Agronomic Performance, Stability Analysis and Evaluation of Anthracnose Disease Resistance of Common Bean Lines Derived by Marker-Assisted Backcrossing in Uganda

The present study focused on evaluating the agronomic performance, stability, and anthracnose resistance of common bean lines derived through Marker-Assisted Backcrossing in Uganda. Eight marker-assisted selection (MAS) backcross-derived bush bean lines with red seed types, alongside two checks, were evaluated in a randomized complete block design replicated two times in five locations for three consecutive crop-growing seasons in 2021 and 2022. The study aimed to identify lines with both high stable yields and enhanced resistance to anthracnose disease for potential release and utilization in future bean varietal development in Uganda. Agronomic traits, including days to 50% flowering, days to 90% physiological maturity, seed yield, seed yield components, and anthracnose disease reaction under natural infestation were assessed. The response to anthracnose disease was further assessed using six isolates of Colletotrichum lindemuthianum representing six different races. Results indicated that the agronomic performances of the MAS backcross-derived bush bean lines were statistically comparable to the recurrent parent NABE14. Specifically, six lines exhibited statistically equal to or higher performance than NABE14 in terms of seed yield, total number of seeds and number of pods per plant. The combined analysis of variance for seed yield showed significant (p Co-42 and Co-5 anthracnose resistance genes in the derived line. In conclusion, UGKT-B157-4, identified as the best-performing and stable genotype, demonstrates promise for release and use in future bean varietal development in Uganda, offering a combination of high yields and enhanced anthracnose disease resistance. The study provides valuable insights into the potential of Marker-Assisted Backcrossing in improving common bean varieties in the region.

Transcriptomic and genetic approaches reveal that low-light-induced disease susceptibility is related to cellular oxidative stress in tomato

The impact of low light intensities on plant disease outbreaks represents a major challenge for global crop security,as it frequently results in significant yield losses.However,the underlying mechanisms of the effect of low light on plant defense are still poorly understood.Here,using an RNA-seq approach,we found that the susceptibility of tomato to Pseudomonas syringae pv.tomato DC3000(Pst DC3000)under low light was associated with the oxidation-reduction process.Low light conditions exacerbated Pst DC3000-induced reactive oxygen species(ROS)accumulation and protein oxidation.Analysis of gene expression and enzyme activity of ascorbate peroxidase 2(APX2)and other antioxidant enzymes revealed that these defense responses were significantly induced by Pst DC3000 inoculation under normal light,whereas these genes and their associated enzyme activities were not responsive to pathogen inocula-tion under low light.Additionally,the reduced ascorbate to dehydroascorbate(AsA/DHA)ratio was lower under low light compared with normal light conditions upon Pst DC3000 inoculation.Furthermore,the apx2 mutants generated by a CRISPR-Cas9 gene-editing approach were more susceptible to Pst DC3000 under low light conditions.Notably,this increased susceptibility could be significantly reduced by exogenous AsA treatment.Collectively,our findings suggest that low-light-induced disease susceptibility is associated with increased cellular oxidative stress in tomato plants.This study sheds light on the intricate relationship between light conditions,oxidative stress,and plant defense responses,and may pave the way for improved crop protection strategies in low light environments.

The role of DNA methylation in the maintenance of phenotypic variation induced by grafting chimerism in Brassica

