the varying temperature treatment group （Group Ⅰ） is higher than that in the constant temperature treatment group （Group Ⅱ）. It suggests that the varying temperature treatment （high temperature in the early period, medium temperature in the middle period, slightly lower temperature in the late period, and slightly higher temperature in the hulling period） is in line with the optimum temperature variation in the hatching of fertilized peacock eggs, contributing the hatching of fertilized peacock eggs.

Screening of Saprolegniasis Preventive Drugs for Golden Trout (Oncorhynchus mykiss) Fertilized Eggs During Hatching

In order to screen the effective preventive drugs for saprolegniasis occurred during hatching of the fertilized eggs of golden trout, effects of two kinds of self-made Chinese herbal compounds（AB） and two kinds of chemical bactericides（peroxyacetic acid andhydrogen peroxide, CD） with three disinfection concentrations were compared in this study. The fertilized eggs of golden trout were randomly divided into 13 groups, each group with two parallels of 1 000 eggs. Drugs at high,medium and low concentration were respectively used to immerse the fertilized eggs35 min for disinfection once a day. The numbers of live and dead eggs were recorded each day before disinfection and the numbers of survival eyed eggs were counted in each experimental group after finishing the whole test. The results indicated that all of four kinds of drugs used here had a certain effect on prevention and control of saprolegniasis during hatching of fertilized eggs of golden trout, and also showed a correlation between the concentrations and effects. After the selection of several drugs, we found that group B-medium, C-medium and D-high exhibited the most remarkable effects on control of saprolegniasis. However, careful consideration should be given to chemical drugs residues and their potential hazard to the eyed eggs. Therefore, Chinese herbal compound B was suggested to be used for prevention and control of saprolegniasis to achieve the healthy aquaculture. And the concentration and operation time of Chinese herbal compound B should be strictly controlled when used in disinfection of fertilized eggs.

Production Cost of Heterobranchus longifilis (Valenciennes, 1840) Fingerlings in the Ponds Fertilized with Poultry Droppings

This study aims to evaluate the production cost of Heterobranchus longifilis larvae reared in fertilized fish ponds. Experiment was carried out in two fish ponds. Each fish pond was filled with 200 liters of water and fertilized with dry poultry droppings (dose: 0.6 g.L-1) and zooplankton was inoculated with 83±13 individuals/L. Six days after inoculation of zooplankton, larvae of H. longifilis of 2 days after post-hatching were directly introduced into ponds with 300 individuals/m3. These larvae used exclusively zooplankton during the first 7 days. From the 8th day, larvae were fed with Coppens feed until 30 days. At the end, the survival rates were ranged between 58.33% and 56.67%. Final mean weights were brought up to all the ponds and were notsignificantly different (p > 0.05). The production costs ranged between 2.55 FCFA and 2.60 FCFA per fingerling with ratio production cost/weight gain around 0.80 FCFA/g in each pond. Then, this production system of fingerlings in fertilized ponds is very efficient and could be popularized in the rural fish farms.

Analysis of Chemical and Biological Soil Properties in Organically and Conventionally Fertilized Apple Orchards

We compared chemical and biological properties of soils in organically and conventionally fertilized apple orchards in Nagano Prefecture (one of the major apple producing regions in Japan). Five apple orchards with different fertilizer management systems were used for this study. The total carbon and total nitrogen contents were higher in the organically fertilized orchard, while the total phosphorus and total potassium were at similar levels in both organically and conventionally fertilized orchards. The bacterial biomass did not differ between the two orchards, but the N circulation activity was clearly higher in the organically fertilized orchard from April to December. Total carbon from 50,000 to 60,000 mg/kg, total nitrogen at about 3000 to 4000 mg/kg, and a C/N ratio of 15 - 20 were suggested to be suitable conditions for a high level of apple production under an organic fertilizer management system.

Correlation and Regressive Model Between Spikelet Fertilized Rate and Temperature in Inter-Subspecific Hybrid Rice

