| Melicope pteleifolia(Champion ex Bentham)T.G.Hartley is a small arbor belonging to the rue family(Rutaceae),which is mainly distributed in southern China and commonly used for urban landscaping.The dried stem or twig of M.pteleifolia is a common traditional Chinese medicine(Sanchaku,caulis seu cacumen Melicope pteleifoliae)in Lingnan area,China.Sanchaku is bitter in taste,cold in property,with rneridian tropism in heart and liver.Sanchakt has many curative effects,including heat-clearing and detoxifying,dispelling wind and eliminating dampness,relieving swelling and pain.It is clinically used for treatment of influenza with fever,tonsillitis,pharyngitis,jaundice hepatitis,epigastric pain,malaria,rheumatic arthralgia,traumatic injury,eczema,dermatitis,haemorrhoids,etc.Moreover,Sanchaku is also an important raw material of Guangdong herbal teas and Chinese patent medicines such as Sanjiu Ganmaoling and Sanjiu Weitai.The distribution areas of M.pteleifolia are wide and scattered,resulted in low production of the herbal material from wild plants.Due to the huge and increasing market demand for M.pteleifolia,the wild resources of M.pteleifolia have been increasingly consumed.At present,many companies have started conducting large-scale domesticated cultivation of M.pteleifolia to fill the market demand.Most companies mainly use the large amount and easily available M.pteleifolia seeds to breed seedlings of the plant.However,the extremely low germination rate of M.pteleifolia seeds was found in practical.In order to improve the seed reproduction efficiency and reduce the cost of seedling cultivation,our research team carried out systematic study to overcome the extremely low germination rate of M.pteleifolia seeds.Based on the preliminary experiments,we proposed the hypothesis that"M.pteleifolia seeds may possess the character of dormancy".Firstly,the whole picture of biological characteristics of M.pteleifolia seeds was established,which help to speculate the dormancy characteristics of M.pteleifolia seeds and provide a direction for dormaney release methods.Secondly,a variety of treatments were tried to screen out a simple and practical method for dormancy breaking,which can also be used to provide ideal materials for studying the mechanism of dormancy release in M.pteleifolia seeds using next-and third-generation sequencing technologies.Thirdly,germination tests under various conditions were eonducted to screen out the optimal harvest time and condition for the germination of M.pteleifolia seeds.Fourthly,a germination test was also carried out in a greenhouse to examine the applicability of the method developed in the laboratory for practical production.Finally,a full-length transcriptome of M.pteleifolia seeds was established as a reference sequence,and comparative transcriptomics analysis was conducted between dormant and dormancy-released M.pteleifolia seeds to elucidate the mechanism of dormancy release in M.pteleifolia seeds.Our main findings are as follows:1.Biological characteristics of M.pteleifolia seedsThe development of M.pteleifolia seeds can be observed by the color of the seed coat turning from white to yellow,then to red,and finally to black.During the period close to maturity,the periearp of M.pteleifolia fruits experienced the proeess of dehiscenee and drying-up.Mature M.pteleifolia seeds are of lower mass and have a thousand-seed weight of 7.53±0.07 g,eontaining relatively less water with the moisture of 11.81±0.14%,which is convenient for seed storage.Mature M.pteleifolia seeds were elliptical spherical shaped,small in size,2.89±0.10 mm in length,2.23±0,14 mm in width,and 1.84±0.10 mm in thickness.The surface of the seeds' coat are covered with a layer of black oily wax.Mature M.pteleifolia seeds consist of seed coat,endosperm and dicotyledonous embryo,which can be classified into the dicotyledon albuminous seed category.The entire germination process of M.pteleifolia seeds was also recorded.The differences of the biological characteristics between M.pteleifolia seeds and the dormant seeds of the other species in Rutaceae were compared,and seed coat with the attachment were speculated to be an important factor in the dormancy of M.pteleifolia seeds.2.M.pteleifolia seeds have combinational dormancyEffects of various pretreal:ments of seed coat on the germination rate of M.pteleifolia seeds were investigated.After concentrated sulfuric acid treatment(soaked in concentrated sulfuric acid for 2,4 or 6 minutes),sodium hydroxide solution treatment(soaked in 30%sodium hydroxide solution for 10,20 or 30 minutes)or scrubbing,the germination rates of M.pteleifolia seeds were significantly improved,indicating that M.pteleifolia seeds had physical dormancy caused by the seed coat.In all seed coat treatment groups,the germination rate of the scrubbing treatment group was the highest(42·67±4.62%),with statistically significant differences eompared