Grafting facilitates the interaction between heterologous cells with different genomes,resulting in abundant phenotypic variation,which provides opportunities for crop improvement.However,how grafting-induced variation occurs and is transmitted to progeny remains elusive.A graft chimera,especially a periclinal chimera,which has genetically distinct cell layers throughout the plant,is an excellent model to probe the molecular mechanisms of grafting-induced variation maintenance.Here we regenerated a plant from the T-cell layer of a periclinal chimera,TCC(where the apical meristem was artificially divided into three cell layers–from outside to inside,L1,L2,and L3;T=Tuber mustard,C=red Cabbage),named rTTT0(r=regenerated).Compared with the control(rsTTT,s=self-grafted),rTTT0 had multiple phenotypic variations,especially leaf shape variation,which could be maintained in sexual progeny.Transcriptomes were analyzed and 58 phenotypic variation-associated genes were identified.Whole-genome bisulfite sequencing analyses revealed that the methylome of rTTT0 was changed,and the CG methylation level was significantly increased by 8.74%.In rTTT0,the coding gene bodies are hypermethylated in the CG context,while their promoter regions are hypomethylated in the non-CG context.DNA methylation changes in the leaf shape variation-associated coding genes,ARF10,IAA20,ROF1,and TPR2,were maintained for five generations of rTTT0.Interestingly,grafting chimerism also affected transcription of the microRNA gene(MIR),among which the DNA methylation levels of the promoters of three MIRs associated with leaf shape variation were changed in rTTT0,and the DNA methylation modification of MIR319 was maintained to the fifth generation of selfed progeny of rTTT0(rTTT5).These findings demonstrate that DNA methylation of coding and non-coding genes plays an important role in heterologous cell interaction-induced variation formation and its transgenerational inheritance.

Aquaporin,beyond a transporter

Aquaporins(AQPs)are one of the most ancient superfamily proteins,which are essential for maintaining the fluid homeostasis of most organisms against various environments.Here,the latest findings for function of AQPs in cell signal transduction in plants are summarized.We also put forward several issues that still need to be addressed in the future.

RcSPL1-RcTAF15b regulates the flowering time of rose (Rosa chinensis)

Rose(Rosa chinensis),which is an economically valuable floral species worldwide,has three types,namely once-flowering(OF),occasional or re-blooming(OR),and recurrent or continuous flowering(CF).However,the mechanism underlying the effect of the age pathway on the duration of the CF or OF juvenile phase is largely unknown.In this study,we observed that the RcSPL1 transcript levels were substantially upregulated during the floral development period in CF and OF plants.Additionally,accumulation of RcSPL1 protein was controlled by rch-miR156.The ectopic expression of RcSPL1 in Arabidopsis thaliana accelerated the vegetative phase transition and flowering.Furthermore,the transient overexpression of RcSPL1 in rose plants accelerated flowering,whereas silencing of RcSPL1 had the opposite phenotype.Accordingly,the transcription levels of floral meristem identity genes(APETALA1,FRUITFULL,and LEAFY)were significantly affected by the changes in RcSPL1 expression.RcTAF15b protein,which is an autonomous pathway protein,was revealed to interact with RcSPL1.The silencing and overexpression of RcTAF15b in rose plants led to delayed and accelerated flowering,respectively.Collectively,the study findings imply that RcSPL1–RcTAF15b modulates the flowering time of rose plants.

脐橙果实发育中有机酸合成代谢酶活性变化的研究

以红桔砧 10年生罗伯逊脐橙为试材 ,研究了脐橙果实发育过程中有机酸代谢有关酶的活性变化及其与有机酸积累的关系。结果表明 :①从 6～ 8月柠檬酸合成酶 (CS)活性迅速增加 ,8月中旬达到高峰 ,随后迅速下降 ,在 10月中旬其活性出现回升后又再次下降 ;② 6～ 8月磷酸烯醇式丙酮酸羧化酶 (PEPC)活性迅速上升 ,8月中旬达到高峰 ,进入果实膨大期后 ,PEPC活性迅速下降 ,在果实膨大末期和成熟期其活性基本不变 ;③在整个果实发育过程中 ,CS、PEPC活性的变化趋势与有机酸含量的变化趋势一致 ,并呈极显著正相关 ,相关系数 (r)分别为 0 916 0和0 8779。这不仅证明脐橙果实中存在通过CO2 固定合成有机酸的途径 ,还说明CS、PEPC是有机酸合成的关键酶 。

Effects of Cinnamic Acid on Bacterial Community Diversity in Rhizosphere Soil of Cucumber Seedlings Under Salt Stress

To investigate the effects of a plant autotoxin, cinnamic acid, on bacterial communities in the rhizosphere soil of cucumber seedlings under salt stress, we used cucumber as the experimental material, cinnamic acid as the autotoxin, and NaCl to apply salt stress. Bacterial communities in the rhizosphere soil were analyzed using polymerase chain reaction （PCR）, denaturing gradient gel electrophoresis （DGGE）, and clone sequencing. Salt stress decreased the diversity of bacterial species in rhizosphere soil of cucumber seedlings at several growth stages. Cinnamic acid exacerbated the effects of salt stress at high concentrations, but alleviated its effects at low concentrations. Cloning and sequencing results indicated that DGGE bands amplified from soil samples showed high homology to uncultured bacterial species. Cinnamic acid at 50 mg kg^-1 soil improved cucumber growth and was the most effective treatment to alleviate the effects of salt stress on bacterial communities.

