Hot topic - CNSH Thực vật - Kỹ thuật nuôi cấy lớp mỏng tế bào (TCL-Thin cell layer)

Hot topic - CNSH Thực vật - Kỹ thuật nuôi cấy lớp mỏng tế bào (TCL-Thin cell layer)

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THIN CELL LAYER CONCEPT

K. Tran Thanh Van

Institut de Biotechnologie des Plantes, UMR 8168-Université de Paris-Sud,
91405 Orsay Cedex, France


Content

1. Introduction
2. Concept and strategy
3. Definition of thin cell layer systems
4. Isolated cells, protoplasts versus thin cell layer systems
5. Comparison of morphogenetic potentials
6. Characteristics of thin cell layer systems
7. Thin cell layer method to overcome the limitation to plant regeneration and its use in plant transformation
8. Concluding remarks
9. References

1. INTRODUCTION

The understanding of the mechanisms which control plant growth and development is of fundamental and applied interests. In order to analyze these mechanisms, we need to be able to program in vitro a given pattern of differentiation before trying to answer to the question of how it is programmed in vitro i.e. in the plant body.
On entire plants, growth, development and senescence result from correlation existing between i) organs, tissues, cells; ii) different cell compartments and iii) cell organelles.
From the embryo stage to the adult stage, plant differentiation proceeds through ?ointertwined? sequences of events leading to the building of complex shapes, structures and functions. All these events occur on a communication network which is highly sensitive to environmental factors whose effects are integrated in the entire plant, in which the perception of stimulis, their processing, their transport to the responsive cells do not lend themselves to a fine analysis although powerful molecular analysis techniques are available.
In animal systems, in contrast, there are well known receptors to specific hormones. Furthermore, to add to the complexity found in plants, there are sophisticated mechanisms of tolerance/resistance which are surimposed onto the basic moving network of correlation.
Other difficulties in analyzing plant differentiation reside in the fact that developmental mutants can be only found in a very small number of species. Although very useful, they are not suitable for the study of specific functions for example a tiny herbaceous species like Arabidopsis thaliana is an unsuitable model for the study of woody species.
As previously mentioned, it is generally admitted that the network of correlation existing between different organs, tissues and cells can modulate plant growth and development. We have experimentally demonstrated that this modulation results in inhibitory processes (Lê Kiêm Ngoc-Tran Thanh Van 1965, Tran Thanh Van and Trinh 1978, Tran Thanh Van 1981, Tran Thanh Van and Bui 2000). The inter-tissue correlation as inhibiting the morphogenetic potentials was demonstrated on Torenia (Tran Thanh Van et al 1974a, Chlyah 1974).

2. CONCEPT AND STRATEGY

The concept of an inhibitory network brought me to the concept of a thin cell layer (TCL) system whereby I have tried to gradually isolate one or a few layers of differentiated cells from the organs/tissue/cells correlation network and have attempted to reprogram them in vitro. ?oDifferentiated? is an important criteria. This allows the investigator to trace back the events at their very beginning. Unlike the nucleus of a mature somatic cell transferred into an enucleated ovule which leads to the development of an embryo and of an adult sheep as in the case of Dolly (which has not been reproduced again by the author Ian Wilmut himself since 1996, Campbell et al 1996), the cells in TCL systems with their nucleus maintained in their own cytoplasm are litterally reprogrammed in order to express all patterns of differentiation ?" not only embryo ?" which can be controlled separately or reprogrammed according to a space/time sequence defined by the investigator and not imposed by the ontogenic program. Even new patterns and functions can be initiated (Tran Thanh Van and Gendy 1996).