To study the sensitivity of inter-subspecific hybrid rice to climatic conditions, the spikelet fertilized rate （SFR） of four types of rice including indica-japonica hybrid, intermediate hybrid, indica and japonica were analyzed during 2000-2004. The inter-subspecific hybrids showed lower SFR, and much higher fluctuation under various climatic conditions than indica and japonica rice, showing the inter-subspecific hybrids were sensitive to ecological conditions. Among 12 climatic factors, the key factor affecting rice SFR was temperature, with the most significant factor being the average temperature of the seven days around panicle flowering （T7）. A regressive equation of SFR-temperature by T7, and a comprehensive synthetic model by four important temperature indices were put forward. The optimum temperature for inter-subspecific hybrids was estimated to be 26.1-26.6℃, and lower limit of safe temperature to be 22.5-23.3℃ for panicle flowering, showing higher by averagely 0.5℃ and 1.7℃, respectively, to be compared with indica and japonica rice. This suggested that inter-subspecific hybrids require proper climatic conditions. During panicle flowering, the suitable daily average temperature was 23.3-29.0℃, with the fittest one at 26.1-26.6℃. For an application example, optimum heading season for inter-subspecific hybrids in key rice growing areas in China was as same as common pure lines, while inferior limit for safe date of heading was about a ten-day period earlier than those of common pure lines.

Analysis of proteomic profiling of mouse embryonic stem cells derived from fertilized, parthenogenetic and androgenetic blastocysts

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation embryos. ESCs exhibit true pluripotency, i.e., the ability to differentiate into cells of all three germ layers in the developing embryo. We used 2-DE MALDI-TOF/TOF to identify differentially expressed proteins among three types of mouse embryonic stem cells (ESCs) derived from ferti-lized, parthenogenetic, and androgenetic (fESC, pESC and aESC, respectively) blastocysts. We detected more than 800 proteins on silver- stained gels of whole protein extracts from each type of ESC. Of these, 52 differentially expressed proteins were identified by the MALDI–TOF/TOF analyzer, including 32 (fESCs vs. pESCs), 28 (fESCs vs. aESCs) and 17 (pESCs vs. aESCs) prominent proteins with significantly higher or lower expression relative to the comparison cells. Among the 32 proteins from fESCs, 12 were significantly increased in expression and 20 were reduced in expression in fESCs com-pared with pESCs. Similarly, 10 of 28 and 8 of 17 proteins were more highly expressed in fESCs and pESCs compared with aESCs, respectively. In contrast, 18 of 28 and 9 of 17 proteins were reduced in expression in fESCs and pESCs compared with aESCs, respectively. Of the eight protein candidates in fESCs, four were in-creased and four were decreased in expression relative to both pESCs and aESCs in the 2-DE analysis. Differential expression of these pro-teins were confirmed by mRNA expression analysis using real time RT-PCR. For three pro-teins, ANXA5, CLIC1 and SRM, Western blot analysis corroborated the expression patterns indicated by the 2-DE results. ANXA5 and CLIC1 were increased in expression and SRM was de-creased in expression in fESCs compared with both pESCs and aESCs. The differentially ex-pressed proteins identified in the present study warrant further investigation towards the goal of their potential application in ESC therapy.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid(LPA)is a small molecule glycerophospholipid,which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.In this study,sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.At first,the maturation medium containing estrus female sheep serum and synthetic oviduct fluid(SOF)were optimized for sheep IVF,and then the effects of LPA were investigated.From 0.1 to 10μmol L^(–1),LPA had no significant effect on the cleavage rate(P>0.05),but the maturation rate and blastocyst rate increased dependently with LPA concentration(P<0.05),and the blastocyst morphology was normal.When the LPA concentration was 15μmol L^(–1),the maturation rate,cleavage rate and blastocyst rate decreased significantly(P<0.05),and the blastocyst exhibited abnormal morphology and could not develop into highquality blastocyst.Besides,the exogenous LPA increases the expression of LPAR2,LPAR4,TE-related gene CDX-2and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10μmol L^(–1).The expression of LPAR2,LPAR4,CDX-2 and OCT-4 from the LPA-0.1μmol L^(–1)to LPA-10μmol L^(–1)groups in early embryos were extremely significant(P<0.05),while the expression of these genes significantly decreased in 15μmol L^(–1)LPA-treated embryos compared with LPA-10μmol L^(–1)group(P<0.05).The inner cell mass in 15μmol L^(–1)LPA-treated embryos was also disturbed,and the blastocysts formation was abnormal.Secondly,the sheep IVF blastocysts were applied to establish embryonic stem cells.The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10μmol L^(–1),while 15μmol L^(–1)LPA decreased OCT-4 and CDX-2 expression in the derived cells.The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1μmol L^(–1)group and LPA-10μmol L^(–1)group extremely significantly increased(P<0.05),but there was significant decrease in LPA-15μmol L^(–1)group compared with LPA-10μmol L^(–1)group(P<0.05).Meanwhile,the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10μmol L^(–1)concentration.This study references the IVF embryo production and embryonic stem cell research of domestic animals.

