| Nicotiana tabacum L. is one of the most important economic crops in our country. Through introducing from abroad and local breeding for a long time, the rich tobacco germaplasm resourceshas been formed. Because of our country is vast and the ecological conditions of diversification, domestic tobacco industry be in the face of a number of problems including the limited cultivated varieties, narrow and weak genetic basis, serious aging of some tobacco species, scarcity quality disease-resistant varieties, unsubstantial basic science ofwide hybridization. There is still have a big gapcompared with the United States, Brazil whichare famous for high quality tobacco production country in the world, which severely limits the development of domestic tobacco industry, in short it is in soreneed of cultivating new varieties.The interspecific hybridization affinity were explored between Nicotiana tabacum L. cv. K326and five disease-resistant wild species:Nicotiana alata Link&Otto, Nicotiana rustica Linnaeus, Nicotiana debneyi Domi, Nicotiana stocktonU Brandege, Nicotiana repanda Willdenow ex Lehmann. Male and female gametophyte development, pistil structure, reciprocal cross compatibility and the overcoming methods were also studied in species mentioned above. The results summarized below:1. Microspore development of six Nicotiana species were almost normal, few of microspore mother cell in meiosis appear anomalies such aschromosome fragment, lagging chromosomes, chromosome bridge, irregular chromatin were observed in a few microsporocytes, which might lead to part of the pollen sterility, but had a little effects on self-seeds setting (except for N. alata); there were a higher percentage of abnormalities in microspore development of N. stocktonii in low-temperature environment, which indicated that N. stocktonii was affected by environmental conditions. Screened out10%sucrose+100ppm boric acid medium as the optimum medium for several tobacco pollen germination medium. Results of the pollen germination in vitro experiment showed that:N. alata pollen and N. stocktonii pollen germination vigor were the lowest, N. repanda was higher, and N. labacum L. cv. K326was highest.2. The megaspore development process and the anatomical morphologies of the style and stigma in Nicotiana tabacum(gla.)S K326(Nta(gla.)S K326) and five wild Nicotiana species were studied by examining paraffin sections and frozen sections. These examinations revealed that the six aforementioned Nicotiana species have two-celled ovaries with free central placentation, anatropous ovules and many ovules per locule. In these species, the stigma is bifid, the style is solid, and there is continuous transmitting tissue between the stigma and style. Female gametophyte and ovule development processes were normal, with only one functional megaspore developing in the embryo sac, which contained seven cells and eight nuclei. The embryo sac matured in flowers at blossom; this maturation and development occurred later than the maturation of male gametes.3. N. tabacum L. cv. K326and five Nicotiana species were reciprocally pollinated. Cross-compatibility was determined by observing pollen germination ratios and pollen tube growth performance by fluorescence microscopy and by calculating seed setting ratios. The results of orthogonal experiments indicated that incompatibility occurred in stigmas in♀Nta(gla.)S K326×♂N. repanda/N. stocktonii crosses and in three-fourths of the styles♀Nta(gla.)S K326×♂N. debneyi/N. rustica/N. alata crosses. Pollen tubes traveled into ovaries in the reverse♀N. debneyi/N. rustica/N. stocktonii/N. repanda×♂N. tabacum L. cv. K326crosses, but incompatibility in stigmas was observed in the♀TV. alata×♂N. tabacum L. cv. K326cross.4. Pollination by different pollination methods and with the addition of different allogenic materials indicated that the compatibility of different crosses was improved by bud pollination. In the♀Nta(gla.)S K326×♂N. alata cross, through repeated pollination and the pollination of old stigmas, incompatibility was overcome, and vigorous seeds were obtained. In all crosses, compatibility could not be promoted by the addition of boric acid, acetone, GA3, or6-BA to pollen. By adding ground N. repanda stigmas and inactivated N. tabacum L. cv. K326anthers to N. repanda pollen, incompatibility in stigmas could be partially weakened in the♀Nta(gla)S K326×♂N. repanda cross, allowing a few pollen tubes to extend1.2cm into the stigma and style.5. Given the stigma incompatibility in the♀Nta(gla.)S K326×♂N. repanda/N. siocktonii crosses, total phenol, amino acids and fatty acids in the stigmas of the aforementioned species were qualitatively and quantitatively analyzed. These analyses demonstrated that. The total phenol and fatty acids in K326are respectively3times and4times higher then wild tobacco, Amino acids content only14.29%of the wild tobacco, orange ketone, anthocyanin, Rhizoma Kaempferiae phenol and rutin content were elevated in all samples. Among amino acids, histidine content was particularly elevated, and among fatty acids, oleic acid (C18:1) and linoleic acid (C18:2) concentrations were particularly elevated. In addition, oleic acid levels were lower than linoleic acid levels (C18:1<C18;2) in the stigmas of N. stocktonii and N. repanda; in contrast, the opposite phenomenon was observed for Nta(gla.)S K326(C18:1>C18:2)-To effectively utilize germplasm resources, improve tobacco quality and enhance tobacco breeding, interspecific compatibility for crosses between N. tabacum L. cv. K326and five wild Nicotiana species and the underlying reproductive physiology of these crosses were examined. |
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