A Study On The Germination Disorder Of Migao Seeds

The success of plant reproduction and the establishment of seedlings are the key to realize the population continuation.Cinnamomum migao H.W.Li is an evergreen tree mainly distributed in Southwest China.It is known as a famous national Miao medicine in Guizhou Province because of its high medicinal value.For a long time,the lack of seedlings and saplings in the natural state has caused the population generation replacement obstacle.As a seed plant,its embryo development and abortion are serious,and the seed germination obstacle under natural and artificial conditions has been considered to be the main reason for the poor natural regeneration of population.However,there is no relevant research on whether it is so.Therefore,in order to better explore and analyze the internal constraints of natural regeneration of C.migao,the seed formation process and germination characteristics were systematically studied by combining morphology,cell biology and physiology,and the physiological mechanism of seed development and germination was explored.Meanwhile,molecular biology technology was used to systematically explain its germination from the transcription level to further reveal the complex regulatory mechanism of seed germination.The main results are as follows:1.The results of 2 years observation on flowering phenology of C.migao from 2018 to2019 showed that the flowering period was from late February to early April every year;C.migao was a panicle with small flowers,and the diameter of corolla was 4.43±0.24 mm;the perianth was 6-lobed and white pilose on both sides,1.15±0.30 mm long and 1.45±0.31 mm wide;there were 9 fertile stamens,all of which were pubescent and arranged in three rounds,with a length of 0.92±0.02 mm,and each anther had 4 pollen sacs;the pistil length was 0.61±0.02 mm,and the ovary had one ovule in one room;the pistil and stamen had a space distance with about 0.49±0.05 mm;the flowering time of single flower was 4-5 day,and the duration of pollen activity and stigma receptivity was short,about 1-2 day.C.migao belongs to facultative outcrossing type,which is self compatible and needs pollination.The flower pollinators mainly include Apis cerana,Calliphora vicina,Eupeodes corollae,Sepsis fulgens,Cophinopoda chinensis,Procladius nigriventris,female of Ephemeridae and Cletus trigonus.2.The results of seed development showed that the fruit(seed)was initially formed in late March,and it was the development period from April to September;the maturity period was concentrated in October to November,and lasted until the first ten days of December.The phenomenon of bumper year and lunar year was existed in the seed setting,which affected the length of mature period.Seed development was significantly positively correlated with TKW,storage substances(soluble sugar,starch,soluble protein and lipids content),antioxidant enzymes(SOD,POD and CAT activities),endogenous hormones(ABA,GA₃,IAA,CTK,ETH,BR,JA and the ratio of ABA/GA₃ and ABA/IAA),but negatively correlated with water content and SA content.3.The transverse diameter,longitudinal diameter and thickness of mature seeds were9.73±0.089 mm,10.73±0.19 mm and 9.31±0.20 mm,respectively.The STKW was 421.34±5.32 g,the moisture content of dry seed was 12.19±1.21%,and the vigor was 98.67±1.15%.The embryo was composed of cotyledons,plumule,hypocotyl and radicle.The endocarp is hard and consists of fibrous layer,sclereid layer and septal fiber layer.Both embryo and endocarp contain germination inhibitors,inhibitory effects of different polar extracts were as follows:endocarp ethyl acetate>methanol>acetone>water,embryo methanol>water>acetone>ethyl acetate;the endogenous inhibitors were analyzed by GC-MS,and 55compounds were identified,mainly including phenols,aldehydes,lipids,fatty acids and ketones.4.The results of dormancy breaking showed that temperature was the most significant ecological factor affecting seed germination,and 10/20?temperature change was more conducive to seed dormancy release;the effect of different chemical reagents on seed germination was H₂O₂>H₂SO₄>Na OH,exogenous hormones GA₃and IAA pretreatment could effectively relieve embryo dormancy,and GA₃ solution concentration of 200 mg·L^-1 had the best effect,which significantly increased germination rate,germination potential and germination index by 91.47%,84.14%and 113.33%,respectively,and shortened the initial germination days by 5 days;15?warm stratification was more conducive to breaking seed dormancy than 4?low temperature stratification.GA₃ pretreatment could accelerate the breaking of seed dormancy.15?