with the germination rates of other treatment groups.Therefore,the scrubbing treatment is preferred as the optimum seed coat pretreatment method.After the pretreatment of seed coat,the germination rate of M.pteleifolia seeds was significantly increased,but was still less than 50%and the germination cycle was long(2 months).Therefore,we proposed that M.pteleifolia seeds may also have physiological dormancy.Then two common methods were adopted for breaking physiological dormancy followed by a germination test.After 15 or 30 days of cold stratification(4?),or 5 days of warm stratification(30 OC),the germination rates of seed coat pretreated M.pteleifolia seeds were not further increased.After 10 or 15 days of warm stratification(30?),the germination rates of seed coat pretreated M.pteleifolia seeds were further increased significantly,and the effect of 15 days of warm stratification(30?)was better(Germination rate:82.00±3.46%).Soaking seed coat pretreated M.pteleifolia seeds in 100,200,300 or 400 mg·L-1 gibberellin solution for 24 hours further significantly increased the germination rates;and the effect of 300 mg·L-1 gibberellin solution treatment was the best(Germination rate:77.33:1:5.03%).These results indicated that M.Pteleifolia seeds also have physiological dormancy.Therefore,M.pteleifolia seeds have combinational dormancy,and the scrubbing treatment followed with 15 days of warm stratification or soaking in 300 mg·L-1 gibberellin solution for 24 hours,can break the combinational dormancy of M.pteleifolia seeds.The applicability of the treatment methods of M.pteleifolia seeds sereened out in laboratory in actual production was validated by a germination test in greenhouse.The germination rate of the seed coat treatment group was significantly higher than that of the seed coat untreated group under greenhouse condition.The germination rates of the combination treatment groups(seed coat pretreatment combined with 100 or 300 mg·L-1 gibberellin treatments)were significantly higher than that of the seed coat treatment only group.The germination rate of the combination treatment group(seed coat pretreatment combined 15 days of warm stratification)was significantly higher than those of the seed coat treatment group and the combination treatment group(seed coat pretreatment combined 5 days of warm stratification).The overall results of the germination tests in greenhouse were consistent with the results in laboratory,indicating that the treatment methods of M.pteleifolia seeds developed in laboratory also have good applicability for actual production.3.Suitable harvest time and germination conditions for M pteleifolia seedsThe effects of harvest time,germination bed,light and temperature on the germination rate of M.pteleifolia seeds were investigated.The germination rate of M pteleifolia seeds picked when the pericarps are naturally dehiscent on the tree is higher.There was no statistical difference between the germination rates of M.pteleifolia seeds grown on different seeding beds,or different light conditions,indicating that germination bed and light have no effect on the germination rate of M.pteleifolia seeds.The germination rate of M.pteleifolia seeds cultured at 30?(12 h)/20 ?(12 h)was significantly higher than those cultured at 20,25 and 30?,indicating that the culture temperature has a significant effect on the germination rate of M.pteleifolia seeds,and the variable temperature condition of 30?(12 h)/20?(12 h)is more favorable for the germination of M.pteleifolia seeds.4.The mechanism of dormancy release in M.pteleifolia seeds by warm stratifieation In this study,both PacBio and Illumina sequeneing were used to establish a referenee sequence for M.pteleifolia seeds.A total of 98,892 isoforms and 93,481 unigenes were obtained as a reference sequence.Then a comparative transcriptome study were performed that identified a total of 89,216 differentially expressed genes(DEGs)with a fold change greater than 2.Among them,54,686 DEGs were significantly up-regulated,and 34,530 DEGs were significantly down-regulated.Based on the results of GO function and KEGG pathway enrichment analysis of these DEGs,it was found that the dormancy release of M.pteleifolia seeds caused by 15 days of warm stratifieation is mainly related to the significant changes of the differentially expressed genes such as abscisic acid receptor,protein phosphatase 2C,gibberellin receptor and DELLA protein in plant hormone signal transduction pathway,adenosine kinase,glycine hydroxymethyltransferase,S_adenosylmethionine synthetase and nucleoside-diphosphate kinase in metabolic pathways,translation initiation factors and heat shock proteins in translational activities and aspartate-semialdehyde dehydrogenase,2-isopropylmalate synthase,ketol-acid reductoisomerase and D-3-phosphoglycerate dehydrogenase in the biosynthesis of amino acids. |
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