Cytological Observation And Polyploid Germplasm Innovation In The Genus Tulipa

Tulipa gesneriana L.,belonging to the tribe Tulipeae in the family Liliaceae,is fascinating bulbous flower all over the world with large,colorful and elegant flowers,and is known as‘the queen of flowers kingdom'.Tulipa is composed of 100¹50 species,and distributes mainly in Central Asia,Europe and North Africa,as well as the Mediterranean area.China is an important distribution area of wild tulip species.There are 18 species?containing one variant?have been found and reported,accounting for approximately 10%of the world's wild tulip resources.These resources mainly distribute in Xinjiang,Yangtze River and Northeast China.Chinese tulip industry has been developing rapidly.However,there are few reports on the hybridization and basic research of ploidy,pollen viability,karyotype and 2n gametes of tulip germplasm resources.In this study,42 tulip cultivars and eight wild tulip species were used as materials to study ploidy level and pollen germination characters.24populations of eight species native to China were cytogenetically analyzed to determine their nuclear type and genetic relationship.The study on the polyploid germplasm innovation of tulip was carried out by using the 2n pollen produced spontaneously and artificially,and by somatic cell chromosome doubling.This research was significant for the development and utilization of wild tulip resources in China and the polyploid breeding of tulip.The mainly conclusions were as follows:There were 24 diploids,17 triploids and one tetraploid identified among the 42 cultivars examined.This work confirmed that all eight species collected in China were diploid?2n=2x=24?,among which chromosome numbers of T.thianschanica,T.sinkiangensis and T.heterophylla were firstly reported.The research showed that the average diameter of somatic cell nuclear of wild tulip species?17.01?m?was significantly smaller than that of cultivars.There was no significant difference in term of the average diameter of somatic cell nuclear between tetraploid?31.82?m?and triploid?28.52?m?cultivars,whereas,both of that were significantly greater than that of diploid cultivars?21.21?m?.The pollen germination rate of the triploid tulip cultivar was the lowest,with the average value of 3.67%;the pollen germination rate of the diploid was the highest,with the average value of 18.12%;and the pollen germination rate of the tetraploid cultivar,‘Judith Leyster',was in the middle,with the average value of 12.33%.The average pollen germination rate of eight wild species was35.54%,which was significantly higher than that of tulip cultivars.The 24 populations of eight wild tulip species were diploid?2n=2x=24?,and the base chromosome number was x=12.There were three types of chromosomes observed,namely Metacentric?m?,Submetacentric?sm?and Subtelocentric?st?,respectively.The arm indexes were all 48.The karyotypes of 24 populations of eight Tulipa species were classified as 3A,3B,4A or 4B,and composed of‘sm'and‘st'or‘m',‘sm'and‘st'chromosomes.The average chromosome length of eight wild tulip species was 9.48?m,among which the value of T.edulis was the smallest?7.29?m?,and the value of T.altaica was the largest?11.60?m?.The average chromosome arm ratio was 3.11,and the minimum value appeared in T.edulis with the value of 2.62,the maximum value appeared in T.sinkiangensis with the value of 3.70.Statistical analysis showed that T.altaica and T.sinkiangensis had higher karyotype asymmetry than others,while the T.edulis had a relatively symmetrical karyotype.Cluster analysis showed that the 24 tulip populations were divided into three main branches,of which three populations of T.edulis were clustered into one branch,indicating that T.edulis had a distant relationship with other wild species.This research found that the average pollen grain diameters of tetraploid,triploid,diploid tulip cultivars and wild species were 95.19?m,71.43?m,59.08?m and 47.18?m,respectively,and there were significant differences between the four kinds of materials.This research evidenced that triploid varieties,‘Golden Parade'and‘Golden Apeldoorn',and diploid varieties,‘Purissima',‘Royal Gift',‘Eskimo Chief'and‘Crispa Fabio',could produce2n pollen spontaneously.We also found that T.schrenkii distributed in China could produce2n pollen spontaneously,and 2n pollen rate reached 13.64%,significantly higher than other six cultivars,which was report for the first time.It was feasible to created new polyploid germplasm by using different ploidy cultivars crossing with tulip cultivars or species which can produce 2n pollen.When diploid cultivars?female parent?crossed with diploid cultivars with 2n pollen,capsule setting rate was high and the polyploid rate in the progeny was low,whereas crossed with triploid cultivars with 2n pollen,capsule setting rate was low and the polyploid rate in the progeny was high.When triploid cultivars were used as female parent,many crossing combinations did not give any mature seeds,and the capsule setting rate was low despite the fact that some combinations was successful,whereas the polyploid rate was higher than other combinations.Tetraploid?2n=4x=48?and pentaploid?2n=5x=60?were found in the progeny.Five of the eight combinations were successful when wild tulip species distributed in China was used as male parent.Triploid,tetraploid,and pentaploid hybrids were obtained from the crosses of‘Synaeda Amor'?2n=2x=24?×T.schrenkii and‘Banja Luka'?2n=3x=36?×T.schrenkii,respectively,which proved that the 2n pollen produced by T.schrenkii were functional.The 2n pollen grains could be produced successfully by diploid cultivars treated with high pressure N₂O gas.Among which,the diameter of the maximum pollen grain of‘Synaeda Amor'increased from 66.72?m to 88.70?m,and the ratio of maximum/minimum pollen diameter increased from 1.22 to 1.65,with the 2n pollen rate of 16.00%.For‘Carnaval de Rio',the diameter of the maximum pollen grain increased from 65.24?m to 97.66?m,and the ratio of maximum/minimum pollen diameter increased from 1.22 to 1.81,with the 2n pollen rate of 26.67%.Four crossing combinations all obtained polyploid progenies with the polyploid rate of 4.02¹7.93%,which revealed that 2n pollen induced artificially was functional in the hybridization process.This study proved that it was feasible to induce polyploid by soaking wild tulip seeds in colchicine.The optimal treatment of seeds was 1.00 g/L concentration for 24 h in T.sinkiangensis with the morphological variation rate of 30.00%.The optimal treatment of seeds was 0.50 g/L concentration for 12 h in T.iliensis with the morphological variation rate of 16.67%.After culturing 10 d in culture medium with 0.05 g/L concentration of oryzalin,seeds of T.sinkiangensis were transferred to the normal culture medium,with the morphological mutation rate of 10.00%,whereas after culturing 30 d in culture medium with0.10 g/L concentration of colchicine,seeds of T.sinkiangensis were transferred to the normal culture medium,with the morphological mutation rate of 16.67%.In contrast,when seeds of T.iliensis were cultured in culture medium with different concentration of oryzalin or colchicine,the induction effects were poor,with the highest morphological mutation rate of 6.67%among 18 treatments.