Study Of Growth And Sexual Reproduction Characteristics Of Vicia Unijuga Var.ganqing In Gannan Alpine Grassland

Perennial vetch（Vicia unijuga var.ganqing）is a legume forage with high nutritive value,bred for use on the Qinghai-Tibetan Plateau of China by the College of Pastoral Agricultural Science and Technology,Lanzhou University.It has a wide adaptability and strong stress resistance,and is an important forage for local pasture improvement in natural grassland.More flowers and less fruit are the main problem of perennial vetch at present.Based on previous work,the present study was conducted over four consecutive years from 2018 to 2021 at three sites;two in Xiahe County（altitude 2880 m）,and Luqu County（altitude 3074 m）in Gannan Tibetan Autonomous Prefecture,Gansu Province,and one in Yuzhong County（altitude 1768m）in Lanzhou,Gansu Province.Through a combination of conventional technology and molecular biotechnology,the differences of leaf structure between perennial vetch,alfalfa（Medicago sativa）and sainfoin（Onobrychis viciifolia）,the flowering process,pollen characteristics and seed yield characteristics of perennial vetch were explored in the laboratory and in the field,to elucidate responses to varying environmental conditions,so as to clarify the reproductive biological characteristics of this forage and its response to environmental conditions.This work aimed to provide theoretical and technical support for the utilization of the high quality forage.The main results are as follows:（1）The difference of leaf structure and photosynthetic function characteristics and their adaptability to altitude between perennial vetch and alfalfa and sainfoin.Compared with alfalfa and sainfoin,perennial vetch leaves showed lower leaf mass per unit area（LMA）,net photosynthetic rate（Pn）,actual photochemical efficiency(Φ_PSII)and total nitrogen content（TN）at all three experimental sites,which indicated that it is a resource-saving species with a‘high input-low’return strategy.The effects of habitat on leaf structure and photosynthetic function characteristics were mainly reflected in the effects of temperature and water availability on leaf gas exchange.The gas exchange and photosynthesis were enhanced by larger sparse and irregular epidermal cells and larger stomata,and the water use efficiency was improved by lower adaxial stomata density.The thickness of palisade tissue,spongy tissue and leaf increased with the increasing altitude,the area of stomata decreased with the increase of altitude,and the thicker epidermal cells were all the advantages of perennial vetch for adaption to alpine conditions.（2）The flowering process and the factors affecting ovule abortion of perennial vetch.The stigma activity and pollen activity of perennial vetch sharply decreased on the 3^rdday after full bloom,and there was basically no activity on the 5^th day.The release of pollen involved the brush mechanism.Bud fade was mainly concentrated in the period from 3 to 5 d after full bloom.The pollen had scattered to the stigma one day before the flower petals opened,and the pollen began to germinate 6 h after reaching the stigma.The pollen tube elongated massively and began to enter the style at 8 h,and reached the ovule at 72 h.Sperm cells reached the micropyle and combined with egg cells at 96 h after flowering.The fertilized egg cells then entered the tetrad stage,and progressed through the proembryo,rod embryo and,globular embryo stages,and reached the kidney embryo stage at 25 d after flowering.At this point,the early stage of embryonic development was basically completed.Flower fall was closely related to the self-pollen blockage of stigma and the folding of pollen tube after entering the ovary.（3）The changes of physiological metabolism in the flowering process of perennial vetch.The contents of total nitrogen,total phosphorus,organic carbon,total potassium,total calcium,total soluble sugar,sucrose,fructose,auxin,gibberellin,abscisic acid,zeatin,kinetin and the ratio of carbon:nitrogen fluctuated significantly at day 3 d and 5 d,and the change trend was basically consistent at the three altitude sites.Transcriptome sequencing showed that homology with Medicago truncatula and Cicer arietinum,was the strongest and the most differentially expressed genes were found between the day before flowering and the day of full bloom.Gene ontology（GO）and Kyoto Encyclopedia of genes and genomes（KEGG）enrichment analysis showed that the genes with higher expression levels at 3 d and 5 d were mainly clustered in metabolic process,cell transformation,hormone regulation,devel opment processes,reproduction-related biological process,enzyme activity,protein transport and nutrition functions.The main expression pathways were carbon metabolism,sucrose and starch metabolism,amino acid metabolism,and plant hormone signal transduction in environmental information pathways.The differential expression of sucrose metabolism was extremely significant and the expression level was higher,followed by the glycolysis pathway and pentose and glucose metabolism pathway.These physiological and metabolic changes jointly regulate the flowering process of perennial vetch.（4）The pollen of perennial vetch is tricolpate trinuclear pollen,with an oval shape in the equatorial view and a triangular shape in the polar view,and the surface ornamentation of pollen is a mesh pattern.The pollen germination rate（Pgr）peaked at92%immediately prior to the flower opening,at which time the anthers were just cracking.The optimum conditions for pollen in vitro culture were found to be 20%Sucrose+5 g L^-1 Agar+5 mg L^-1 Boric acid+40 mg L^-1 Ca Cl₂ under 70%humidity and 25°C,and Pgr under this regime was 90%.It was found that humidity had the greatest influence on the Pgr.This was consistent with the fact that perennial vetch showed higher pollen activity at high altitude with high humidity and the blooming stage was around 25°C.（5）Atmospheric temperature and soil moisture were found to be the main environmental factors affecting the growth and sexual reproduction of perennial vetch.At the Luqu County site at 3074 m altitude,perennial vetch mainly displayed fewer reproductive branches,and mainly sexual reproduction.At this altitude the limited resources were allocated more towards reproductive growth,with lower aboveground biomass and relatively higher reproductive success rate.At the Xiahe County site at2880 m altitude,perennial vetch displayed the highest reproductive success rate and harvest index.The traits were bounded between the other two sites,relatively more resources increased the nutrient input in the reproductive stage.At the Yuzhong County site at 1768 m altitude,there were sufficient resources to promote asexual reproduction;thus,perennial vetch displayed more branches,higher plant height and aboveground biomass,relatively lower resource input for reproductive growth,and the lowest relative sexual reproductive success rate and harvest index.In summary,this study provides an evaluation of the adaptability and a detailed description of sexual reproductive biology characteristics of perennial vetch to growing conditions in alpine grassland.This information has important value for the efficient seed production expansion and to inform extension initiatives promoting the use of perennial vetch on the Qinghai-Tibetan Plateau.