河北省重点实验室植物转基因中心官方网站

Key issues and the solutions in plant tissue and cell culture

There are two critical issues in plant tissue and cell culture aiming at expressing totipotency of cell, one is the initiation of cell division and another is the differentiation of regenerated plant from callus after long-term culture. The genotypes that is easy to culture usually is easy to initiate cell division and induce plant differentiation. However, the genotype expected to manipulate is generally not the type of easily to culture.

To manage the initial cell division, firstly it should choose young explant with potential of cell division, secondly choose the suitable types and regulate the level of hormone, as well as the types and composition of medium. To induce cell division of monocotyledon, it is necessary to use auxin in MS or AA medium at dark condition. To induce cell division of dicotyledon, it is necessary to use cytockine in B5 or N6 medium at dim light. Despite of as nitrogen supply, ammonium N also has some effects like auxin, but culturing isolated cells or small size cultures should use ammonia nitrogen, and NH₄⁺ should not be used because it is toxic to cell membrane. For nitrate nitrogen, it shows some effects as cytockine to the cultures with low nitrate reduction ability. As carbon supply, glucose can facilitate cell division. Culturing with light also shows the effects like cytockine. At present, it is not difficult to initiate cell division. In the past publications, most of the plants can be successfully manipulated with suitable explant, medium and hormones, except for the mini spore of cotton. The most difficulty is to make the divided cell into desired embryogenic state to maintain cell division. In this case, it usually needs subcultures aimed at improving the quality of the cultures. The key is to use the above chemical and physical factors appropriately to initiate and maintain cell division.

To differentiate plant from the long term cultures, it is important to slow down or reduce the rate of cell division. The difficulty of differentiation is usually occurred in the long term culture. Cell line cloning, protoplast culture, mutant selection, transformation and transformants selection, etc, generally need long term culture process. During the long term culture, it needs more subcultures, thus, the ratio of vigorous dividing cells would be significantly increased in the cultures developed. The more vigorous dividing cells in the culture, the more difficult to regenerate plant. To the vigorous cultures, cytokinins, nitrate nitrogen, light, low temperature and active carbon, etc., can slow down and reduce the cell division. The cell with strong dividing capability is extremely easy to develop resistance to the cell division inhibiting substances which result in failure of differentiation. Hence, differentiation procedure usually needs to change to use different substances to ensure to slow down the cell division of culture successfully. So long the strong cell division is depressed, cells in the culture would be differentiated and finally will be developed into regenerated plants.

Due to the complexity and variability of cells, the knowledge and skills of plant tissue and cell culture are usually very empirical, artistic and irregularity. To solve these problems, the author proposed the ideas and methods of cell state regulation（The Origination of Cell State Hypothesis and Its Significance to Life Science，www.pgec.org）. It changed the plant tissue and cell culture from experiences to a predictable and inferential level. This makes the plant tissue and cell culture to some extent like having its own “Mendel’s law”.

Wang Haibo

Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences,

Shijiazhuang, 050051, China