Delaying Leaf-senescence Via PPF1 Gene Transformation And Characterization Of Several Mutants Related To Reproduce In Rice

(â… )As a law of nature for plants, senescence is not always welcomed with respect to theproduction of higher plants. Therefore, manipulation of the leaf senescence developmentmay result in agricultural benefits. Improving photosynthesis and suppressing senescencefor a longer functional period on the basis of a rational life time, were believed to beeffective measures to regulate the relationship of source-sink in plants. In the presentresearch, PPF1, a gene which was previously demonstrated to influence the process ofplant development through controlling chloroplast development, was introduced into riceby Agrobacterium-mediated transformation. In addition, many factors involved intransformation procedures were also investigated.1 After co-cultivation of over 970 scutellum calli with Agrobacterium tumefaciens,approximately 280 hygromycin-resistant calli were obtained, and 54 independentplantlets derived from different calli were regenerated.2 PCR and Southern blot assay demonstrated that the target gene was integrated intothe genome of 42 plantlets with a positive ratio of 77.7ï¼…. Most of the positive plantswere improved in chlorophyll content and photosynthetic index.3 A leaf-senescence-delaying line, designated as line 269, was generated from T₃generation plants. PCR and Southern blot analysis confirmed that the foreign DNAfragment was successfully integrated into rice genome. By classical linkage analysis,the introduced PPF1 gene, which was expressed stably in line 269, was proved to beinheritable as a single Mendelian factor and to be causation of theleaf-senescence-delaying phenotype.4 Several physiological characters, such as chlorophyll content, net photosynthetic rate,and stoma conductance were significantly or extremely improved in line 269 as aconsequence of expression of the foreign gene. Meanwhile, the transgenic line wasimproved significantly in panicle weight, 1000-grain weight, seed-setting rate, as wellas yield per plant. Thus, we considered that expression of PPF1 gene in ricesuppressed the leaf-senescence process and hence increased the production, mightby controlling chloroplast development.5 Several important factors affecting rice transformation frequency mediated byAgrobacterium were investigated with 3 indica rice varieties and 2 japonica ricevarieties. Agrobacterium strain Agl1 and EHA105 were proved to have positiveassistant affection on co-transformation, and re-suspending the agrobacterium thalliwith MS culture media can significantly improve the resistant calli rate; Desiccationculture has an significant influence on both the resistant calli rate and the differentiation rate, but different receptors may have different methods for desiccationculture; Adding DMSO and pro to the differentiation media, and NAA to the rootingmedia, can, to some extent, improve the regeneration rate and the surviving rate,respectively. A lot of useful transgenic plants were obtained by using this protocol.(â…¡)Reproduce is a vital process for plants and several factors were involved in it.According to the angiosperm's embryo genesiology, abnormalities of plant reproducewere mainly showed in male-sterility, female-sterility, or male-female-sterility. Sometimesmalformation of the floral organ also gave obstacle to plant produce. In recent year, lots ofmutant related to plant reproduce have been identified and characterized. So we havelearned lots of factors participated in this procedure, though the molecular mechanism stillremains uncertain. In the present study, we want to characterize several mutants relatedto rice produce through anatomical, cytological observations, genetic analysis andmolecular localization of the target gene.1 We report a female sterile material originating from spontaneous mutation, andcharacterize it as a novel type of female sterile mutant in rice through anatomical,cytological and fluorescence microscope observations. Finally, the target gene was finemapped on rice chromosome.1.1 The mutant morphology was the same as the wide type rice 202R except for its' lowerseed setting rate. The seed setting rate of 202R by selfingwas above 90ï¼…, while thatof the mutant did not exceed 5ï¼…. When the mutant was crossed as male parents, theseed setting rate was normal. However, when it was crossed as female parents, wecould not obtain a single seed. Investigations also showed that pollen grains of themutant were normal in its morphology and in its staining gradation when compared tothe wild. type. So we deduced, that the mutant, designated as fs-202R, was a femalespecific sterile mutant and the mutation did not affect the male gametophyte function.1.2 We found almost all the mature embryo sacs were of typical polygonum type andmore than 90ï¼…of the embryo sacs had complete inner components. Furtherobservations of the development ofthe embryo in the mutant revealed that the eggcell could not complete the fertilization with the sperm cell, and that the embryo andthe endosperm were undetectable, though the disintegration of the egg cell whichdid not complete the fertilization was noticed, suggested that the sterility of themutant might Occur in the course of pollination.1.3 