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The cell state hypothesis like a “Mendel’s law”in plant tissue and cell culture

1996年我应邀到韩国做“组织培养中的细胞状态调控”的报告，美籍韩裔科学家Ok Young Lee-Stadelmann博士（对我人生有重要影响的学术导师之一）帮我做翻译，她向听报告的师生们介绍说“这是组织培养中的孟德尔定律”。在此，我把恩师称之为“组织培养中的孟德尔定律”以英文的形式介绍给大家。

The cell state hypothesis like a “Mendel’s law” in plant tissue and cell culture

Wang Haibo

Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, 050051, China

Keywords: cell state; tissue and cell culture; plant regeneration; aging; canceration

Before Mendel law proposed, people cannot predict the offspring by sexual cross breeding. Similarly, plant tissue and cell culture also could not be inferred in more than100 years. This paper shows the way to solve the problem.

In plant tissue and cell culture aiming at expressing totipotency, inducing callus is generally not a problem with a dynamic explant, the main barrier is the difficulty to regenerate plant, particularly to the callus regenerated from protoplast or microspore. Whether a plant can be regenerated or not depends on what type the callus belongs to. The type of the callus is determined by its major compositional cells.

Through a long-term research of tissue and cell culture in wheat, rice, corn, cotton, soybean and so on, I have found that different cell types in culture are the various displays of cell states. Seven typical cell types were proposed, which are helpful to clarify the pattern of the relationship of cell state changing: 1) embryogenic cell (S_e), 2) conservative cell (S_a), 3) stimulating cell (S_b), 4) multiplicative cell (S_t), 5) conservatively degenerated cell (D_a), 6) radically degenerated cell (D_b), and 7) hyperactively dividing cell (S_c)(Fig.1).

Fig.1. The typology of typical cell states.

S_e: embryogenic cell, S_a: conservative cell, S_b: stimulating cell, S_t: multiplicative cell, D_a: conservatively degenerated cell, D_b: radically degenerated cell, S_c: hyperactively dividing cell. These cells were drawn up by the observation under microscope in order to highlight their characteristics. The cell shapes in the diagram are schematically represented for each of the cell states.

Among these, the cell types of S_e, S_a, S_b and S_t can be changed into each other by self and artificial regulation, and they also can be degenerated to the long curved (D_a) or expanded (D_b) cells in opposite directions (Fig. 2). The S_c cell is very similar to the S_t cell, but gained a hyperactive dividing capability beyond the normally control. The other cell types are the transitional states of these typical types. When a callus is composed of hyperactively dividing cells or mainly of degenerated cells, it can not regenerate any plant at all. When a callus is mainly composed of conservative cells, it will regenerate plants via organogenesis. When a callus is mostly composed of the cells between the stimulating and the conservative states, it will regenerate plant via somatic embryogenesis (Fig.3). The key of plant tissue and cell culture is to regulate the states of cells in callus. Thus, I put a hypothesis forward to explain and to guide these issues theoretically, which named The Cell State.

Fig.2. The changing pattern and the relationship of different cell states.

The S_e cell can develop to all kinds of cells via cell division. Both S_a and S_b cells can still divide with some degrees of differentiation in opposite directions. They formed the specialized tissues with different functions. The S_t cells, like stem cells, have strong dividing capability responding the rapid growing and wound repairing. These four cell states are interchangeable and can also be degenerated to long curved (D_a) or expanded (D_b) cells and can be mutated to S_c cells (with hyperactively dividing capability out of the control of organism, eg. cameration) as showing in this diagram. What shown in dotted line direction has not been fully implemented. Note that except for D_a, D_b and S_c, all of the four cell states (S_e, S_a, S_b and S_t) allow the successful tissue and cell culture.

Fig. 3. Some common types of cells and calli of wheat.

The pictures are taken from calli induced from immature embryos of wheat under microscope and stereoscope. a and b: the degenerated cells from the surface of calli of A and B, respectively. c and e: the cells from the calli of C and E, respectively. d: the cells in a slice of callus D. Shown inside Petri-dishes is shoot regeneration from different types of callus.