Effect of preparation program on maternal anxiety of mothers fertilized through in vitro fertilization

Objective: To determine the effect of preparation to play a maternal role in mothers fertilized through in vitro fertilization on maternal anxiety. Methods: A total of 60 mothers undergoing in vitro fertilization were assigned into intervention group and control group. Pregnancy concerns and stress questionnaires were research tools. Questionnaires were completed in both groups before organizing preparation program. Then, the preparation program was held for four sessions for the mothers in the intervention group, and the subjects in both groups were immediately investigated again after completion of preparation program and one month later. Data analyses about pregnancy stress and concerns of mothers and its dimensions were performed by repeated measure (analysis of variance), Mann-Whitney, Friedman and Wilcoxon tests. Analysis of demographic variables was performed by using independent t-test and Chi-square test in SPSS 21. Results: The score of pregnancy stress and concerns before the intervention was not significantly different between the two groups. There was a significant difference in the intervention group in the score of pregnancy stress and concerns before and after the intervention (P<0.001), before and one month after the intervention (P<0.001), immediately after and one month after the intervention (P<0.001) which was not significant in the control group. Conclusions: Maternal preparation program can be effective in reducing maternal stress and concerns.

USING CYTOGENETIC ANALYSIS OF MOUSE EGGS FERTILIZED IN VITRO FOR MONITORING THE MUTAGENICITY OF CHEMICALS ACTING ON GERM CELLS——1.Method for preparation of slides of chromosome of mouse eggs at the first cleavage metaphase and the preliminary use

In this experiment,the sperms were obtained from(C57×CBA) F1 male mice and the eggs from NIHstrain female mice.Using the fertilization techni-que in Vitro,the slides of chromosome of mouse eggsat the first cleavage metaphase were prepared.

Effects of Addition of Lactobacillus plantarum and Enterococcus faecium Inoculants to High-Nitrogen Fertilized Timothy (Phleum pratense L.) on Fermentation, Nutritive Value, and Feed Intake of Silage

The objective of this study was to examine the effects of addition of inoculant to high-nitrogen (N) fertilized timothy on fermentation, nutritive value, and feed intake of silage. The silage of timothy cultivated with a N fertilizer rate at two levels (high level (H), standard level (S)) was prepared with (SI, HI) or without (SC, HC) an inoculants. The CP content of H increased by 38 g&middotkg-1 DM compared with that of S, and the WSC and ADF contents decreased compared with those of S. Regarding the fermentation of silage, the pH and NH3-N ratio was significantly lower in the silage with inoculant (SI and HI), showing improvement of the fermentation compared with those of the silage without the addition (SC and HC). The CP content was significantly higher in HC and HI than in SC and SI, and the NDF content was the lowest in HI among the four treatments. The CP digestibility of HC and HI was significantly higher than those of SC and SI. The EE digestibility of SI was significantly higher than that of SC, and that of HI was significantly higher than that of HC. The DCP contents were significantly higher in HC and HI than in SC and SI. The TDN content was the lowest in SC and highest in HI. The DCP intakes of HC and HI were significantly higher than those of SC and SI. When the N fertilizer rate was increased, the DCP content and DCP intake of the silage increased, and the addition of inoculant improved the fermentation and increased the TDN content.

The Influence of Mycorrhizal Fungi on the Growth of Apple and Sour Cherry Maidens Fertilized with Different Bioproducts in the Organic Nursery

Apple/Malus x domestica cv. ＇Topaz＇/M26 and sour cherry cv. ＇Debreceni Botermo＇/Prunus mahaleb L. maidens were fertilized with different organic fertilizers and soil amendments in an organic nursery. A mycorrhizal inoculant, Micosat, was added to the non-fertilized control and to combinations fertilized with various bioproducts. Under assessment was the effect of nutrient delivery on the growth and development of maiden fruit trees focussing on tree height, thickness of the tree trunk, the number and the length of lateral shoots in each treatment combination. It was found that the introduction of mycorrhizal fungi into the soil without fertilizer application significantly improved the growth and development of apple and sour cherry maidens. Mineral fertilizer application （NPK） combined with mycorrhizal inoculation had a negligible effect on the above features. In the presence of bioproducts such as granular manure, Humus Active ＋ Aktywit PM, BF Amin, Vinassa and Florovit Eko, the mycorrhizal fungi were more biologically active than in the absence of these products.

How many donor colonies should be cross-fertilized for nursery farming of sexually propagated corals?

