Determination Of Compatibility Of Pineapple Hybridization And Preliminary Study On The Application Of CRISPR/Cas9 Knockout Technology In Pineapple

Pineapple(Ananas comosus L.)is one of the main tropical fruits in the world.It is native to the tropical regions of Central and South America.China has been introducing and cultivating for nearly 400 years.Pineapple has many problems such as serious self-incompatibility and high cost of tissue culture seedlings,which seriously affects its variety improvement and seedling breeding.Therefore,this study used the main pineapple varieties and the same genus as materials to carry out hybrid affinity determination and hybrid seed nursery experiments;On the other hand,on the basis of the previous research of this research group,CRISPR/Cas9 gene editing technology was used to knock out the genes related to pineapple somatic embryo development AcHB13 and AcKAN2.Below are key research findings:1)Take ’Shenwan’(A.comosus cv.’Shenwan’)and ’Bali’(A.comosus cv.’Comte de paris’)as the female parent,’MD-2’(A.comosus cv.’MD-2 ’),’ cain ’(A.comosus cv.’ Smooth Cayene ’),three Taiwanese farmer varieties were used as male parents,and the hybrid affinity of the main pineapple varieties was determined.the result shows:Among the combinations with ’Shenwan’ as the female parent,the ’Shenwan’ × ’MD-2’ combination has the best hybrid affinity,seed setting rate(number of seeded small fruit number of hybrid small fruit)and affinity index(The number of seeds total ovules of seeded small fruits)were the highest,reaching 97.2% and 0.07;The rate is 0).In the hybrid combination of’Bali’ as the female parent,the seed setting rates of ’Bali’ × ’Tai Nong 16’ and ’Bali’ × ’MD-2’are similar(57.7% and 58.3%).The sum index was 0.04 and 0.03,and the hybrid combination of ’Bali’ × ’Tai Nong 11’ showed no seed formation(seed formation rate was0).2)When crossing between species,The seeding rates of ’Shenwan’ × ’Jade Linglong’(A.nanus cv.’Yulinglong’),’Shenwan’ × ’Green Red Bud Pineapple’(A.bracteatus var cv.’Green Tricolor’),’Shenwan’ × ’Tachibana’(A.erectifolius),’Tachibana’ × ’Shenwan’,’Jade Linglong’ × ’Shenwan’ are 88.2%,77.1%,74.6%,73.9%,71.2%,43.4%,and the hybrid affinity index was 0.128,0.103,0.077,0.087,0.078,0.123,and finally obtained 45,80,52,70,9,55 interspecific hybrid plants;When crossing within species,’Caine’ × ’Shenwan’,’Shenwan’ × ’MD-2’,’MD-2’ × ’Shenwan’,’Shenwan’ × ’caine’,the seed rate is 74.1% 73.0%,70%,37.5%,the cross affinity index was 0.056,0.076,0.060,0.042,and finally obtain 11,11,17,9 intraspecific hybrid plantsand.3)In order to compare the affinity between parents of different kinship,self-inbred combinations(’Shenwan’ × ’Shenwan’)and cross-breeding combinations of different cultivated species(Queen’s’ Shenwan ’ × Caine’s’ stingless caine)’)And interspecies hybridization(pineapple’ Shenwan ’ × dwarf pineapple’ Yu Linglong ’)pollination microscope observation.The results showed that the pollen of the self-crossing combination can germinate on the stigma,the germination rate is about 40%,but the pollen tube grows slowly,and the pollen tube has stopped growing after 7 hours of pollination,with an average length of about 0.49 mm;The pollen germination rates of different types of crosses and combinations of interspecific hybrids were as high as 85% and 95%.After 7 hours of pollination,the pollen tubes were 2.62 mm and 7.13 mm long,reaching the middle and bottom of the style until they entered the ovule to achieve fertilization.4)The seeds began to germinate around 15 days after germinating and about 25 days after germination began to take root and spread leaves.In the experiment of four different germination methods,the results show that the "combination germination method" has the best effect,with a germination rate of 66.50%,germination time of 13.60 days and hybrid seedlings up to 3.18 cm at 40 days.5)Using an efficient multi-target CRISPR/Cas9 gene editing vector system,the knockout vectors pYLCRISPR/Cas9-AcHB13,pYLCRISPR/Cas9-AcKAN2 for editing the genes related to pineapple somatic embryo development were constructed.Transformed pineapple callus through Agrobacterium tumefaciens-mediated transformation.After 3rounds of resistance screening of 30 mg/L,40 mg/L,and 50 mg/L kanamycin,30 resistant plants were obtained,waiting for subsequent PCR detection.And sequencing verification.