3. DEFINITION OF THIN CELL LAYER SYSTEMS

The TCL system consisted of explants of small sizes which are excised from different plant organs (stem, leave, root, floral inflorescence, flower primordia or floral organs, cotyledon, hypo-/epicotile, apical zone or embryo. They are excised either longitudinally -the explants are designed as longitudinal TCLs (lTCL)- or transversally- designed as transverse TCLs (tTCL). The lTCLs (1mm x 0.5 or 10 mm) include only one tissue-type for example a monolayer of epidermal cells (which could be peeled off the organs) or several (3-6) layers of cortical cells whereas the tTCLs (0.2/0.5 mm or a few mm of thickness) include a small number of cells of different tissue-types (epidermal, cortical, cambium, perivascular and medullar tissue as well as parenchyma cells). (For TCL method, see Tran Thanh Van and Gendy, 1996). The common trait of lTCL and tTCL is to be ?othin? i.e. an inoculum with as small a number of cells as possible. The trait of being ?othin? is of paramount importance because candidate marker molecules of differentiation could be localized in situ in the target cells or the responsive cells. Such localization allows to circumscribe the responsive cells.

4. ISOLATED CELLS, PROTOPLASTS VERSUS
THIN CELL LAYER SYSYEMS

One can raise the question of why not using isolated cells which apparently is simpler than a TCL system which is a multicellular system. We have to remember that an isolated cell-excised from a pluricellular organism- is not a single-cell-organism. A multicellular system such as the TCL system as defined above bears some inherent temporal/ spatial organization, a sort of ?omemory? which is lost in an isolated cell per se. Moreover, isolated cells or even protoplasts are not simple. Soon after their isolation, protoplasts built a cell wall; both protoplasts and isolated cells form clumps of newly formed cells hence with a temporal/spatial organization which is different from the one existing before their isolation from the donor tissue/organ.