Coral reef restoration approaches have often utilized adult colonies as sources for fragments (i.e. clones) to be transplanted. Although restoration through this method is fast and cheap, it has been pointed out that it may reduce genetic diversity of the restored population. Low genetic diversity is a concern for reef restoration when seed fragments are raised asexually from only a few donor colonies. This can lead to lower fertilization rates among seed fragments, and reducing the longterm benefits of reef restoration in particular areas. Additionally, low genetic diversity can compound the effects of increased ocean temperature and other environmental stressors, further jeopardizing the health of a reef. An alternative approach through sexually propagated coral cultures and out-plantings can alleviate this problem. Sexually produced offsprings are more genetically diverse. They can be produced in far greater numbers than coral fragments and do not imply destructive methods. Ongoing research at the Akajima Marine Science Laboratory in Okinawa, Japan has helped to improve the production and maintenance of sexually propagated larval cultures. Our results show that crosses between gametes from 6 or more colonies will provide the highest fertilization rate (>95%). Based on the results, we suggest the use of 6 or more donor colonies for practical gamete fertilization in sexually derived coral culture.

Differentiating between fertilized and unfertilized eggs prior to incubation based on oxygen flux measurement

One unresolved challenge in the egg industry is how to efficiently and non-invasively detect unfertilized eggs prior to incubation.This detection ability would not only significantly improve hatching rates and reduce costs but also conserve incubator space and prevent poor-quality embryos from contributing to the spread of infections.This study demonstrates a procedure for distinguishing between fertilized and unfertilized eggs prior to incubation by studying the respiratory differences between fertilized and unfertilized eggs using the Non-invasive Micro-test Technique(NMT).A customized micro-testing examination platform,NMT Egg Testing System(NMT-ETS)was constructed for the real-time monitoring of the intensity and rate of oxygen exchange between the egg and its external environment.The results from this study revealed that at room temperature,there is a significant difference in gas exchange rates between fertilized and unfertilized eggs.The results indicate that the oxygen flux of fertilized eggs exceed 20 pmol/(cm^(2)·s),whereas unfertilized eggs show a much lower oxygen flux.Based on the results,the NMT method can be used to effectively distinguish between fertilized and unfertilized chicken eggs.

Murine fertilized ovum, blastomere and morula cells lacking SP phenotype

In the field of stem cell research, SP (side population) phenotype is used to define the property that cells maintain a high efflux capability for some fluorescent dye, such as Hoechst 33342. Recently, many researches proposed that SP phenotype is a phenotype shared by some stem cells; some progenitor cells,; that SP phenotype is regarded as a candidate purification marker for stem cells. In this research, murine fertilized ova (including conjugate; single nucleus fertilized ova), 2-cell stage; 8-cell stage blastomeres, morulas; blastocysts were isolated; directly stained by Hoechst 33342 dye. The results show that fertilized ovum, blastomere; morula cells do not demonstrate any ability to efflux the dye. However, the inner cell mass (ICM) cells of blastocyst exhibit SP phenotype, which is consistent with the result of embryonic stem cells (ESCs) in vitro. These results indicate that the SP phenotype of ICM-derived ESCs is an intrinsic property; independent of the culture condition in vitro,; that SP phenotype is one of the characteristics of at least some pluripotent stem cells, but is not shared by totipotent stem cells. In addition, the result that the SP phenotype of ICM cells disappeared when the inhibitor verapamil was added into medium implies that the SP phenotype is directly associated with ABCG2. These results suggest that not all the stem cells demonstrate SP phenotype,; that SP phenotype might act as a purification marker for partial stem cells such as some pluripotent embryonic stem cells; multipotent adult stem cells, but not for all stem cells exampled by the totipotent stem cells in the very early stage of mouse embryos.

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic,Androgenetic and Fertilized Blastocysts

MicroRNAs（miRNAs） are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions.In this study,miRNA and messenger RNA（mRNA） profiles were examined by Illumina microarray in mouse embryonic stem cells（ESCs） derived from parthenogenetic,androgenetic,and fertilized blastocysts.The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression.Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs（aESCs） and fertilized ESCs（fESCs）,a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs（pESCs） and fESCs,and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs.In addition,a total of 575,5 and 376 miRNA-mRNA target pairs were observed in aESCs vs.fESCs,pESCs vs.fESCs,and aESCs vs.pESCs,respectively.Furthermore,15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR.Finally,transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3,Dyrklb,and Prrg2 mRNA,inhibition of miR-363 increased the expression of Nfat5 and Soatl mRNA,and inhibition of miR-883b-5p increased Nfat5,Tacstd2,and Ppapdc1 mRNA.These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.