Effects of Rest Grazing on Organic Carbon Storage in Stipa grandis Steppe in Inner Mongolia, China

This study was aimed to evaluate the potential effects of rest grazing on organic carbon storage in Stipa grandis steppe of Inner Mongolia, China. Using potassium dichromate heating method, we analyzed the organic carbon storage of plant and soil in Stipa grand＆ steppe after rest grazing for 3, 6, and 9 yr. The results indicated that as the rest grazing ages prolonged, the biomass of aboveground parts, litter and belowground plant parts （roots） of the plant communities all increased, meanwhile the C content of the biomass increased with the rest grazing ages prolonging. For RG0, RG3a, RG6a, and RG9a, C storage in aboveground vegetation were 60.7, 76.9, 82.8 and 122.2 g C m2, respectively; C storage of litter were 5.1, 5.8, 20.4 and 25.5 g C m^-2, respectively; C storage of belowground roots （0-100 cm） were 475.2, 663.0, 1 115.0 and 1 867.3 g C m^-2, respectively; C storage in 0-100 cm soil were 13.97, 15.76, 18.60 and 32.41 kg C m^-2, respectively. As the rest grazing ages prolonged, the organic C storage in plant communities and soil increased. The C storage ofbelowground roots and soil organic C was mainly concentrated in 0-40 cm soil body. The increased soil organic C for RG3a accounted for 89.8% of the increased carbon in vegetation-soil system, 87.2% for RG6a, and 92.6% for RG9a. From the perspective of C sequestration cost, total cost for RG3a, RG6,, and RG9a were 2 903.4, 5 806.8 and 8 710.2 CNY haq, respectively. The cost reduced with the extension of rest grazing ages, 0.15 CNY kg^-1 C for RG3a, 0.11 CNY kg-~ C for RG6a and 0.04 CNY kg℃ for RG9a. From the growth characteristics of grassland plants, the spring was one of the two avoided grazing periods, timely rest grazing could effectively restore and update grassland vegetation, and was beneficial to the sustainable use of grassland. Organic C storage for RG9a was the highest, while the cost of C sequestration was the lowest. Therefore, spring rest grazing should be encouraged because it was proved to be a very efficient grassland use pattern.

Dissipation of chlorpyrifos in pakchoi-vegetated soil in a greenhouse

The dissipation of chlorpyrifos in pakchoi-vegetated soil was investigated in the summer and autumn in a greenhouse and field, respectively. The dissipation of chlorpyrifos in pakchoi-grown soil was comparatively described by fitting the residue data to seven models （lst-order, 1.5th-order, 2nd-order, RF lst-order, RF 1.5th-order, RF 2nd-order, and bi-exponential or two-compartment models）. Statistical analysis was performed using the SPSS 11.5 statistical package. The bi-exponential model was selected as the optimal model according to the coefficient of determination r^2. The dissipation half-lives （DT50） of chlorpyrifos in pakchoi-vegetated soil at the recommended dose in the summer and autumn, calculated by the bi-exponential model, were 0.6 and 1.2 d in a greenhouse, 0.4 and 1.0 d in a field, respectively; the corresponding values at double dose were 1.2 and 2.1 d in a greenhouse, 0.5 and 1.3 d in a field, respectively. The kinetic data indicate the dissipation of chlorpyrifos in pakchoi-grown soil in a greenhouse is slower than that in a field, and dissipates slower in the autumn than in the summer.