The cell state, here, comprehensively reflects a cell in composition, structures, morphology, functions and potentials. In the cell state hypothesis, every cell has its state which is abstracted as being controlled by a series of “cell state factors”. These factors include the elements not only in genetics and physiology, but also in chemistry, physics, composition and structures (Fig.4).

Fig. 4. The roles of cell state in a living system.

Biotic or abiotic hinge on whether there is a system of substances with “cell structure”, which minus entropy formed and itself boomed in nature dominated by the first and second laws of thermodynamics. The formation of biotic depend on whether this kind of unique system formed by substance composition and comprehensive effects of chemistry and physics. In this system, the steady and regular programmed activities (including network communication) determined by structures is the foundation of genetics, whereas the dynamic and regular programmed activities belong to physiological phenomena. If the structures determining the steady and regular activities are changed by physiological influences, genetic variations will appear. This is the secret of evolution. The cell state is determined by genetic and physiological factors and affects the state and quality of organisms.

No matter where the factors come from, their roles can be imagined or concluded as having physiological and biochemical effects. The cell state factors can be divided into three categories: 1) stimulating factors (E), 2) conservative factors (I), and 3) degenerate factors (D). The E factors mainly promote or maintain the cell at a stimulating state. They usually promote the biosynthesis for cell division. While I factors chiefly make or keep the cell at a conservative state. They induce cell differentiation and produce basic materials for macromolecular biosynthesis. Life activities are based on the existence of E and I factors. It is that these two kinds of factors supporting and pushing the cell cycle. Both of these two factors can be further classed into internal and external types. The internal factors include the relevant genes, endogenous active substances, the effects of cell structures and their change. The external factors can be considered as all the exogenous elements. The D factors accumulated in the cell can show some similar effects as I factors when they are at low level, but their nature is to damage the cell structures and to disturb or injure the cell activities. The mechanism of cell state can be expressed in a formula as below:

S represents the cell state coefficient（abbreviated as S value）.

∑ denotes the total value of the same type of factors.

[ ] mean the level of the composite effects of the same kind factors.

“₀” and “_S” indicate internal or external factors respectively.

Cell division capability can be controlled by regulating S value. Cell divides when S = 1 or S≈1. When the S value is close to 1, the higher level of [∑E ] and [∑I ] , the stronger the cell division. But when the S value is away from 1, the cell must be at differentiated state. These changing can be figuratively expressed as a balance, and the typical cells can be expressed as the relevant balances (Fig.5).

Fig. 5. Balance model of cell state regulating.

The cell state can be understood as a balance controlled by the levels of [∑E], [∑I] and [∑D]. The size of the colored rectangles shows the levels of the related cell state factors ( E₀ - light green, E_s – dark green, I₀ – light red, I_s - dark red, D – brown ), and the force to the fulcrum of the balance reflects the cell division potential or capability. Different balances figuratively express the different cell types. Cells can be divided into balanced and unbalanced groups. Zygote is at the state when [∑E₀] / [∑I₀] =1, it is the almighty cell. S_e and S_t in this diagram, shown the successful culture to the wanted states, while S_a, S_b shown the cell states which cell division are not fully induced. S_c shown the typical state of canceration, while D_a andD_b shown the typical degeneration oppositely. The initiation and improvement of cell division are to make the S value to S_e or S_t, and the differentiation is to make the cell state unbalanced, to make the S value to S_a, S_b in opposite direction, or even to D_a and D_b.

To analyze the cell division potential, we can also refer to another formula:

D_p: Cell division potential.

S_i: Cell division potential coefficient. When S≥1, S_i=1/S, and when S＜1,S_i=S.

△∑D :The increased ∑D but cannot give cells lethal damage.

In most cases, auxin and reduced nitrogen (ammonium-N and ammonia-N) can be regarded as E factor-substances; cytokine, oxidized nitrogen (nitrate-N) can be used as I factor-substances; and unfavorable matters to the cells or metabolites from itself can be listed as D factor-substances. Generally, MS or AA medium can increase the S value of the cultures, on the other hand, N6 or B5 medium will decrease the S value. Cell state regulating became operational with these discoveries.

