Studies On Floral Organ Mutants Of Petunia With Morphological, Anatomical And Molecular Approaches

In the past decades, genetic studies in several floral organ mutants of Arabidopsis, Antirrhinum and Petunia have led to many models established for the determination of floral organ respectively, such as ABC, ABCD and ABCDE. The understanding of human beings to flower development, floral organs formation was restrained to some extents due to the limited flower or floral mutants, furthermore very few were known to the antheroid petal and multi-floral organs. Up to date, only dol, blind, gb and phoenix floral organ spontaneous mutants were discovered in petunia and they were usually used to investigate the interaction of those floral organ identification genes. The theory of flower development was improved by the studies in petunia, in which some man-made floral mutants were constructed through isolating many MADS-BOX genes transferring to the wild type or spontaneous mutant or insert mutagenesis. Though much more studies were concentrated on the class B genes, there were quite few literatures about petal number to be referred. Petunia hybrida, either as a model plant for plant molecular biology research or as a widely used garden flower, plays an important role in scientists' studies and people's life. Using blind-like floral mutant and the double flower petunia plants constructed by us, the structural and molecular analysis were conducted and the main results were as follow:1. The blind-like mutant was discovered among the inbred lines and followed by propagation of cutting and in vitro micro-propagation, the traits were found to be stable. The main varied morphological characteristic of blind-like mutant is multi-flower and exhibiting antheroid structure on the top of petals. Lots of double flower petunia plants were obtained by in vitro macro-propagation and in vitro culture, chromosome doubling. Numerous stamens and many petals can be seen in double petunia flower. There was dramatic genetic variation among somatic variants regenerated from callus of double petunia after long-term subculture.2. Paraffin section microscopy and Scanning electron microscopy analysis indicated that there are antheroild structure in the petal of blind-like mutant and the inner petals of double petunia. There are difference in the formation between double petunia and Prunus mume, some of the excessive inner petals have the clinandrium-like structure. The antheroid structure on the top of petal of blind-like mutants was formed as soon as the stamen initiating.3. A normalization cDNA library was constructed using RNA extracted from flower during different developmental stages of double petunia. The cDNA library quality was tested by isolation and analysis of a novel gene PHCYP51. A subtractive library between single and double petunia was constructed, and some candidate cDNA fragments were obtained through anti-northern blot. Utilizing the cDNA library and the first strain cDNA, we isolated ten MADS-BOX genes' coding sequence and compared with that of the single petunia, which indicated some of them exhibit sense mutant and nonsense mutant between them.5. The primary genetic analysis of the blind-like mutant and double petunia indicated that the blind-like mutant maybe resulted from transposon insert mutagenesis and the recessive allele is responsible for the single-flower phenotype. RT-PCR analysis according to the ten MADS-BOX genes among the floral organs from single, double and blind-like petunia indicated that the expression pattern of some of them is different. Through tissue in situ hybridization analysis, we discovered that there was no difference in PHAP2A expression pattern among wild type, double and blind-like petunia from the flower meristem to the floral organ formation, on the other hand, PMADS3 was differentially expressed in all the floral organs of blind-like mutant, in stamen and pistil of wild type and in the inner petals, stamen and pistil of double petunia.6. The sense and RNAi vectors of the genes that isolated from cDNA library combined with RACE method according to subtractive cDNA and some MADS-BOX genes were constructed. Some of them have been transferred to petunia and tobacco plants. Through investigating the phenotype of those transgenic plants, we discovered that double flower transgenic tobacco plants, which carded RNAi structure of S3 fragments. Further investigation demonstrated that the excessive petals were transformed from stamen. Comparing with wild type plants, the transgenic plants showed various shapes in anther and some of the transgenic plants with excessive petal in one solitary flower, moreover, the plants in which all the stamen transformed into petals were complete male sterile.Aaccording to the similarity between antheroid structure of blind-like mutant, inner petal anatomical observation and MADS-BOX genes expression pattern of double flower petunia, we proposed that the blind-like mutant was resulted from ecotopic expression of class C genes in petal. Since the formation of double flower tobacco attribute to the RNAi function of gene isolated from petunia, and the transgenic plants with severe phenotypes have much more excessive petal, which is same as to double flower petunia, we think that the possible formation of double flower petunia resulted from (1) the numerous proliferation of stamen number and (2) the loss of function of petal identified genes in the part of stamen and those stamen transformed into petals.