An Ethylene-inhibited NF-YC Transcription Factor RhNF-YC9 Regulates Petal Expansion in Rose

The speed of flower opening is closely related to their ornamental period.Ethylene functions as a negative regulator involved in the regulation of the petal expansion process.In this study,we isolated a NF-YC transcription factor gene,RhNF-YC9,fromrose petals.RhNF-YC9 expression was induced at the early stages of flower opening but was inhibited by ethylene treatment.Silencing RhNF-YC9 decreased the speed of petal expansion from stage 2 to stage 5.The expressions of 11 cell expansion-related genes involved in cell wall loosening,cell turgor modulation,and cytoskeleton remodeling were significantly down-regulated in RhNF-YC9-silenced petals.We also found that silencing RhNF-YC9 decreased the expression of gibberellin acid(GA)biosynthetic gene RhGA20ox while significantly increasing the transcripts of GA catabolic gene RhGA2ox,reducing the accumulation of GA4 and GA7.The influence of ethylene treatment on the expression of RhGA20ox and RhGA2ox showed the same trend.These results together suggested that RhNF-YC9 positively regulated the speed of petal expansion and mediated the crosstalk between ethylene and GA.Our findings revealed a new insight into the function of NF-YC transcription factors involved in ethylene-regulated petal expansion.

Degradation of chlorpyrifos in laboratory soil and its impact on soil microbial functional diversity

Degradation of chlorpyrifos at different concentrations in soil and its impact on soil microbial functional diversity were investigated under laboratory condition. The degradation half-live of chlorpyrifos at levels of 4, 8, and 12 mg/kg in soil were calculated to be 14.3, 16.7, and 18.0 d, respectively. The Biolog study showed that the average well color development （AWCD） in soils was significantly （P 〈 0.05） inhibited by chlorpyrifos within the first two weeks and thereafter recovered to a similar level as the control. A similar variation in the diversity indices （Simpson index lID and McIntosh index U） was observed, but no significant difference among the values of the Shannon-Wiener index H＇ was found in chlorpyrifos-treated soils. With an increasing chlorpyrifos concentration, the half-life of chlorpyrifos was significantly （P ≤ 0.05） extended and its inhibitory effect on soil microorganisms was aggravated. It is concluded that chlorpyrifos residues in soil had a temporary or short-term inhibitory effect on soil microbial functional diversity.

Cloning of vacuolar H^+ -ATPase subunit c genes from Japanese iris, and functional characterization in yeast

Five different isoforms （IrlVHA-c1-c5） of V-ATPase subunit c （VHA-c） were cloned from a Japanese iris （Iris lactea Pall. var. chinensis Fisch. Koidz） cDNA library using degenerate primers PCR and the 5＇-RACE technique. The sequence analysis showed the open reading frame （ORF） of the IrlVHA-c1 c5 to be 495 bp, corresponding to a protein of 164 amino acids. Among the five isoforms, IrlVHA-c1 and IrlVHA-c2 are completely homologous. The IrlVHA-c protein is localized at the vacuolar membrane as indicated by a green fluorescent protein （GFP） marker. Its over-expression in yeast could enhance yeast tolerance to NaCl stress. These results show that there are at least five genes encoding different isoforms of IrlVHA-c in Japanese iris and IrlVHA-c is important for the function of V-ATPase.

A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers

Fresh-cut roses(Rosa hybrida)are one of the most important ornamental crops worldwide,with annual trade in the billions of dollars.Gray mold disease caused by the pathogen Botrytis cinerea is the most serious fungal threat to cut roses,causing extensive postharvest losses.In this study,we optimized a detached petal disc assay(DPDA)for artificial B.cinerea inoculation and quantification of disease symptoms in rose petals.Furthermore,as the identification of rose genes involved in B.cinerea resistance could provide useful genetic and genomic resources,we devised a virusinduced gene silencing(VIGS)procedure for the functional analysis of B.cinerea resistance genes in rose petals.We used RhPR10.1 as a reporter of silencing efficiency and found that the rose cultivar‘Samantha’showed the greatest decrease in RhPR10.1 expression among the cultivars tested.To determine whether jasmonic acid and ethylene are required for B.cinerea resistance in rose petals,we used VIGS to silence the expression of RhLOX5 and RhEIN3(encoding a jasmonic acid biosynthesis pathway protein and an ethylene regulatory protein,respectively)and found that petal susceptibility to B.cinerea was affected.Finally,a VIGS screen of B.cinerea-induced rose transcription factors demonstrated the potential benefits of this method for the high-throughput identification of gene function in B.cinerea resistance.Collectively,our data show that the combination of the DPDA and VIGS is a reliable and highthroughput method for studying B.cinerea resistance in rose.

