PLANT CELL TISSUE CULTURE

Organogenesis

Relies on the ability to most cells to "dedifferentiate" and revert to a being "stem-cell".

  i.e. actively dividing with no clear variation in cell type.

In plants, this is referred to as the formation of MERISTEMS and in culture is typified by the formation of callus tissue.

Various explants or suspension cell cultures can be used to induce meristemic tissue when cultures with are treated with

HIGH AUXIN AND CYTOKININ LEVELS.

 By changing the in hormones in the culture medium it is possible to induce callus cultures to re-differentiate to form viable plants.

 

Plant Meristems  -

 

To some degree equivalent to mammalian “stem cells”

 

Plant cells are truly totipotent. (toton = latin = entire)

 

In mammals only the fertilised egg is truly totipotent.

 

Really pluripotent (pluris = latin = many several).

 

In animals the mature embryo is a miniature variant of the adult animal.

 

Remember that meristems are active sites of hormone, particularly auxin, production.

 

Embryonic embryo development

 

Zygotic Embryo Development - Covers development from the time of fertilisation until seed formation occurs.

    The major roles for the embryogenic process are:

1.    Establish the basic body plan.

 

2.     Set aside meristematic tissue for postembryonic elaboration of structure (leaves, roots flowers, etc.)

 

3.    Establish an accessible food reserve for the germinating embryo until it becomes autotrophic.

 

Following fertilization, the embryo develops within an embryo sac which is surrounded by the maternal diploid tissue of the ovule. 

 Stage 1

 

Axis – apical / dorsal produced early in development.

Evidence for this polar movement of auxin.

How is this established? From maternal cells?

 

Apical Cell – produces embryo

Basal Cell – produces filamentous suspensor, 6- 8 cells long.

 

The suspensor connects embryo to maternal tissue. 

 

Stage 2

 

Division lead to the formation of a 16-cell ball ….Pro-embryo

Unknown signals from the Proembryo establishes the HYPOPHYSIS

This is the uppermost cell of the suspensor…

 

The hypophysis gives rise to the quiescent organising centre of the root meristem.

 

First defined shoot meristem marker is WUSCHEL (WUS) produced at the centre of the 16 cell pro-embryo.

 

Central cells divide radially to form to “heart shape”

 

 

Stage 3

 

Heart stage embryo ~250 cells

 

1.    Radial patterning emerges in the globular stage as the three tissue systems (dermal, vascular, and ground) of the plant are initiated.

 

2.    The dermal system will form from the protoderm and contribute to the outer protective layers of the plant.

 

3.    The vascular system functions in support and transport and arises from the procambium cells that differentiate in the centre of the globular embryo.

 

4.    Ground tissue forms from the ground meristem and surrounds the developing vascular tissue; however, the ground and vascular systems form independently.

 

Position rather than clonal origin appears to be the critical factor in embryo pattern formation

Suggest cell-cell communication

E.g. microsurgery experiments on somatic carrot embryos demonstrate that isolated pieces of embryo can often replace the missing complement of parts.

 

 

 

Shoot Meristem.

 

Meristem divide to form two types of cells.

(a)                       stem cells remaining at the centre

(b)                      daughter cells which are dispersed to the periphery.

 

Meristems may be functional for long periods of time so that stem / daughter balance must be precise.

 

Basic body plan has been established by a combination of WUS and CLAVATA3 (CLV3).

 

WUS is a transcription factor – which tells cell adjacent to stem cells to express CLV3

 

Secreted CLV3 interacts in CLV1 / CLV2 receptors which limits WUS expression. 

 

 

Control Circuit – Negative feedback Loop

 

STEM                                               CLV3

 


ORGANISER CENTRE                 WUS          via CLV1/CLV2

 

 

 

-        WUS knockout – meristems shuts down.

-        CLV3 knockouts – the meristem enlarges

 

Cell-to-cell communication is important, as if the organiser centre cells are killed, adjacent cell will differentiate. 

 

 

 

Root Meristem

 

Hypophysis will become the root quiescent centre…

 

 

 

 


…..which organises the meristem.

 

Organisation in established by cell-to-cell communication mediated by auxin.

 

Evidence….embryo’s lacking auxin response factor – MONOPTEROS fails to form hypophysis and no root tissue develops.

 

Is a similar feedback loop to WUS/ CLV3 involved in roots?

Not known but organisation is the same.

 

 

From meristems to organs

 

Leaves are typically produced directly from primary shoot meristems. …..daughter cells enter “zone of competence” where new organs form.

 

Leaf primordial derive from ~12 primordium founder cells..located on the sub/epidermal layer at the periphery of the meristem.

 

Leaf primordial formation is auxin mediated.

 

Cells between stem and periphery end up being “stem”. 

 

SHOOTMERISTEMLESS (STM) –

Prevents cells entering differentiation pathway but repressing “ASYMMETRIC LEAVES1” (AS1).

 

 

 

 

 

 

 

 

 

 

 

 

 


STM1 minus plants, AS1 is expressed in the meristem which shuts it down

 

AS1 minus mutants KNAT expression persists in primorida resulting in leaves with a stem like appearance.

 

AS1/STM1/KNAT are all transcription factors.

 

 

 

Problem with Culture: How do you mimic such a complicated process?

·       Choosing the right explant.

o      Inter-nodes

o      leaf pieces

o      zygotic embryos

o      basal or apical meristems

o      single cell suspension cultures

·       Altering the culture conditions.

o      changing hormone levels

o      changing micro-nutrients

o      changing pH

o    changing osmoticum

 

Problem with tissue culture : Somaclonal variation

Tissue culture involves successive rounds of plant division.

With increasing time in culture, the regenerated plant displays abnormalities e.g. albinism.

Genetic analysis revealed considerable chromosomal re-arrangements.

Not due to "active division" as not observed with meristematic tissue.

But callus tissue is also stressed due to non-physiological levels of hormones are used.

 
But what is the cause of this somaclonal variation?

 

       Two main mechanisms are suspected..

1.    Plant genomes are full of repetitive DNA.

This repetitive DNA contains many examples of mobile genetic elements.

The function of many of these has been lost over time........BUT some are activated during cell division and by
stress.

2. Could somaclonal variation therefore be due to

Recombination between homologous regions.

To avoid somaclonal variation,

 

1.    Must avoid or reduce the period of tissue culture.

2.    Somatic embryogenesis

 

 

Surprisingly, certain plant tissues can be induced to form "somatic embryos" which are fundamentally very similar to "zygotic embryos" which form within the seed. 

      A very attractive feature as

 

 

SOMATIC EMBRYOGENESIS

Somatic embryogenesis dependent only on auxins.

Very dependent on the right explants

-         carrot single cell suspension cultures

-         zygotic embryos

 

Appropriate auxin transport and distribution are needed for embryo development and pattern formation.

How is this controlled?

Activation tagging has identified “WUS” as a key element.