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5. COMPARISON OF MORPHOGENETIC POTENTIALS BETWEEN ISOLATED CELLS/PROTOPLASTS AND
THIN CELL LAYER SYSTEM
Both isolated cells and protoplasts end up within a short interval of time via successive cell divisions, in a multicellular mass of cells among which one is ?oselected? or ?oinduced? or possibly ?oreleased? from the ?oinhibition network? of the surrounding cells, to undergo somatic embryogenesis after restructuring their cell wall. Although being quasi ?oisolated?, it is surrounded by other cells, therefore exchanging with them and with the culture medium via a network of communication through this particular newly-structured cell wall, factors such as extracellular proteins (among those chitinase, peroxydase were identified) and whose inducing effect was shown (De Vries et al 1988, Mo et al 1996). Compared to the small number of patterns displayed by isolated cells/ protoplasts which are, in most cases, callus, somatic embryos mixed with non embryogenic cells, tracheids and roots, TCL systems, in contrast, display a greater number of patterns including new ones (Tran Thanh Van 1973a, Tran Thanh Van 1973b, Tran Thanh Van 1981, Tran Thanh Van et al 1974b). Not only are they revealed, but they can be programmed either separately (pure program) or combined according to various temporal/spatial sequences not normally expressed in vivo or in vitro when more voluminous explants are used; for example: in vitro differentiation i) from the subepidermal cells, of roots first followed by direct formation of flowers without vegetative buds or stem and without a callus phase, ii) of fertile cônes in Sequoia sempervirens (Tran Thanh Van et al 1985b), iii) of precocious flowers in Soja Biloxi (Jullien and Wyndaele 1992), iv) of unicellular hairs from the cotyledon epidermal cells of Torenia, v) of pseudo embryo structures (PES) on tTCL of Digitaria which start as somatic embryos i.e. with one-cell-origin but they develop rapidly into adult type plants and not into plantlets (Bui et al 1998c). This program offers an advantage in genetic transformation over the vegetative bud program in which the one-cell-origin is not always the case. Moreover, with somatic embryos going through the plantlet stage of growth, the expression of the inserted gene(s) at the adult stage can be modified due to methylation, co-suppression or somatic variation processes.
6. CHARACTERISTICS OF THIN CELL LAYER SYSTEMS
Facing to the paradoxe of the availability, on the one hand, of refine physiological, biochemical, molecular techniques allowing scientists to identify and isolate gene(s) that confer new properties and, on the other hand, the lack of appropriate experimental plant model, we have tried to conceive TCL systems in which target cells/responsive cells are the very ones cells that are excised and exposed to well defined environmental factors and to well defined compounds-at least at the beginning- of the culture medium.
The trait of being ?othin? is important as the localization of the target/responsive cells could be easily be made in vivo during the time course and in space by using, for example fluorescent markers of morphogenetic differentiation as well as the observation of their changes during the time course and in space without destroying the plant material using a tunneling microscope. Since tremendous progresses have been made in the identification of genes implied in the synthesis/metabolism of auxins, ABA, ethylene, cytokinins for citing only these genes and their mechanisms of regulation, target cells (at the perception site) and responsive cells can be identified using an appropriate choice of makers. Besides the fact that one can assume that the endogenous content of the so called endogenous factors is minimum and the ?otransport? processes are presumably less complex than in a more voluminous explant, the trait of being ?othin? offers another advantage. Target cells and responsive cells are in close contact with the wounded cells which released cell-wall fragments or oligosaccharides by the hydrolytic enzymes upon the excision of the TCLs from the donor tissue/organ. The influence of oligosaccharides, more precisely of oligogalacturonides?" combined or not to growth substances, on the differentiation processes including nodulation in roots of Leguminous, are already shown (Tran Thanh Van et al 1985a, Eberhard et al 1989, Mohnen et al 1990, Tran Thanh Van and Mutaftschiev 1990, Lerouge et al 1990, Marfa et al 1991, Spiro et al 1998).
The time interval for morphogenesis to occur is relatively short (an average of 14 days from the beginning of culture). The frequency is high: 100% (or close to) of TCLs respond. The intensity of the organ neoformed, therefore the ratio between the number of responsive cells and the number of cells present in one TCL is high. For example, on one Nicotiana tabacum lTCL (1mm x 10 mm including 3-6 cortical cell layers, excised from "floral branches") the following organs are differentiated de novo and directly on the surface of the TCL (without an intermediate callus):
-# 50 flowers for flower program
-# 500-700 vegetative buds for bud program
-# 15-20 roots for root program.
It has been hypothesised that the differentiation of flowers could be due to the particular physiological stage (floral) of the donor organ (floral branches), however the de novo differentiation of root and also of vegetative bud from the same physiological stage could not be explained according to the same hypothesis. In fact, it has been shown that RNA transcripts found on lTCL during the induction of the flower program are not expressed in the lTCL at time zero of the culture (Meeks-Wagner 1989). The above mentioned patterns, including ?oflower?, are ?oinduced?, i.e. the nucleus of the differentiated cells in its own cytoplasmic environment is ?oreprogrammed?.