Effects of short-term heat stress on PSII and subsequent recovery for senescent leaves of Vitis vinifera L. cv. Red Globe

Heat stress occurs frequently in energy-saving sunlight greenhouses(ESSG) at the late growth stage. Three-year delayed cultivation(DC) of the Red Globe cultivar of Vitis vinifera L. was used to clarify the physiological mechanisms of short-term heat stress on PSII and subsequent recovery from heat stress. By November, the photosynthetic function had declined and the fall in transpiration rate(E) with heating time increased the possibility of heat damage. In July, the most obvious increase was in the relative variable fluorescence at J point at 40°C, and in November it changed to K point. The 5 min of heat treatment resulted in a significant increase of the relative variable fluorescence at 0.3 ms(W), and after 10 min of heat treatment, the number of reactive centres per excited cross section(RC/CS), probability that a trapped exciton moves an electron into the electron transport chain beyond Q–(at t=0)(Ψ) and quantum yield of electron transport at t=0(φ) decreased significantly(P<0.05), suggesting that the reaction centre, donor and acceptor side of photosystem II(PSII) were all significantly inhibited(P<0.05) and that the thermal stability of the photosynthetic mechanism was reduced. The inhibition of energy fluxes for senescent leaves in November was earlier and more pronounced than that for healthy leaves, which did not recover from heat stress of more than 15 min after 2 h recovery at room temperature.

Inheritance Analysis and Identification of SSR Markers Linked to Late Blight Resistant Gene in Tomato

Late blight caused by Phytophthora infestans is the most serious disease of tomato production in China. Studies on the genetics of resistance and identification of molecular markers are very useful for breeding late blight resistant varieties. The objective of this paper was to study the inheritance of late blight resistance and identify simple sequence repeat （SSR） markers associated with resistance allele in tomato （Lycopersicon esculentum Mill）. The results came from an F2 progeny of 241 plants derived from a cross between 5~ inbred line that is susceptible to late blight and a resistant accession CLN2037E. The late blight responses of F2 plants were tested by artificially inoculation of detached-leaflets in plate and natural infection assayed under greenhouse conditions. Both methods showed that the resistance is dominant and inherited as monogenic trait. Genetic mapping and linkage analysis showed that the late blight resistance gene Ph-ROL was located on chromosome 9 with a genetic distance of 5.7 cM to the SSR marker TOM236.

Cytokinin induced shoot regeneration and flowering of Scoparia dulcis L.(Scrophulariaceae)-an ethnomedicinal herb

Objective:To develop an improved protocol for micropropagation of ethnomedicinally important Scoparia dulcis(S.dulcis) L.Methods:Explants were inoculated on MS basal medium supplemented with kinelin and 6-benzylaminopurine for shoot bud induction.To enhance the shoot induction,various auxins like 3-indoleacetic acid or 3-indolebutyric acid or a-naphthylacetic acid were tested along with 2.32 M KI and 4.44 μ M BAP.The regenerated shoots were rooted in half strength MS medium supplemented with various concentrations of IAA,IBA or NAA.After roots were developed,the plantlets were transplanted to pots filled with vermiculate and sand and kept in growth chamber with 70%-80%humidity under 16 h photoperiod.After acclimatization,the plantlets were transferred to the garden and survival percentage was calculated.Data were statistically analyzed and means were compared using Duncan's multiple range test(P<0.05).Results:An in vitro method was developed to induce high frequency shoots regeneration from stem,mature leaf and young leaf explants of S.dulcis.Shoot induction on young leaf explants was most successful in MS medium supplemented with combination of two cytokinins(2.32 μ M KI and 4.44 μ M BAP) 2.85 μ M IAA,10%CM and 1 483.79 μM adenine sulfate.A single young leaf explant was capable of producing 59 shoots after 13 days of culture. Flower was induced in medium supplemented with combination of KI and BAP.Conclusions: Cytokinins are the key factor to induce the direct shoot regeneration and flowering of S.dulcis.

