Manipulation Of Auxin And Cytokinin Level In Cotton Concurrently Increases Fiber And Seed Yield

Cotton is one of the most valuable commercial crops and China is the largest cotton producer and consumer in the world. In addition to the fiber used in textile manufacturing, cotton seed is an important source of oil and protein, and the seed hulls is used for cattle feed and mushroom production. Although great progress has been made in the improvement of fiber qualities and yield by traditional breeding methods, unfortunately, cotton seeds get smaller with the increasing fiber yield. Thus, it is an important task for cotton breeders to simultaneously improve the cotton seeds and fiber productivity.Genetic modification is a powerful way to put certain desirable traits into organism and provides a new method for cotton breeding. In previous study we demonstrated a successful stretagy to significately increase cotton fiber yield by spatiotemporal manipulation of auxin biosynthesis in cotton ovule (FBP7::iaaM). However, a shortcoming of this strategy is that the seed of the transgenic cotton became, more or less, smaller than the wildtype. Thus, we need to find a way to solve this problem and concurrently increase the yield of both fibers and seeds.Cytokinins are a group of phytohormones that regulate cell division and influence numerous developmental and physiological processes including leaf senescence, vascular development, cell differentiation at shoot and root apical meristem, abiotic and biotic stress responses. Cytokinins also can increase the fixation and absorption of carbon in source tissues of plant, coordinate the transport and metabolism of nutritive plants of sink and source tissues, facilitate fruit setting and promote assimilate accumulation during the development of fruit and seed. Importantly, recent studies have revealed that cytokinin is a key regulator for seed yield. Exogenous application of cytokinin compounds could improve the yield of seed cotton and lint fiber. However, large-scale commercial applications of cytokinins in crops are practically infeasible due to high costs and time consumption.Promoting cytokinin biosynthesis is an effective way for enhancing the endogenous level of cytokinins in plants. The ipt gene encodes isopentenyl transferase, the enzyme catalyses the rate-limiting step in cytokinin biosynthesis. ipt has been widely used for the enhancement of cytokinins in transgenic plants. However, constitutive overexpression of the gene is usually associated with adverse effects on growth and development of the plants. To increase endogenous cytokinins in cotton, our lab had tried to generate transgenic cottons using a constitutive promoter, CaMV35S, to control the expression of ipt. However, the transformed calli never further developed into plant regenerants.Another approach to elevate the endogenous cytokinins is suppressing deactivation of cytokinins. Cytokinin oxidase/dehydrogenase (CKX) that catalyzes the catabolism of cytokinins to inactive products that lack the N6-unsaturated side chain is a crucial negative regulator controlling endogenous cytokinin contents in plant kingdom. Suppressing expression level of CKX results more cytokinins in inflorescence meristems, thus, more grains.To enhance endogenous level of cytokinins in cotton, in this paper, the down-regulation of CKX gene (GhCKX) cassettes were under control of a constitutive promoter, CaMV35S, and two ovule-specific promoter (D113 and Pv), respectively. After evaluation of these CKX down-regualted cottons, we stacked GhCKX down-regulation gene cassettes together with FBP7::iaaM to improve the seed and fiber yield of transgenic cotton. Main results are as following:1. Down-regulation of GhCKX in cottonUsing Agrobacterium-mediated transformation methods,17 independent transgenic 35S::GhCKXRNAi (CR) cottons were generated. According to the gene suppression level, transgenic lines could be divided into three groups: slightly-suppressed (gene expression level was decreased by less than 30%), moderately-suppressed (decreased by 30-70%), and severely-suppressed (over 70%,) group. The GhCKX severely-suppressed cottons showed a typical cytokinin overproduction phenotype, e.g., stunted shoot, shorten internode, smaller leaves and floral organs, and even sterility. Contrarily, no developmental abnormalities were observed in the slightly suppressed cottons, the moderately down-regulated lines showed a slightly decreased, bore more squares and bigger flowers.2. Down-regulation of GhCKX enhanced endogenous cytokininsTo confirm that regulation of GhCKX changes the level of endogenous cytokinins in transgenic cottons, we determined the content of 13 different cytokinins of GhCKX moderately-suppressed