Creation And Identification Of Interspecific Clubroot Resistant Hybrids Between Brassica Oleracea And Brassica Rapa

Clubroot is a worldwide soil-borne disease caused by Plasmodiophora brassicae Woron.All the susceptible varieties of cruciferae can be infected by the pathogen.Brassica oleracea is the main cruciferous crop,and has a larger cultivated areas in our country.In recent years,due to the influence of clubroot,production and quality of Brassica oleracea significantly has been reduced.At present,the clubroot resistance of main cultivated cultivars of Brassica oleracea in China are not ideal.Breeding resistant cultivars is a economical and effective way to control clubroot in production.However,due to the lack of disease-resistance resources in Brassica oleracea,the clubroot resistance breeding of Brassica oleracea is greatly limited.B.rapa L.ssp.rapa and B.rapa L.ssp.pekinensis are the member of the Brassica rapa family in cruciferae and contain more clubroot-resistant materials.B.rapa L.ssp.rapa and B.rapa L.ssp.pekinensis have been studied more carefully for clubroot resistance gene and have been widely used in disease-resistant breeding.Distant hybridization can be used to transfer the resistant genes from Brassica rapa to Brassica oleracea by embryo rescue technique,which can improve the resistance in Brassica oleracea.Through continuous backcrossing,finally the clubroot resistance gene of Brassica rapa will be transfered to Brassica oleracea,and this will lays a solid foundation for the clubroot resistance breeding of Brassica oleracea.In this study,Brassica oleracea,B.rapa L.ssp.rapa and B.rapa L.ssp.pekinensis of Brassica rapa were used as research materials.The hybrids of Brassica oleracea×B.rapa L.ssp.rapa and Brassica oleracea×B.rapa L.ssp.pekinensis were obtained by artificial bud pollination and embryo rescue.The morphological identification,cytological identification,pollen fertility analysis and clubroot resistance testing of hybrid F₁ and BC₁ obtained from distant hybridization were performed.The main research results were as follows:1.The clubroot resistance of 69 cruciferous materials were tested by naturally induced inoculation methods in the field.The results showed that there were three immune-resistant materials,three high-resistant materials,11 resistant materials,11tolerant materials,and 41 susceptible materials.Six materials with better economic traits and strong resistance were selected for artificial inoculation identification in the controlled condition.Based on the results of indoor and field studies,two B.rapa L.ssp.rapa species WJ-1,WJ-2 and two B.rapa L.ssp.pekinensis CR-YX,CR-CM which have strong and stable resistance for clubroot were selected as the resistant source of distant crosses.2.This study was conducted with B.oleracea D₃,E₄ and 545 as female parents and B.rapa L.ssp.rapa WJ-1 and WJ-2 as male parents.By comparing the compatibility of these crosses,the frequency of pods per flower between E₄ and WJ-1 was 90%,higher than others,and highest frequency of immature embryos survived/buds pollinated in combination of D₃×WJ-1,was 3.09%,and plantlets numbers of four combinations of D₃×WJ-1,E₄×WJ-1,E₄×WJ-2,545×WJ-2 were5,0,0,0 respectively.These plants were identified as true hybrids which had the anticipated chromosomes?2n=19?by morphological observation and cytological identification.The pollen fertility of these plants derived from interspecific hybridization was aborted.Artificial inoculation in the controlled conditon was used to identify clubroot resistance of the tissue culture seedlings.The results showed that the five F₁ obtained from the combination D₃×WJ-1 were resistant to the clubroot.3.This study was conducted with B.oleracea D₃,and B₁ as female parents and B.rapa L.ssp.pekinensis CR-YX and CR-CM as male parents.According to the flowering time,4 hybrid combinations were made.Only eight plantlets were obtained from combination of D₃×CR-YX.The chromosome number of hybrid F₁ shoot tip cells was 19.Hybrid F₁ was determined as a true hybrid through cytological identification and morphological observation.Compared with the parents,the anther of hybrid F₁was abnormal and pollen was not fertile.The F₁ was tested for the clubroot resistance by indoor artificial inoculation.Three materials GY?-10,GY?-12 and GY?-20 were resistant to physiological race 4,and five materials were susceptible to physiological race 4.4.In this study,colchicine was used to treat F₁ btween Brassica oleracea and B.rapa L.ssp.pekinensis.Compared with the normal materials,the doubled materials had larger leaves and flower buds,and the fertility was restored.The chromosome number of material which was treated by colchicine was 38.Using F₁ generation from Brassica oleracea×Brassica rapa as the backcross female parent and the Brassica oleracea as the backcross male parent,the numbers of backcross plants obtained from the cross combinations GY?-12Q×R₁ and GY?-12Q×235 were 1 and 4 respectively.The number of viable embryos/pollinated flower buds in combination GY?-12Q×235was the highest at 10%,and higher than any other combinations in this experiment.The backcrossing lines were identified as true hybrids by observing morphological characteristics.Pollen fertility analysis showed that only the pollen of GY?-12Q-6-1was viability of 5%.The clubroot resistance of BC₁ was tested by artificial inoculation in the controlled condition.The identification results were as follows:GY?-12Q-6-1,GY?-12Q-6-2,GY?-12Q-6-3,GY?-12Q-6-4 were resistant to physiological race4,GY?-12Q-4-1 was tolerant to physiological race 4.In summary,a total of 13 hybrids F₁ were obtained in this experiment,which were crossed with Brassica oleracea×B.rapa L.ssp.rapa and Brassica oleracea×B.rapa L.ssp.pekinensis.All hybrids F₁ were identified as true hybrids by cytology and morphology anlysis.The pollen of hybrid F₁ is not fertile by TTC staining.Using artificial inoculation methods to identify the clubroot resistance of F₁ hybrids,the results showed that 8 lines of F₁ were resistant to physiological races 4,and 5 lines were susceptible.Backcrossing with hybrid F₁ and Brassica oleracea,a total of 5 BC₁lines were obtained.All BC₁ lines were identified as true hybrids by morphology.Only the pollen of GY?-12Q-6-1 had viability by TTC staining.4 lines of BC₁ were resistant to physiological races 4 and 1 lines was tolerant to physiological races 4.