Dynamic Relationship Between Biologically Active Soil Organic Carbon and Aggregate Stability in Long-Term Organically Fertilized Soils

Biologically active soil organic carbon （BASOC） is an important fraction of soil organic carbon （SOC）, but our understanding of the correlation between BASOC and soil aggregate stability is limited. At an ecological experimental station （28° 04＇-28° 37＇ N, 116° 41＇-117° 09＇ E） in Yujiang County, Jiangxi Province, China, we analyzed the dynamic relationship between soil aggregate stability and BASOC content over time in the red soil （Udic Ferrosols） fertilized with a nitrogen-phosphorus-potassium chemical fertilizer （NPK） without manure or with NPK plus livestock manure or green manure. The dynamics of BASOC was evaluated using CO2 efflux, and soil aggregates were separated according to size using a wet-sieving technique. The soils fertilized with NPK plus livestock manure had a significantly higher content of BASOC and an improved aggregate stability compared to the soils fertilized with NPK plus green manure or NPK alone. The BASOC contents in all fertilized soils decreased over time. The contents of large aggregates （800-2 000 μm） dramatically decreased over the first 7 d of incubation, but the contents of small aggregates （〈 800 μm） either remained the same or increased, depending on the incubation time and specific aggregate sizes. The aggregate stability did not differ significantly at the beginning and end of incubation, but the lowest stability in all fertilized soils occurred in the middle of the incubation, which implied that the soils had a strong resilience for aggregate stability. The change in BASOC content was only correlated with aggregate stability during the first 27 d of incubation.

Is Nitrous Oxide Reduction Primarily Regulated by the Fungi-to-Bacteria Abundance Ratio in Fertilized Soils?

The production of nitrous oxide (N2O) is a widespread trait in fungi and is of interest because denitrifying fungi lack the N2O reductase gene (nosZ) that regulates N2O reduction to nitrogen gas (N2). The adaptive ability of soil fungi is better than that of bacteria in acidic soils. We investigated the N2O reduction potential, described by the N2O product ratio (Nn2O), N2O/(N2O+N2), in soils of different types of fields under crop cultivation with different fertilizer inputs and a bare fallow field with no fertilization as a control. The fungi-to-bacteria abundance ratio (Rf/b) was negatively correlated (P < 0.01) with the natural pH of the soil;however, the high value of Rp/b measured in vineyards was due to the large inputs of manure. When the denitrification potential was measured at natural pH values of soils, Nn2O was negatively correlated (P < 0.01) with soil pH. When the denitrification potential was measured after short-term modifications of soil pH, however, no significant correlation was found between Nn2O and the modified pH. Based on stepwise multiple regression analysis, soil pH and residual nitrate (NO3) were the key factors regulating N2O reduction in soils at natural pH values (R^2 = 0.8& P < 0.001), whereas the key factor was the soil residual NO, alone (R^2 = 0.83, P < 0.001) when the soil pH was modified. When the effect of the soil chemical properties was weakened, a high Rf/b value had the potential (P < 0.01) to affect N2O reduction;however, the role of fungi was offset by the presence of denitrifying bacteria. These results provide evidence that compared to the indirect effects of Rf/b、the direct effects of the soil chemical properties have a greater effect on N2O reduction in fertilized soils.

A live birth of activated one-day-old unfertilized oocyte for a patient who experienced repeatedly near-total fertilization failure after intracytoplasmic sperm injection

Total or near-total fertilization failure after intracytoplasmic sperm injection （ICSl） is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnancy and live birth after calcium ionophore A23187 （A23187） activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia, and who had experienced repeated near-total fertilization failure after ICSI. In the second ICSI cycle, only one oocyte was fertilized while nine were unfertilized. Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer. A clinical pregnancy was achieved and a healthy baby was born. To our knowledge, this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte. This indicates that ＂rescue oocyte activation＂ on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.

The egg cell is preferentially fertilized in Arabidopsis double fertilization

In flowering plants(angiosperms),fertilization of the egg cell by one sperm cell produces an embryo,whereas fusion of a second sperm cell with the central cell generates the endosperm.In most angiosperms like Arabidopsis,a pollen grain contains two isomorphic sperm cells required for this double fertilization process.A long-standing unsolved question is whether the two fertilization events have any preference.A tool to address this question is the usage of the cyclin-dependent kinase a1(cdka;1)mutant pollen,which produces a single sperm-like cell(SLC).Here,we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC.Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell,precursor of the two sperm cells.Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2%of the ovules,single fertilization of the egg cell occurred.Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery,which fertilized the central cell,resulting in 20.7%double-fertilized ovules.This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell.Taken together,our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.