The hist one varia nt SI_H2A.Z regulates carote noid biosynthesis and gene expression during tomato fruit ripening

The conserved histone variant H2A.Z is essential for transcriptional regulation;defense responses;and various biological processes in plants,such as growth,development,and flowering.However,little is known about how H2A.Z affects the developmental process and ripening of tomato fruits.Here,we utilized the CRISPR/Cas9 gene-editing system to generate a sl_hta9 sl_hta11 double-mutant,designated sl_h2az,and found that these two mutations led to a signi fi cant reduction in the fresh weight of tomato fruits.Subsequent messenger RNA(mRNA)-seq results showed that dysfunction of SI_H2A.Z has profound effects on the reprogramming of genome-wide gene expression at different developmental stages of tomato fruits,indicating a ripening-dependent correlation between SI_H2A.Z and gene expression regulation in tomato fruits.In addition,the expression of three genes,SIPSY1,SlPDS,and SlVDE,encoding the key enzymes in the biosynthesis pathway of carotenoids,was signi fi cantly upregulated in the later ripening stages,which was consistent with the increased contents of carotenoids in sl_h2a.z double-mutant fruits.Overall,our study reveals a role of SI_H2A.Z in the regulation of carotenoids and provides a resource for the study of SI_H2A.Z-dependent gene expression regulation.Hence,our results provide a link between epigenetic regulation via histone variants and fruit development,suggesting a conceptual framework to understand how histone variants regulate tomato fruit quality.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Molecularly tagged genes and quantitative trait loci in cucumber with recommendations for QTL nomenclature

Cucumber,Cucumis sativus L.(2n=2x=14),is an important vegetable crop worldwide.It was the first specialty crop with a publicly available draft genome.Its relatively small,diploid genome,short life cycle,and selfcompatible mating system offers advantages for genetic studies.In recent years,significant progress has been made in molecular mapping,and identification of genes and QTL responsible for key phenotypic traits,but a systematic review of the work is lacking.Here,we conducted an extensive literature review on mutants,genes and QTL that have been molecularly mapped or characterized in cucumber.We documented 81 simply inherited trait genes or major-effect QTL that have been cloned or fine mapped.For each gene,detailed information was compiled including chromosome locations,allelic variants and associated polymorphisms,predicted functions,and diagnostic markers that could be used for marker-assisted selection in cucumber breeding.We also documented 322 QTL for 42 quantitative traits,including 109 for disease resistances against seven pathogens.By alignment of these QTL on the latest version of cucumber draft genomes,consensus QTL across multiple studies were inferred,which provided insights into heritable correlations among different traits.Through collaborative efforts among public and private cucumber researchers,we identified 130 quantitative traits and developed a set of recommendations for QTL nomenclature in cucumber.This is the first attempt to systematically summarize,analyze and inventory cucumber mutants,cloned or mapped genes and QTL,which should be a useful resource for the cucurbit research community.

Flower Development and Anatomy of Agapanthus praecox ssp. orientalis (Leighton) Leighton

Floral buds of Agapanthus praecox ssp. orientalis were observed under dissecting and optical microscope to characterize floral organs development and to study relationships between anther development and microsporogenesis. Floral organs differentiation was comprised of 6 distinct stages including nought differentiation, inflorescence bud differentiation, floret primordia differentiation, tepal primordia differentiation, stamen primordia differentiation, and pistil primordia differentiation. Six tepals differentiated almost simultaneously which cross arranged in space and appeared in hexagonal distribution pattern. Six stamens were differentiated inside the tepals at the same time. Finally, 3 carpel primordia differentiated and formed syncarpous pistil. The whole process of floral bud differentiation took approximately 40 d with the first 3 stages developing more slowly than the later 3 stages. Morphology and color of the anther underwent obvious changes during the period between stamen primordia differentiation and anther maturation. Microspores also underwent significant development during this same interval. The relationship between the process of microsporogenesis and anther development has already been made clear by the sauash techniaue.