A high frequency /intensity combined to a short time interval and the directness in the organ/embryo neoformation are also confirmed on other species: Populus (25 times more efficient using tTCL of 0.4-0.5 mm compared to 1 cm explant (Lee-Stadlemann et al 1989), Garcinia mangostana (50 times higher in tTCL of 3 mm than with half leaf explant (Goh et al 1994), Aranda Deborah (Lakshmanan et al 1995, Heliconia (Goh et al 1995) Rhynchostylis gigantea (Bui et al 1999c), Lilium longiflorum (Nhut 1998, Bui et al 1999a, Nhut et al 2000a, Nhut et al 2000b, Nhut et al 2001a, Nhut et al 2001b, Nhut et al 2001c), Brassica napus (Pua et al 1989, Charest et al 1988, Krismaszewska and Keller 1985), Panax giseng (Ahn et al 1996), sunflower (Pelissier et al 1990), Pelargonium (Gill et al 1992) etc...
7. TCL METHOD TO OVERCOME THE LIMITATION TO
PLANT REGENERATION AND ITS USE IN
PLANT TRANSFORMATION
The high potential in embryogenesis/organogenesis displayed by TCL systems was also observed on Petunia (Mulin and Tran Thanh Van 1989), sugar beet (Detrez et al 1988), sunflower (Pélissier et al 1990), and on species known as recalcitrant to in vitro regeneration such as i) some leguminous Psophocarpus tetragonolobus (Trinh et al 1981, Tran Thanh Van et al 1986), Soja biloxi (Jullien and Wyndaele 1992), Vigna unguiculata (Bui et al unpublished data), Phaseolus vulgaris (Cruz-de-Carvalho et al 2000), ii) some woody dicot species (Hardwickia binata Roxb (Das et al 1995), Paulownia (Bui et al unpublished data), Citrus (Carimi et al 1999), Poncirus (Bui et al 1999b), Pinus radiata (Villalobo et al 1985), Sequoiadendron giganteum, Sequoia sempervirens (Tran Thanh Van et al 1985b, Monteuuis 1991), Vitis (Hieu, unpublished data), iii) some woody monocot species: Bambusa glaucescens (Jullien and Tran Thanh Van 1994), Cocos nucifera (Sugimura and Salvana 1989, Blake 1983, Gupta et al 1984), iv) herbaceous monocot species: Iris pallida (Schricke et al 1988), Sorghum bicolor (Gendy et al 1996), Digitaria sanguinalis (Bui et al 1988c), Oriza sativa (Nhut et al 2000), Rhynchostylis giganteum (Bui et al 1999c), Phalaenopsis amabilis (Tran Thanh Van et al 1974a), Paphiopedilum auroreum, P. holdeni, P. maluki, P. delenatii (Tran Thanh Van et al unpublished data), Musa and plantain (Okole and Schulz 1996), Zea mays (Conger et al 1987), Hordeum vulgare (Becher et al 1992).
The high density of organs/embryos neoformed from a reduced number of cells in a TCL system results from a high number of cells which are ?oinduced? in the morphogenetic process. This allows i) a better isolation/identification of putative markers of differentiation, ii) a higher yield of transgenic plants. This high potential in regeneration extended to recalcitrant species is a key factor to the success in plant improvement. It is a well-known fact that positive results in genetic manipulation has been limited due to the low regeneration rate even on species-easy-to-regenerate. Furthermore, TCL systems with the closer contact between wounded cells and responsive cells add the advantage of the wounding effect on Agrobacterium virulence induction in transformation via agroinfection.
As for the fundamental aspects of the study of the differentiation processes, the TCL system has been successfully used as a model system to analyse biochemical and molecular markers of differentiation (Thorpe et al 1978, Kay and Basile 1987, Meeks-Wagner et al 1989, Tiburcio et al 1989, Neale et al 1990, Richard et al 1991, Tran Thanh Van 1991, Tran Thanh Van and Gendy 1993, Fu et al 2000, Biondi et al 2001).
Concerning the applied aspects, transformation techniques were successfully used on different TCL systems to speed the transgenic plant obtention (Trinh et al 1987, Pua et al 1987, Charest et al 1988, Bui et al 1998c). Due to the fact that the target cells are situated on or close to the surface of the explant, transformation by using agroinfection or biolistic using DNA or Agrobacterium coated particules give a high rate of transcient expression of GUS. Figures 1, 2, 3, 4 illustrate different stages of transformation performed on Amaranthus tTCLs (Jeanneau et al 2000).
The small size of the TCL systems may favor the use of methods of in planta Agrobacterium-mediated gene transfer by infiltration. However, co-culture and selection regimes as well as Agrobacterium concentration must be worked out in order to be adapted to the small size of the lTCL or tTCL explants.
8. CONCLUDING REMARKS
In order to answer to the question of how plant cells sense and integrate signals from a wide range of environmental factors and process these signal(s) for differentiation, our strategy is to select signal(s) which induce specific response in defined target cell(s). In TCL systems, the target/responsive cells could be easily located instead of being scattered all through a heterogenous organism or an organ fragment or a callus.
We have demonstrated on the TCL system that one can reprogram differentiated cells into multi-programmable patterns with a specific spatial /temporal sequence.
Some makers for differentiation selected among putative molecular markers of plant growth and development reported in the literature could be tested on experimental systems on which the reversibility/irreversibility phase during which one can /or can not any more change one morphogenetic program to another. Up to now, as far as we know those phases have been determined only on TCL systems due to the fact that i) different morphogenetic patterns can be ?oreprogrammed? on ?odifferentiated cells?, ii) histological / cytological study has been conducted on TCL systems during their programmed differentiation (Dien and Tran Thanh Van 1974, Nassogne et al 1985, Kay and Basile 1987, Creemers-Molenaar et al 1994) iii) biochemical and molecular markers have been determined on TCL. The performance of TCL systems was also reviewed by other authors (Hicks 1981, Compton and Veilleux 1992).

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