2. Plant regeneration

Question 1

How has plants’ ability to regenerate been exploited in industry?

Answer 1

Plant regeneration properties - microporpagation - can regenerate whole body from individual tissues -> most plant cells are totipotent

Question 2

Explain plant in vitro regeneration

Answer 2

In vitro regeneration - process of regenerating a whole plant from a certain tissue - placed in nutrient-rich medium - process relies on the inherent totipotency of plant cells = plant cloning

Explant take - sterilized - placed into nutrient rich medium - added hormones auxin and cytokinin - explant dedifferentiates - forms callus (mass of unorganized cells) - organogenesis -> plantlet

Question 3

What types of plant cells exist?

Answer 3

• Zygote: only embryonic stem cell - only totipotent stem cell

Specified in specific niches:
- Root meristem
- Shoot meristem
-> apical / lateral meristems

Additional cambial stem cells - produce phloem and xylem tissues in secondary growth

Question 4

Explain the structure of root meristem

Answer 4

Consists of non-stem cells, stem cells and organizing center (OC) = quiescent center

Highly regulated signalling to maintain cells in different pluripotency states

Question 5

Explain the structure of shoot meristem

Answer 5

Consists of the bud - in the center central zone (CZ) + peripheral zone (PZ)

CZ - slowly dividing reservoir of stem cells - self-renew + differentiate cells into PZ
PZ - more differentiated, more rapidly dividing cells - eventually differetiate into organs

Question 6

Are the same genes used for root and shoot meristem maintenance?

Answer 6

No, genes for root and shoot meristem formation and maintenance are related but not identical

Question 7

Explain what are cambial stem cells

Answer 7

Cambial stem cells - in cambium - thin layer of meristematic tissue between the xylem and phloem in vascular plants - conatin xylem and phloem precursors - develop in secondary growth - cambial stem cells have indeterminate growth - can continue dividing through lifetime

Question 8

What is plant secondary growth? How does it differ from primary growth?

Answer 8

Secondary growth - increases the diameter of the stem and root - cambial stem cells are responsible for the secondary growth

This is different from primary growth, which increases the length of the plant and is driven by the apical meristems

Question 9

What is plant primary growth?

Answer 9

Primary growth - increases the length of the plant and is driven by the apical meristems

Question 10

Why is the definition of toti/pluripotency with respect to regeneration so vague in plants?

Answer 10

Definition of toti/pluripotency with respect to regeneration so vague in plants because stem cells in plants are organised differently than animals - normally niche specific stem cells don’t perform totipotency but they can if needed (micropropagation)

Question 11

Do plant tissue stem cells have similar function to animal tissue stem cells?

Answer 11

No, in plants niche specific stem cells can become any other tissue - fate not restricted - animal stem cells are restricted to the niche

Question 12

Why lifestyle is an important consideration when reflecting on different “nature” of stem cells in different phyla and kingdoms?

Answer 12

Because plants don’t move - are expected to regenerate at higher capacity what they loose - need to change capacity, not damage

Question 13

What are the two types of regeneration trajectories that reveal regenerative capacity of a plant?

Answer 13

Regeneration after trauma - triggered by wounding - site specific
- maintain tissue context
- from single, isolated cells

Regeneration without trauma - triggered for development or environmental cues - not site specific, broader systematic changes

Question 14

Explain plant regeneration without trauma

Answer 14

Plant regenerationw without trauma:
- plant development - indeterminate - # of organs not pre-determined
- in perennial plant growth resumes after dormancy periods
- this regeneration mediated by apical and lateral meristems and re-capitulates organogenesis and differentiation from embryogenesis
=> this is not regeneration - normal plant life

Question 15

Explain plant regeneration after trauma

Answer 15

Regeneration after trauma in vascular tissue:
- several plant vascular tissues resume proliferation and replace severed connection
- distinct vascular cells undergo trans-differentiation into missing vascular cells
- other cell types can resume division but produce scar tissue but not regenerate

But trauma in root meristems dealt differently

Question 16

Explain the role of PIN1

Answer 16

PIN1 - auxin transport in plant cells

Question 17

What role does PIN1 play in regeneration

Answer 17

Pin1 - transporter - transports auxin
- auxin directionally transported to repair wounded tissue

Question 18

What is the pathway triggered in plant wounding?

Answer 18

WIND1 ?? check on ipad slide notes

Question 19

Would plants be able to regenerate if their stem cell niches were compromised?

Answer 19

Yes, stem cell niches would be restored by new cells —- mechanism must be ensured by plant nature because if they get severely damaged cause can’t move to escape

Question 20

What drives root stem cell niche regeneration?

Answer 20

Root stem cell niche (SCN) regneration driven by not only by root SCN genes

Question 21

What is the role of plant stem cells in tissue repair and maintenance?

Answer 21

Tissue repair: plant stem cells regenerate even if stem cell niche cut off

Maintenance: stem cells maintained by meristems - stem cell niches - stem cells divide and produce new cells

=> plant stem cells not needed for regeneration - they can dedifferentiate to become stem cells - with only one condition - need to be closer to the tip than 200um - otherwise lost potential to dedifferentiate into stem cells

Question 22

Which cell types are involved in tissue repair?