Twenty minutes after flowering, the speed of the pollen tube's elongation betweenthe mutant and 202R diverged. The former was apparently slower than the latter.The pollen tubes in 202R reached the ovary, the basal ovule and the micropyle 30 min, 45min, and 60min after flowering, respectively, while those in the mutant wereabnormally blocked in the style transmitting tissue during the same period with thefollowing main troubles: (1) swelled tip of the pollen tube like air bladder; (2) bendedtip of the pollen tube and conversed elongation; (3) agglomerated tip of the pollentube and distorted elongation. (4) branched tip of the pollen tube. As a result, nopollen tubes were found entering the embryo sac in the mutant, even 10h afterpollination. Thus, we concluded that the mutant's female sterility character was aconsequence of the disruption of the double fertilization caused by the arrest of theelongation of the pollen tube.1.4 F₁ plants of all cross were fertile and F₂ plants segregated with a ratio of 3:1 (fertile:sterile), which suggested the mutation was controlled by a single recessive geneand designated as PTBFS(t).1.5 The F₂ population of cross G630/fs-202R (102 sterile plants) were used in genemapping. Unkage analysis revealed that the PTBFS(t) gene was flanked with thefollowing SSR makers on CHR.5, RM153, RM122, RM13, RM413, RM592, RM405,RM437, RM169 and RM289, with the genetic distance of 28, 26, 1.5, 1.5, 0, 3.1,8.9,27.9 and 32.1CM, respectively. So the PTBFS(t) gene was first located betweenRM413 and RM405 with 4.8 CM in genetic distance and 890K in physical interval.Fine mapping was conducted in a F₂ population (2382 sterile plants) generated ofthe same cross and the PTBFS(t) gene was then located to a 130K region betweenRL85 and RL167. Combining with the mapping results in F₂ of cross 9311xfs-202Rand duo1 xfs-202R, the PTBFS(t) gene was narrowed down to a physical region of72K between RL33 and RD19 on CHR.5.1.6 The 72K region was analyzed by using TIGR Rice Browse and 10 TIGR Rice Lociwere identified. All the loci were then confirmed be genes with transcriptional activityby employing an in-silicon hybridization strategy. In addition, we also found lociLOC_Os05g05270, LOC_Os05g05320 and LOC_Os05g05340 were matched tocertain ESTs or proteins which were previously demonstrated participating in thepollination procedure, so we believe these three genes might be responsible for themutation and finally take them as the candidates for the PTBFS(t) gene.2 We report a floral organ mutant originating from spontaneous mutation, andcharacterize it through anatomical, cytological observations and by genetic analysis.2.1 In the mutant, the paleas and lemmas are overdeveloped and the spikelets are openwith one or two flowers setting on the axis. Between paleas and lemmas, severalpalea and lemma-like structures were noticed and the number of anther and pistilwas abnormal. The seed setting rate of the mutant was low and 3ï¼…of the spikeletsproduce twins in one bract and we named it SRS2. 2.2 Anatomical observations for floral structures of SRS2 revealed that almost 60ï¼…ofthe spikelets generated twin-flowers in the bract and the palea and lemma-likestructures originated from lodicules were acted as the boundaries of the twin-flowers.The mutant flower, with fewer anthers and more pistils, was obviously decreased inthe pollen fertile rate.2.3 Tile mutant floral morphogenesis differs from one or twin flowers a spikelet wouldfinally generate. In single-floret mutant, the floral primordia abnormally developed ina triangle and then produce series of abnormalities during the development ofanther primordial, Iodicule primordia and the carpel primordia. In double-floretmutant, the mutant floral primordial was shrinked from one side and formed twoprimordia, then the palea and lemma-like structures acted as the boundaries of thetwo floral primordia were formed. Consequently, the stamen primordial, the Iodiculeprimordia and the carpel primordia for each floral primordia were development.2.4 F₁ plants of all cross were normal in floral structures and F₂ plants segregated with aratio of 3:1 (normal: abnormal), which suggested the mutation was controlled by asingle recessive gene and designated as SRS2(t).3 We report here a male sterile material originating from the progenies of rice cross,and present the result of phenotypic characterization, genetic analysis, and genemapping.3.1 The mutant was decreased in plant height when compared with normal plant. Theseed setting rate of controlled self-pollination was zero while that of uncontrolled was10ï¼…. In the mutant floral bract, the anther was transparent with color in white, thoughthe number of stamens and pistil was normal. KI stain revealed that no pollen grainswere observed within the anther, so we named it ms-np (male sterility with nopollens).3.2 F₁ plants of all cross were fertile and F₂ plants segregated with a ratio of 3:1(fertile: sterile), which suggested the mutation was controlled by a single recessivenuclear gene.3.3 SSR analysis indicated that three makers on CHR.6, RM3. RM541 and RM343,were flanked with the mutant gene and the genetic distance was 15.2. 15.2 and7.9CM, respectively. So the ms-np gene was preliminary located between SSRmarker RM541 and RM343 on CHR.6 in rice.