lines and severely-suppressed line by LC/MS. The concentration of IP, ZT, ZR and IPR was always higher than wild type in the leaves, stem shoots, flower buds, and ovules. Total cytokinins in plants of the moderately-suppressed lines, CR-3 and CR-6, increased 20.4% and 55.5% respectively, and that of the severely-suppressed line (CR-13) enhanced 134.2%, compared to that of the wild type. With the decline of the gene expression, the contents of cytokinins increased significantly, confirming that down regulation of GhCKX elevated the level of endogenous cytokinins in cotton.3. Moderate down-regulation of GhCKX delayed leaf senescence and increased leaf photosynthesisThe chlorophyll content of upper, middle and lower leaves in and severely-suppressed line were higher than that in the wild type at the time of 90,105 and 120 DAS (Days after sowing). Meanwhile, the abscised leaf number was much lower than that of the wild type. Photosynthetic rate of leaves in moderately-suppressed lines was higher, showing a 10.7% and 12.2% increase than wild type. However, down-regulate of GhCKX inhibited root development with less and shorter lateral roots, especially, in the severely-suppressed lines.4. Moderate down-regulation of GhCKX increased both seed and lint yieldBased on the expression level of GhCKX coupled with phenotype observation in transgenic cottons, one GhCKX slightly suppressed line, CR-7, five moderately down-regulated lines, CR-3,-4,-5,-6, and-8, and two severely suppressed lines, CR-11 and CR-13, were planted in field condition to observe their agronomic performance. The averaged boll number of the five moderately down-regulated lines was produced 19.0% more bolls than the wild type. Moreover, the seed cotton weight per boll, and seed index (the weight in grams of 100 seed) of the moderately down-regulated lines were hiher than the control. On the contrary, the number of the boll and the seed size in the severely suppressed cottons, CR-11 and CR-13, were lower and smaller compared to the wild type. To further assess the performance of the moderately down-regulated GhCKX cotton in yield improvement, CR-6 (T4) was selected as the best line into field trail using the randomized complete block test.The seed cotton yield per plot of moderated suppressed line (CR-6) increased 17.2%, and the seed yield and lint yield increased 15.4% and 20.0% respectively, as compared to the wild type into field trail using the randomized complete block test. On the contrary, the number of the boll and the seed size in the severely suppressed cotton CR-13 were lower and smaller, and the lint yield and and seed yield decreased compared to the wild type. As to the quality of the fiber, there were no notable alterations in fiber length, fiber strength and micronaire in transgenic cottons, indicating that increase of endogenous cytokinins in cotton has little influence on qualities of transgenic fibers.5. Seed-specific suppression of GhCKX improved seed development and increased seed cotton yieldSeed-specific suppression of GhCKX (Pv::antiGhCKX and D113::antiGhCKX) also elevated the level of endogenous cytokinins in the embryo. The averaged value of 100 seeds weight of Pv::antiGhCKX and D113::antiGhCKX transgenic lines was increased by 10.2% and 6.5% respectively compared to the wild type.6. Combining FBP7::iaaM with 35S::GhCKXKNAi improved yield of lint and seed concurrently with fine fiber qualityFBP7::iaaM and 35S::GhCKXRNAi genes were stacked together by cross of CR-6 and FM 9-8. Homozygous lines that hold two gene cassttes (FR) were idetified from F4 148 plants. The stacking line showed higher photo synthetic rate, lower number of abscised leaves and bigger bolls like displayed by CR-6. The weight of 100 seeds of homozygous lines (FR-3) increased by 14.8% and 5.58% compared with FBP7::iaaM transgenic plant and wild type respectively. The number of fiber initials per seed, lint percentage and lint index of homozygous lines (FR-3) were all increased. The results showed that 35S::GhCKXRNAi gene cassette confered the enlargement of seed size and successfully overcame the shortcoming of smaller seeds in FBP7::iaaM cotton, meanwhile, FBP7::iaaM produced more fibers per seed and increased fiber yield. Our data demonstrated that gene stacking by crossing FBP7::zaaM with 35S::GhCKXRNAi transgenic lines is a promising strategy for fiber yield and seed yield improvement concurrently.Taken together, our data demonstrated that moderately enhancing endogenous cytokinins by suppression of CKX is a feasible and effective strategy for yield improvement for cotton. Futhermore, stacking the elite transgenes together, such as FBP7::iaaM and 35S::GhCKXRNAi, can further increse the yield of fibers and seeds.