But in some cases, paradoxes to the conclusions might be encountered. As some exceptions, if the culture is sensitive to NH₄⁺, ammonium can not be used as E factor, and when the culture has strong nitrate reducing power, nitrate-N cannot show any effects as I factors. NH₄⁺ mainly play a role as E factor for the relatively big callus, but to some extent it will show obvious effects as D factor in single cell and protoplast culture, because it is toxic to cell membrane. In this case, ammonia-N, amino acids for example, should be recommended instead. And when the culture has strong nitrate reducing power, nitrate-N effects as I factor will be badly weaken.

For the variation range, different cell states distribute in a dolphin-like diagram (Fig. 6). According to this diagram and the formulas or the balances mentioned above, and the understanding of the functions to cells about chemical and physical factors, the cell states of cultures can be successfully regulated towards the desired position. Hence, with these achievements, plant tissue and cell culture can be manipulated and managed by ratiocination. And these findings will also provide referential ideas to animal cell culture.

Fig. 6 The dolphin-like distribution of the cells with different states.

The changes of the cell states follow the dolphin-like distribution instead of a linear distribution. The D_b and D_a states are distributed in the head and the tail, respectively, while S_e is in the abdomen and S_c is in the back. According to the cell state formula, when the S value of the cells moves far away from 1, the cells are more likely in the degenerated states and thus less likely to divide. The cells in the head or tail (D_a and D_b) are no longer dividing. However, if the S value of the cells approach 1, the cells are more likely to divide, but the closer the cell states to the back of the dolphin, the stronger dividing the cells shown, the more difficult their dividing capability can be controlled. It should be considered that the values of the dividend and divider of the formula represent the level of corresponding factors and relate to the capacity of cell division.

Factors affecting cell state include all kinds of cell state factors and also the cell division latest occurred, because when a cell divide it can subverts the state from the original. In this hypothesis, E and I factors are the basis of living phenomena, but D factors usually play the tragedy maker in the living processes. In an organism, where D factors heavily accrue, the cell goes to aging or even death. Therefore, the explant to initiate callus should be chosen as young as possible. However, D factors can also be accumulated in culturing cells, especially in a long duration culture. When D factors accumulated where there are a lot of cells having strong evolutional capability or potential, some of them will be mutated or be stressed/selected into hyperactively dividing types. Only when the cell changed into this kind of type, it can dilute the degenerate substances in the cell and maintain S value as a living cell should have via cell division. This may be the mechanisms of canceration which have not been revealed yet and the reason of Sc cells appearing. Gene will be mutated and the balance of genes expression will be broken with the suppression of D factors, which result in the disorder of cell vitality. The subculture approaches in fact, never give the cells in culture equal opportunities to develop, the strong dividing cell will always be aggressive and getting more and more, so the cultures finally will be changed into vigorous growing types. In this situation, the differentiation approach used before will not work as effective as original, high level of I factor-substances should be used to slow down the vigor of the cultures in one or several times of subculture and then to select the compact clusters from them to regenerate plants.

Subculture plays a very important role in cell state regulating in tissue and cell culture. Before protoplast isolation, the cells should be regulated to strong dividing types, which the protoplast will be easy to isolate and to culture. But for the regenerated callus from protoplast or callus induced from microspore, they are usually growing too vigorously and difficult to regenerate plant. To deal with this kind of problems, the cell state should be regulated to Se type. The large scale cell culture also can be managed by cell state regulation, when the cells are needed to growing well, their state should be regulated to St type, but when the cells are wanted to produce more secondary metabolism products, the cell state should be regulated to Sa or Sb types.

The establishment of the theory of the cell state also has very important significance for human health. Life span will be consumingly impacted by the speed and the amount of D factors accumulation in the development of the organism. Reducing the level of D factors will lead to a better result for leaving aging and lesion. An organism will be restored to health or rejuvenated if the D factor-substances can be eliminated (Fig. 7). Most of all, cancer will be no longer occurred when D factor substances are getting into nonentity.

Fig. 7. The hypothetical cell state regulation model for the control of lesion.

In this model, the cells in or close to the abdomen (S_a, S_b, S_e, even to S_t) are juvenile and healthy. They not only have dividing capability but also can be controlled easily. The S_e cells in particular do not show any degenerate signs. Successful efforts to make the states of the cells to approach the abdomen will reverse the cancerous and aging.