The Mechanism Of Seed Deterioration And Vigour Testing Methods For Forage Species

Seed vigour is one of the most important indicators reflect seed quality.Deterioration/aging is the main factor that causes decrease of vigour.Investigate the mechanism of forage seed deterioration,vigour testing methods and main factors affected testing are of great importance in effectively evaluating quality of forage seeds,improving natural grassland and establishing artificial grassland.This study used seeds of several important forage species including:Medicago sativa,Trifolium repens,Elymus nutans,Avena sativa,Festuca sinensis and Broms inermis as the experimental material.The flow cytometry were firstly used and along with other techniques,combine with laboratory and field test to investigate the following contents:the mechanism of vitro degradation of forage seeds,DNA changes,and rate of DNA replication(represented by 4C/2C ratio);relationship between DNA replication and seed vigour;effect of the semi-permeable layer in grass seed on evaluation of vigour using conductivity method;and the feasibility of radicle emergence rate for seed vigour determination in grass species.The following main results obtained:1.The appropriate method for determination of seed DNA by flow cytometry was explored using 30 forage seeds.Results showed that small seeded plants are more suitable use whole seeds,while the medium and large seeded plants are more suitable using embryos as material to determine genome size.In the selection of isolation buffer,the suitable isolation buffer of leguminous seeds was LB01.For grass species,the small-and medium-seeded plants was LB01,while the large-seeded plant was MG.The genomes size of the 30 forage species tested varied significantly,ranging from 423 Mbp to 9772 Mbp.2.Study on damage of genetic material in seed deterioration with dry seeds of Elymus nutans as material using flow cytometry showed that as seed deterioration increased,DNA degradation increased,the nuclear debris content increased,the correlation analyses showed that the percentage of debris was significantly negatively correlated with germination rate(R~2=0.5)(P<0.05).An increase in specific bands resulted in an increase in genomic DNA,and the genomic DNA content of heavily aged seeds(DNA content was 140.82 pg/3C)was three times than that of controlled seeds(46.94 pg/3C).3.The study of DNA replication in the process of water absorption with different germination percentage tested using alfalfa and white clover as materials,showed that there was no difference in the DNA replication rate of dry seed even with different aging levels.As seed water absorption time increased,DNA replication was fast in the higher germinated seeds than the lower germinated seeds,besides,there was a significant difference in DNA replication in seeds with different vigour level ahead of the germination,for instance,the germination of Medicago sativa were 78%,67%and 68%,while DNA replication rates(4C/2C)were 35%and23%,13%,respectively;the germination percentage of Trifolium repens were 89%,48%and 14%,while 4C/2C were 34%,19%and 17%,respectively(P<0.05).4.The relationship between 4C/2C and vigour of 10 Medicago sativa(germination 68%-81%)and Trifolium repens(70%-95%)seed lots showed that DNA replication in early water absorption(24h)4C/2C has a significant positive correlation with field emergence(R~2=0.77;0.71 for Medicago sativa and Trifolium repens,respectively)(P<0.01);Further analysis with acceptable germination of commercial seed lots showed that 4C/2C ratio and field emergence also has a significant positive correlation with R~2 being 0.81 and 0.77,respectively(P<0.01),which was significantly higher than the relationship between standard germination and field emergence.This results indicates that DNA replication can be used for seed vigour evaluation of the tested species.5.The response of the seed semi-permeable layer to the evaluation of vigour by using electrical conductivity showed that there was no correlation between seed germination and electrical conductivity for intact seeds of Festuca arundinacea and Bromus inermis with a semi-permeable layer,nevertheless,after piercing seed coat,a significant negative correlation obtained(R~2 were both 0.90)(P<0.01).For Avena sativa seeds without seed semipermeable layer,both intact and pierced seeds had a significant negative correlation(R~2 were 0.79 and 0.90,respectively),which revealed that semi-permeable layer in grass seeds limited electrical conductivity test in vigour assessment.Further determination of 15 commercial Avena sativa seed lots(with germination rate≥80%)showed that electrical conductivity was significantly negatively correlated with the field emergence of both two sowings dates(R~2=0.60,P<0.01;R~2=0.53,P<0.05).Thus,the conductivity method is considered as suitable method for evaluation vigour of Avena sativa in which a seed semipermeable layer is absent.6.The relationship between radicle emergence rate and seed vigour of 15 seed lots of Avena sativa(standard germination rate 80%~93%)and Elymus nutans(70%~84%)showed that mean just germination time(MJGT)was significantly negatively correlated with the seedling emergence in two sowings dates(R~2=0.52,0.52;0.58,0.74,respectively)(P<0.01)of both species.The radicle emregence(RE)of Avena sativa seeds after 52h water absorption was significantly positively correlated(R~2 was 0.62 and 0.63)(P<0.01)with field emergence,while Elymus nutans at 76 h after water absorption was positively correlated(R~2 was 0.64 and 0.67)(P<0.01).This results indicated that MJGT and RE can effectively evaluate vigour of the tested species.