Reproductive Biology Of Crofton Weed (Eupatorium Adenophorum) And Its In Vitro Rapid Propagation System Establishment

Crofton weed [Eupatorium adenophorum Spreng. = Ageratina adenophora (Spreng.) King & H.E. Robins. = E. glandulosum H.B.K. non Michx.], a worldwide noxious invasive weed, is an erect perennial herb to scrambling subshrub native to Mexico and Costa Rica of Central America. Because of its stress tolerance, large number of cypselas, and marked ability to regenerate from stems and roots, crofton weed now become one of the most harmful invasive weeds in China. Although many control measures have been extensively examined and implemented, this weed is extremely difficult to control or eradicate, and still spreads rapidly in China. Therefore, considerable efforts are being put forward to find new and innovative methods for integrated management of crofton weed, for which a greater understanding of the biology and reproduction of crofton weed is needed. In this thesis, we studied the reproductive biology, ecological physiology, including environmental factors effect on seeds germination and suitable regions distribution, and finally an in vitro fast regeneration system was built for this weed. The main results were concluded as follow: (1) Fresh collected pollen cultured in vitro with the use of 14 culture media (including an improved Monnier's medium and a BK medium) did not germinate, nor did pollen germinate on the stigma, and no pollen tubes were seen in the style. Using paraffin section and semi-thin section, the floral organ differentiation, megasprogenesis, microsporogenesis and the embryogenetic process were observed in crofton weed. Crofton weed had four anther chambers. The anther wall was composed of epidermis, endothecium, one layer of midldle layer and amoeboid tapetum. The cytokinesis of microspore mother cell in meiosis produced the tetrad which was tetrahedral. The ovule became anatropous as the integument grew, surrounding the nucellar lobe, a structure containing the dividing megaspore mother cell (MMC) surrounded by a layer of nucellar epidermis. Instead of beginning a meiosis division, the MMC underwent two mitotic divisions to produce four cells, which constituted a four-nucleate embryo sac, and the endothelium began differentiation from the epidermal cells of the integument. After another mitotic division, an eight-nucleate embryo sac was produced. Thereafter, one of the two cells near the chalazal end developed embryo while the other degenerated, and the left cells near the micropylar end continued proliferation to form endosperm. These results provide additional evidences for the previous conclusion that crofton weed reproduces by apomixes, and the mode is diplospory. (2) Laboratory studies were conducted to test the effects of environmental factors on seeds germination of crofton weed. Crofton weed seeds were treated by 5 g/L mixed salts solutions composed by NaCl, Na₂CO₃, and Na₂SO₄. Results showed that salt stress inhibited seed germination, radicle and hypocotyl elongation. Solutions with Na₂CO₃ have been shown to have greater inhibition on germination than other solutions. The inhibition of root elongation was more prominent than that of hypocotyl. Approximately 80% of seeds germinated at a low level of salinity (< 50 mmol/L NaCl), and some seeds (18.0±5.6%) germinated even at 200 mM NaCl. These data suggest that even at high soil salinity conditions, crofton weed can germinate. Maximum germination (84.3±5.0%) was observed at pH 6.0. Germination was 33.6% to 20.0% over a pH range from 5.0 to 7.0, with a trend toward higher germination under neutral-to-acidic conditions. Germination of crofton weed decreased with increasing water potential, and germination was totally inhibited at -0.8 MPa. Germination decreased from 80.2% to 3.0% as osmotic potential increased from 0 (distilled water) to -0.7 MPa, indicating that germination can still occur under moderate water stress conditions. Seeds germinated over a range of 10 to 35℃with optimum germination at about 20℃. High temperature markedly restricted germination, with only 19.6% at 35℃. A wide range of regimes supported germination. However, no seeds germinated at high salinity stress (e.g. 200 mmol/L) with high or low temperatures (e.g. 30, 10℃). The interactive effects of salinity (S) and temperature (T) on germination (G) were described by the following response surface function:G=1.5004+9.9930·T-0.4080·S-0.2707·T²+0.0063532·T·S-0.0003266·S², which could be helpful to predict the crofton weed germination time in regions with different salinity levels.(3) Estimates of climatic and soil factor tolerances for crofton weed were developed based on the seed germination studies and climate characteristics in the distribution regions of China reported by previous literatures. Tolerance range estimates were finally used in conjunction with spatial grids for climate and soils to create a potential invasive distribution map for China. The suitability maps suggest that the future range of crofton weed in China will be restricted largely to the Yunnan-Guizhou Plateau (includes major parts of Yunnan and Guizhou provinces, the southwestern part of Sichuan province and the western part of Guangxi province), southern China, and coastal regions in south-east China. Crofton weed also tends to be a sporadic problem in other regions, where the climatic and edaphic conditions are suitable for the seed germination.(4) A callus induction and plantlet regeneration system for crofton weed was developed by studying the influence of explant type (leaf, stem, and nodal segment) and different concentrations of plant growth regulators. The leaf was a better explant for callogenesis compared to the stem. The highest callus induction frequency (87.2%) was obtained from leaf segments on Murashige and Skoog's medium (MS medium) supplemented with 0.5 mg/L (2,4-dichlorophenoxy) acetic acid and 2.0 mg/L 6-benzylaminopurine (BA), and 71.6% differentiation along with a multiplication rate of 4.1 adventitious shoots per callus was achieved with a combination of 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L BA. In addition, MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L BA was the best medium for axillary shoot regeneration from nodal segments. Rhizogenesis of cultured shoots was satisfactorily obtained in half-strength MS without any growth regulators. The regenerated rooted plantlets were successfully acclimatized in soil where they grew normally without showing any morphological variation. These studies provide the prerequisite system for the development of genetic engineering in the future and propagating crofton weed rapidly for further study.