On Plant Reproductive Cell-Mediated Transformation

On a basis of evaluation for the mechanism of different plant transformation systems for exogenous DNA delivering process, regeneration of target delivered and integration of exogenous DNA, this contribution puts emphasis on analyzing the plant reproductive cell-mediated transformation system including discussion of the principle of cell-mediated transformation method rely on the results in plant reproductive biology and molecular cytobiology. After exploring research many times for eliminating autofluorescence in cotton pollen grain and ovary, the movement mechanism of exogenous DNA delivered by pollen-mediated transformation or pollen tube pathway was studied and evaluated using some experimental biological methods, such as confocal laser scanning microscopy and scanning electron microscopy. In addition, the biomechanics analysis for the exogenous DNA delivered by the pollen tube pathway of cotton was conducted. Following aspects will be tackled:1. Plant reproductive cell-mediated transformation system belongs to the technical area of plant improvement by the plant genetic engineering, in which sexual cell or tissues concerning with sexual reproduction process is regarded as transforming receptor. Pollen-mediated transformation and pollen tube pathway is followed with interest at home and abroad, as these methods are considered kinds of practical protocols for plant genetic engineering industrialization without genotype dependence.2. Mature pollen grain not only can be transformed in certain concentration water solution, but also in gaseous medium. For the microprojectile bombardment-mediated transformation, the pollen exine can be penetrated by the microprojectile, while bombarded pollen was vigorous and fertile seeds were obtained when bombarded pollen grains were pollinated into stigmata. Exogenous DNA delivered in pollen grain, with the male germ unit (MGU) together, was moved into wallless area of female germ unit (FGU) following the pollen tube growth, and taken part in generative cell fusion. The movement line of exogenous DNA is pollen tube internal-pathway. Exogenous DNA can move into zygote cell nucleus during either generative cell fusion or zygote cell division process. For the plant with 3-cell pollen, exogenous DNA can move into dividing male generative nucleus. Therefore, exogenous DNA delivered by the pollen-mediated transformation has 2 or 3 chances moving into nucleus.3. For the cotton pollen tube pathway, exogenous DNA can move along the external-pathway of pollen tube, from the crevice between pollen tube and transmitting tissue cell wall in the top of ovary, surface of placenta, micropyle, cannel cell of nucleus into embryo sac. And generative cell in fusion status was transformed directly. It is different with former researchers that the way of exogenous DNA delivered by pollen tube pathway is pollen tube-external pathway not pollen tube-internal pathway or only just nucellus pathway, transforming receptor cell is generative cell in fusion status not zygote. The explanation is more reasonable and supported by experimental results. The peculiarity of this method is that exogenous DNA was moved into cell at walll ess area, however, the cell was not touched and hurt. Exogenous DNA delivered by pollen tube pathway has 2 chances moving into nucleus same as the pollen-mediated transformation.Plant cell wall and nuclear membrane are two bigger protective screens for the exogenous DNA inserting host genome. Pollen-mediated transformation and pollen tube pathway not only makes exogenous DNA surmounting above obstacles skillfully through pollen tube internal or external -pathway, but also makes transformed cell overcoming the difficulties during regeneration with the aid of natural embryo development. In this paper, the status of reproductive cell-mediated transformation in the transformation system was discussed the development of plant reproductive cell genetic engineering was also prospected.