Analysis Of The Effect Of Exogenous Spraying Of Silver Nitrate On The Formation Of Hermaphrodite Flowers In The Stamens Of The Whole Female Muskmelon

The all-female melon has absolute advantages in the preparation of hybrids.Using the all-female as the female parent can reduce labor costs and improve seed production efficiency,which is of great significance for ensuring the purity of the hybrid.However,in the process of self-reservation,male attraction is the main factor restricting seed production.Although some chemical reagents can be used to induce male production,the application methods have different effects on male attraction.In this study,a full-female thick-skinned melon WI998 was used as a material to design a two-factor experiment to study the male attraction effect.The two leaves 1heart,3 leaves 1 heart,4 leaves 1 heart,and 5 leaves 1 heart of the melon seedlings were applied externally.AgNO₃ at concentrations of 150,300,450,and 600 mg/L,observed morphology,microscopic observation,flower bud differentiation,transcriptome sequencing and analysis of differentially expressed genes,and real-time quantitative PCR and other methods to spray AgNO₃ to induce all female melon WI998)The effect of stamens forming bisexual flowers is comprehensively studied.The results are as follows:1.Effects of spraying AgNO₃ at different seedling ages on the induction of amphoteric flowers in all female melonThe all-female melon WI998 produced stamens at different seedling ages（2 leaves,3 leaves,4 leaves,5 leaves,1 heart）with different concentrations of AgNO₃（150,300,450,600 mg/L）to produce stamens to form bisexual flowers.After the data analysis and statistical analysis of the average male attraction rate,average initial flowering node position,average end flowering node position and average number of bisexual flowers per plant,the gender differentiation was obtained.The effect of bisexual flower differentiation of female melons.The results show that the four-leaf stage is the best period for male attraction of all female melons WI998;the concentration of 450mg/L AgNO₃ is the most suitable concentration.2.Morphological observation of bisexual and female flowers induced by AgNO₃Compared with female flowers,the stamens of all female muskmelon wi998 treated with different concentrations of AgNO₃（150,300,450,600 mg/L）at different seedling ages（2,3,4,5 and 1 heart）can be clearly seen after induction,and the observation shows that the ovary of most female flowers is much larger than that of the female flowers.3.Microscopic observation to induce males to produce amphoteric flower pollen viability and identify the amount of pollenStamen pollen produced by amphiphilic flowers induced by different concentrations of AgNO3（150,300,450,600 mg/L）at different seedling ages（2 leaves,3 leaves,4 leaves,5leaves,1 heart）using acetate magenta staining After treatment,three fields of vision were randomly selected for microscopic observation.The results showed that:4 leaves and 1 heart,the maximum amount of viable pollen obtained by treatment with 450mg/L AgNO₃ was 210.33 on average;followed by 2 leaves and 1 heart with 450mg/L AgNO₃ treatment yielded an average amount of pollen of 102.67;5-leaf 1 heart with 450mg/L AgNO₃ treatment resulted in an average pollen amount of 87.67;3-leaf 1 heart with 600mg/L AgNO₃ treatment The average pollen amount is 78.33;while the 5-leaf 1-heart treatment with 600 mg/L AgNO₃ has the least amount of viable pollen,with an average of 15.33.4.Determination of seed viabilityThe bisexual flowers self-introduced with 450mg/LAgNO₃ in 4 leaves and 1 heart were selfed,and the seeds obtained after selfing were tested for germination rate.The results showed that the germination rate could reach 99%and the seed viability was high.5.Transcriptome analysis and qRT-PCR verification of all female muskmelon treated with AgNO₃ and untreated controlIllumina Hi Seq4000 sequencing technology was used to perform transcriptome sequencing on flower buds with a diameter of 2 mm obtained from the whole female melon WI998 treated with 450 mg/L AgNO₃ and untreated control.The control strain of the whole female melon WI998 without AgNO₃ treatment was 2 mm.The average size of flower buds obtained21,856,345 Clean reads,compared to the reference genome was 41,339,275,accounting for94.56%of Clean reads,the number of reads compared to the unique position of the reference genome was 40,881,088,accounting for 93.52%of all reads,the whole female melon WI998without The average GC content of the 2mm-sized flower bud sequencing data of the AgNO₃-treated control strain was 43.87%;the 2mm-sized flower buds obtained by the whole female melon WI998 treated with 450mg/L AgNO₃ averaged 23,638,454 Clean reads,and the reference genome was 44,370,008,accounting for Clean reads 93.85%,the number of reads compared to the unique position of the reference genome is 43,894,460,accounting for 92.84%of all reads.The average GC content of 2mm-sized flower bud sequencing data obtained by the whole female melon WI998 after 450mg/L AgNO₃ treatment is 43.47%.The differentially expressed genes of the whole female melon 4 leaf 1 heart treated with 450mg/L AgNO₃ and the untreated control（the diameter of the flower bud is 2 mm）were compared.A total of 17 differentially expressed genes were detected,of which 11 genes were down-regulated and 6 genes were up-regulated.GO functional classification found that 22Unigenes are classified into biological processes,15 Unigenes are classified into cytological processes,and 15 Unigenes are classified into molecular functions.GO function classification analysis method was used to annotate all the differentially expressed genes.13 genes were screened out for qRT-PCR analysis.The results of qRT-PCR analysis and sequencing results are roughly the same,only the expression level of gene MELO3C009337（probable aquaporin PIP2-4）is quite different.It contains 9 differential genes related to proteins and 4 differential genes related to enzymes.A differential gene MELO3C018449 was screened,annotated as ABA8’-hydroxylase（CYP707A）,which is a member of the cytochrome CYP450 gene superfamily,but whether it is a key gene that determines the conversion of the entire female line into males needs to be confirmed by further experiments;analysis It was found that the ABA synthesis pathway is related to the inducing of stamens to form bisexual flowers.