Answer 22

Tissue repair is done by the cells of the tip - up to 200um from the tip - still have the capacity to dedifferentiate and give rise to new cell types

Question 23

How does auxin flux contribute to repair of severed vascular tissues?

Answer 23

When tissue damaged - directional auxin transport to the site - new vasculature differentiates

Local auxin increase plays a pivotal role in stimulating vascular tissue regeneration - orchestrates cellular processes that lead to the formation and differentiation of new vascular tissues:
- auxin as a morphogen
- for cell reprogramming
- promotes proliferation
- hormone corsstalk with gibberellins and cytokinins
- promotes vascular fate

Question 24

Why can the root tip regenerate even if all stem cells are excised?

Answer 24

Bceause plant cells are pluripotent - can dedifferentiate and redifferentiate into a new cell type to replace the lost - this mechanism evolved because plants are not dynamic - can’t run way form predators - need good regeneration systems to survive

Question 25

Explain what is lateral root development

Answer 25

**Lateral roots** grow out **from the primary root** - develop **from pericycle cells** - layer of cells found just inside the root endodermis, adjacent to the vascular tissues (xylem and phloem)

Question 26

What is callus?

Answer 26

**Plant callus** - **mass of undifferentiated, proliferating cells** - stuck in unidfferentiated state - forms when plant tissues are subjected to certain conditions - particularly in response to wounding or during in vitro culture

Question 27

How is callus formation related to lateral root development?

Answer 27

These two processes **use similar pathway** - callus initiates a regeneration pathway similar to lateral root development

Question 28

Explain the mechanism of callus formation

Answer 28

**Callus resembles tip of root meristem** - callus formation **requires lateral root genetic programme** - depending on **auxin** **level** -> lateral root growth vs callus formation - **strong auxin triggers regeneration (callus)** - weaker auxin lateral root formation

Question 29

Explain callus induction experiments in vitro

Answer 29

**Callus induction** by **genetic experiment** - **alf4-1**: wt, het, homo -> alf4-1 **needed for root development** - otherwise callus - **ABA +/-** -> similar results - **doesn't affect callus induction** - no mersitem activation in callus formation In **callus formation** - a **blob** formed In root growth - directional growth But overall **callus formation requires lateral root genetic programme** - **auxin** signalling

Question 30

Do all plant cells regenerate by callus formation?

Answer 30

**No**, **only those** tissues which **have lateral root growth ability** - o**thers dedifferentiate and respecify** into the needed tissue

Question 31

What are the barriers to plant regeneration?

Answer 31

**Barriers** to plant regeneration: - **epigenetic**: does epigenetic modification change in regeneration - **ploidy**: are all cells of all ploidy equally competent to regenerate

Question 32

What is required to form shoot from callus tissue?

Answer 32

**Epigenetic changes** are needed - specifically in **WUS gene**

Question 33

Plants are not observed to form tumours, what are the mechanisms that block this?

Answer 33

**Plants don't get cancer** because of **epigenetic specifics** + **cell divisions very controlled** + have **more DNA damage response genes** **Also, elephants**, even tho larger and higher chance of cancer - don't get cancer as oftne as expected becaus have more DNA damage response genes

Question 34

Explain the significance of the finding that all callus, irrespective of the tissue of origin, goes through a developmental pathway resembling lateral root development

Answer 34

Lateral leaves, lateral roots take different mechanisms to grow but callus formation which used lateral root growth mechanism is done both in leaves and roots - ??

Question 35

What does it tell you about the origin of organising centre of a callus with respect to its cell types

Answer 35

??

Question 36

What is endoreplication?

Answer 36

**Endoreplication** - **modified cell cycle** - cell undergoes **DNA replication without subsequent cell division** - increase in the cell's nuclear DNA content - **polyploidy**

Question 37

How are endoreplication and trichome formation related?

Answer 37

**Endoreplication** is a **natural** process in differentiated **plant cells** - BUT **endoreplication may prevent regeneration** from all plant cell types -> **suppression of endoreplication** **prevents** differentiation and/or maintenance of **differentiation pathway for trichomes** - transformation into trichomes can't be sustained Low ploidy is aborting trichomes ===> endoreplication drives trichome formation

Question 38

Is endoreplication a barrier to regeneration in differentiated cells?

Answer 38

**Yes**, the **more endoreplication the more restricted/locked in the differentiated state** - loss of division potential, terminal differentiation Only can be **reversed in case of wounding / hormonal signalling** read more

Question 39

Regeneration in plants summary

Answer 39

Question 40

How could it be proved that endoreplication is a barrier to plant cell regeneration in toher tissue contexts?

Answer 40

Create **inducable deletions of endoreplication genes** -> edit non-regenerating cells to see if would regenerate

Question 41

How could it be proved that non-pericycle like cells are not capable of regeneration?

Answer 41

Try over-expressing needed genes for regeneration

Question 42

Does the statement that endoreplicated cells are unable to regenerate contradict the finding that leaf margin cells are able to resume division after trauma?

Answer 42

Didn't form callus - different callus type - not all can form any tissues Callus = scar tissue in this case ?? read