【作者】 张伟；

【导师】 何业华； 栾爱萍；

【作者基本信息】 华南农业大学 ， 农业硕士（专业学位）， 2020， 硕士

【摘要】 菠萝(Ananas comosus L.)是世界主要的热带水果之一,原产中南美洲热带地区,我国已有近400年的引种栽培历史。菠萝存在着严重的自交不亲和现象和组培苗成本太高等诸多问题,严重影响其品种改良和种苗的繁育。因此,本研究以菠萝主栽品种及同属种类为材料,进行了杂交亲和性测定和杂交种子育苗试验;另一方面,在本课题组前期的研究基础上,利用CRISPR/Cas9基因编辑技术对菠萝体细胞胚发育相关基因AcHB13、AcKAN2进行了敲除实验。主要研究结果如下:1)以‘神湾’(A.comosus cv.‘Shen Wan’)、‘巴厘’(A.comosus cv.‘Comte de paris’)为母本,‘MD-2’(A.comosus cv.‘MD-2’)、‘卡因’(A.comosus cv.‘Smooth Cayene’)、3个台农系列品种为父本,进行了菠萝主栽品种的杂交亲和性测定。结果表明:以‘神湾’为母本的组合中,‘神湾’בMD-2’组合的杂交亲和性最好,结籽率(有籽小果数/杂交小果数)和亲和指数(种子数/有籽小果总胚珠)都最高,达到97.2%和0.07;但‘神湾’ב台农16’和‘神湾’ב台农17’出现了不结籽现象(结籽率为0),在‘巴厘’为母本的杂交组合中,‘巴厘’ב台农16’和‘巴厘’בMD-2’的结籽率相近(57.7%和58.3%),亲和指数为0.04和0.03,‘巴厘’ב台农11’的杂交组合出现了不结籽现象(结籽率为0)。2)在种间杂交时,‘神湾’ב玉玲珑’(A.nanus cv.‘Yulinglong’)、‘神湾’ב绿红苞凤梨’(A.bracteatus var cv.‘Green Tricolor’)、‘神湾’ב立叶凤梨’(A.erectifolius)、‘神湾’ב三色凤梨’(A.bracteatus var cv.‘Tricolor’)、‘绿红苞凤梨’ב神湾’、‘玉玲珑’ב神湾’结籽率分别为88.2%、77.1%、74.6%、73.9%、71.2%、43.4%,杂交亲和指数为0.128、0.103、0.077、0.087、0.078、0.123,最终分别获得了45、80、52、70、9、55株种间杂种植株。在种内杂交时,‘卡因’ב神湾’、‘神湾’בMD-2’、‘MD-2’ב神湾’,‘神湾’ב卡因’、结籽率分别为74.1%、73.0%、70%、37.5%,杂交亲和指数为0.056、0.076、0.060、0.042,最终分别获得的11、11、17、9株种内杂交植株。3)为比较不同亲缘关系亲本间亲和性,进行了自交组合(‘神湾’ב神湾’)、不同栽培种类间杂交组合(皇后类‘神湾’×卡因类‘无刺卡因’)及种间杂交组合(菠萝‘神湾’×矮凤梨‘玉玲珑’)3个组合的授粉显微镜观测。结果表明:自交组合的花粉在柱头上可萌发,萌发率约为40%,但是其花粉管生长缓慢,授粉7 h后花粉管已停止生长,平均长约0.49 mm;而不同类型间杂交以及种间杂交组合的花粉萌发率高达85%、95%,授粉7 h后花粉管长2.62 mm、7.13 mm,到达花柱的中部和底部,直至进入胚珠实现受精结籽。4)种子从催芽15 d左右开始萌发,萌发后约25 d后开始生根展叶。在4种不同发芽育苗方法试验中,结果表明;“组合发芽育苗法”效果最佳,其发芽率达66.50%、发芽时间达13.60 d、40 d时的杂交苗高达3.18 cm。5)利用高效的多靶点CRISPR/Cas9基因编辑载体系统,构建了编辑菠萝体细胞胚发育相关基因AcKAN2、AcHB13的敲除载体pYLCRISPR/Cas9-AcHB13、pYLCRISPR/Cas9-AcKAN2。通过根癌农杆介导转化菠萝愈伤组织,经过3轮30 mg/L、40 mg/L、50 mg/L的卡那霉素抗性筛选后得到30株抗性植株,等待后续PCR检测及测序验证。更多还原

【Abstract】 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.更多还原