Somatic Embryogenesis Improvement And Laticifer Differentiation In Anther Culture Of Hevea Brasiliensis

Hevea brasiliensis belongs to Euphobiaceae. It is a perennial cross pollination tree and is widely planted in tropical areas. Natural rubber produced by Hevea is one of the most elemental industrial raw materials and strategic goods, which plays a key role in national economy. Anther culture is one of the most important biotechnological means to improve Hevea genetic property. However, its low embryogenic frequency prevents the large scale applications of juvenile-type clones and genetic transformation. In the present paper, a series of work was carried out to improve somatic embryogenesis of anther culture in Hevea. The internal and external factors influencing somatic embryogenesis were investigated and cultural conditions were improved. Based on the advantage of increasing the embryogenic frequency, the technique of isolated microspore culture was performed in Hevea in this paper. Transgenic technique is one of the most important approaches to crop genetic improvement and was also used in this paper. An expression vector harboring a key embryogenic gene was constructed and a transformation system was studied by Agrobaterium mediation. We found laticifer cells exist in callus cultures derived Hevea anthers for the first time. The correlation between laticifer cell differentiation and somatic embryogenesis was investigated, which may provide a hint for the improvement of somatic embryogenesis. The main results of this research were as follows:(1) The morphology and the histology of anther calli of Hevea were systemtically studied for the first time, and significant difference between embryogenic callus (EC) and non-embryogenic callus (NEC) were observed. EC was loose, friable, and unsmooth with many small spherical protuberances on its surface. EC cells were small, globular, and tightly attached to each other, and had a big nucleus. NEC included two different types, NECⅠand NECⅡ. The NECⅠwas compact, hard, and smooth, while the NECⅡwas soft, water-soaking. Histologically, both NEC cells were large and irregular in shape, and had thin cytoplasm. However, all of the NECⅡcells had no nucleus and some of NECⅠcells had an extremely small nucleus. Furthermore, the NECⅡcells were vacuolizated and had wider extracelluar gap than the NECⅠones. EC induction rate and its embryogenic rate were significantly correlated. The differentiation ability and the growth rate of the EC were still strong in the late cultural stages.Physiological and biochemical mechanism of embryogenesis of EC of the rubber tree was investigated for the first time. Starch content of EC was higher than that of NEC in all cultural stages, and EC cells accumulated more starch in its late cultural stages, which may provide material basis for further embryoid development. The high protein content may be a key factor of embryogenesis of EC. The high carotenoid content of EC indicated its cells have a strong antioxidative capacity preventing its cells brown and death. EC cells of the cultivars having high embryogenic ability had stronger differentiating ability and metabolized more animato compared to those having low embryogenic ability, which makes the former consume more carbon source. Therefore, the depletion of carbon source may activate a signal of embryoid development. The high content of CAT and SOD in EC of different cultivars and their embryogenic ability were significantly correlated, suggesting that CAT and SOD were the basis of embryogenesis.(2) Nine types of somatic embryos derived from anther calli were systematically classified, and double-embryoids were reported for the first time and they can develop into normal plantlets.Cultural conditions of somatic embryogenesis were improved. Plantlet regeneration was significantly improved by incubating the embryoids under weak light of 500 Lux for 10 to 15d after cultured for 2 months in the dark. The embryogenesis rate was significantly improved by additon of 1 mgL-1 rare earth in the induction media.(3) A protocol of isolated microspore culture in Hevea brasiliensis has been carefully studied in the present paper. Two different isolation methods of indirectly grinding anthers and directly grinding anthers were tested and the former was optimal with lower contamination, less impurity, and better culturing effect compared to the latter although it had a low efficiency. Different pretreatments were also studied and Starvation Medium B pretreatment for two days before the microspore incubation was the optimized one. The effects of different carbon sources and pH and components in induction media were investigated. The results showed maltose as carbon source and pH6.6 and modified N6 medium supplemented with 2,4-D 0.5 mgL-1 and KT 0.5 mgL-1 in induction media had an optimal induction effect with 8.33% differentiation rate of microspores which differentiated mainly through B way. Among five cultivars only Haiken2 obtained cell mass and microcalli from microspores, suggesting the differentiating ability of isolated microspores of Hevea is genotype-dependent. An electroporation test using microspores as receptors were carried out and the results of transient expression was confirmed the exogenous gene was expressed in microspores of Hevea by GUS staining.(4) The calli of Hevea variety Haikenl were genetically transformed using Agrobacterium EHA105 habouring pBIBBM that contained theβ-glucuronidase(GUS) and Kanamycin-resistant gene and Baby Boom gene. The results showed that the Kanamycin concentration at 50 mgL-1 was the best selective pressure, and the transformation rate of the cocultivation temperature 20℃was greatly higher than that of 26℃. This research optimized the genetic transformation system of Haiken2 by Agrobacterium mediation and obtained Kanamycin resistant callus lines and transgenic embryoids, which provided the prerequisite for getting the transgenic plantlets.(5) Laticifers are highly specialized cells present in over 20 plant families. They are well defined in planta. In vitro development of laticifers was also observed in some plants, but uncertain in the callus cultures of rubber tree, one of the most economically important latex producing plants. In the present study, we provide evidence that laticifer cells present in the callus cultures of rubber tree by histochemical and immunohistochemical studies. They present in the callus mainly as separate non-elongated form, very different from the morphology of laticifer cells in planta, excluding their origin from explants, although a few elongated laticifers were observed. These laticifer cells may be the initial characteristic of articulated laticifers.The occurring frequency of laticifer cells in the callus was genotype-dependent and negatively correlated with the somatic embryogenetic ability. The PR107, RRIM600, Reyan8-79, and Reyan7-33-97 had more laticifer cells in their callus cultures and lower cmbryogenesis compared to Haiken2, and laticifer clusters were only observed in the callus of the former four cultivars, suggesting that the presence of laticifer cells in the callus inhibit somatic embryogenesis in tissue culture of rubber tree. An improving cultural condition such as regulation exogenous hormone might be undertaken to reduce laticifer cells of calli and enhance their embryogenesis, which may provide a hint of improving anther culture of Hevea.