+200 mg·L^-1 was the best combination for breaking seed dormancy,and after 60 days of stratification,the germination rate of seeds reached the maximum value of 77.33±4.16%,the germination rate,germination potential and germination index increased by 73.11%,89.96%and 128.13%respectively compared with that of 0 day stratification.5.The physiological changes during stratification showed that mobilization of storage materials and the increase of enzyme activity activated the seed life and promoted the release of dormancy of C.migao.The germination rate of the 2 stratification treatments was significantly positive correlated with the soluble protein and POD,and negatively correlated with the starch and lipids content.The physiological changes of the four stages of germination showed that the germination of the seeds was positively correlated with the content of soluble sugar and soluble protein in the storage materials,and negatively correlated with the content of starch and lipids;in addition,it was negatively correlated with antioxidant enzymes MDA and SOD.And it was positively correlated with the contents of GA₃,IAA,CTK and BR,and negatively correlated with the contents of ABA,ETH,JA and the ratio of ABA/GA₃ and ABA/IAA.6.Illumina Hi Seq technology was used to analyze the transcriptome of four stages of seed germination.In FFS and CCS groups,43558 and 40532 differential expression genes(DEGs)were obtained.In CCS group,DEGs were mainly accumulated in the decomposition of storage materials(lipids,starch and sucrose),energy supply,antioxidant capacity and hormone synthesis,glyceride metabolism,starch and sucrose metabolism,pentose phosphate pathway,citric acid cycle(TCA cycle),oxidative phosphorylation and glycolysis/gluconeogenesis pathways.During seed germination,the key genes of triglyceride decomposition such as LIP,MGLL,ADH and ALDH,fatty acid degradation key genes such as ACSL,ACOX and MPF2,key genes in starch and sucrose metabolism pathway such as ISA,AMY,Inv,HK,FK,SS and SPS were significantly up-regulated in LK and MF stages,and the expression levels of genes related to lipid degradation reached earlier than those related to starch and soluble sugar metabolism peak value.Lipid,starch and other substances were decomposed gradually with the germination of seeds,and then entered the energy supply channels such as pentose phosphate,glycolysis,TCA cycle and oxidative phosphorylation.In LK stage,the expression of GPI,G6PDH,PGLS and pfp related to the above pathway increased8-to 10-fold,the expression of GADPH,pgm?and PK increased by 9-to 14-fold,and the expression of MDH,mae B and ALDH increased 8-to 13-fold,ACLY,ACO and CS expression increased 6-to 10-fold,which accelerated the material energy supply in LK stage.Among the metabolic pathways related to seed antioxidant capacity,including peroxisome and phenylpropane biosynthesis,68 genes related to POD and other related genes were enhanced at LK stage,which was conducive to maintaining stable environment during seed germination.Endogenous hormone synthesis is related to tryptophan metabolism,zeatin biosynthesis and diterpene biosynthesis.In the LK and MF stages of seeds,CYP707A,a key gene for ABA decomposition,was up-regulated 11.1-fold,and the key genes of hormone synthesis such as GA,IAA and CTK were significantly up-regulated.In conclusion,the pollination of C.migao could be completed normally at flowering stage,but the flowering phenology was earlier than that of early research;the seeds can grow and develop smoothly under the coordination regulation of internal physiology;mature seeds have physiological dormancy caused by endogenous inhibitors and hormone levels,and the dormancy release effect shows that the dormancy type was physiological shallow dormancy,and this may be the main reason for the obstacle of seed germination;dormancy and germination process of the seeds of C.migao are closely related to the internal physiological regulation,among which the balance of hormones played a key role in the release of dormancy and germination.