Types of tissue culture

Question 1

what are the eight types of plant tissue culture

Answer 1

1. whole plant
2. embryo
3. root
4. cell suspension
5. protoplast
6. callus
7. microspore
8. meristem

Question 2

whole plant culture

Answer 2

• maintains entire plant in sterile conditions

Question 3

applications of whole plant culture

Answer 3

1. arabidopsis - seed surface sterilized, sown on medium. water, support, minerals, and light are only requirements
2. horticulture - some seeds only germinate in specific conditions

Question 4

embryo culture

Answer 4

• a type of whole plant culture, but with embryos
• avoids dormancy problems
• avoids embryo/endosperm incompatibility in hybrids

Question 5

what is embryo rescue

Answer 5

obtain interspecific hybrid plants prior to abortion of embryos due to post-fertilization barriers

Question 6

protoplast culture

Answer 6

• cells are stripped of middle lamella and cell wall by pectinases and cellulases
• protoplast remains alive
• good for increasing plant ploidy and producing hybrids

Question 7

protoplast culture specifications

Answer 7

• medium requirements critical (bc of lack of cell walls)
• protoplast vulnerable to osmotic concentrations
• protoplast fusion requires media that enables cells to stick together

Question 8

what is the difference between callus culture and liquid suspension culture?

Answer 8

callus culture - undifferentiated or unorganized mass of cells on agar
suspension culture - single or groups of cells are suspended

Question 9

how is callus culture established?

Answer 9

a) diversity of cell types - less differentiated cells are more responsive to callus induction on simple media
b) physiological status of explant - nutrient status, hormonal content, dormancy status
c) genotype

Question 10

Removed

Question 11

callus culture features

Answer 11

1. compact - cells tightly joined and the tissue mass is one solid piece
2. friable - cells loosely joined and individual cells readily seperable

Question 12

callus

Answer 12

a tissue that develops in response to injury caused by physical or chemical

Question 13

callus forms due to:

Answer 13

1. removal of cells within the explant from organizational controls
2. provision of mineral nutrients and growth regulators for autonomous and indeterminate cell growth

Question 14

callus culture growth patterns

Answer 14

1. growth patterns leading to organized development - morphogenisis
2. growth patterns leading to continued proliferation of unorganized callus -maintenance

Question 15

callus culture- adventitious organogenesis and somatic embryogenesis

Answer 15

adventitious organogenesis - regeneration
somatic embryogenesis - development of embryos from somatic tissue

Question 16

somatic embryogenesis

Answer 16

stimulation of callus or suspension cells to undergo a developmental pathway that mimics the development of zygotic embryo

Question 17

zygotic embryogenesis

Answer 17

formation of an embryo as a result of double fertilization of the ovule

Question 18

microspore derived embryogenesis

Answer 18

production of embryos from microspores
- result requires haploid doubling

Question 19

meristem culture

Answer 19

meristem can produce an entire shoot and ultimately a whole plant

Question 20

micropropagation

Answer 20

creating multiple clones of a plant using plant tissue culture

Question 21

stages of microspore culture

Answer 21

i) inititation- explants placed in tissue medium for shoot inititaion
II) multiplication - cultures maintained on initiation medium for this stage (shoots multiply by 3X/month)
III) rooting - auxin only, focus is producing root tissue
IV) hardening off - acclimatizing plants to new conditions

Question 22

micropropagation advantages

Answer 22

• from one to many plantlets rapidly
• genetically identical clones
• multiplication in controlled lab conditions
• continuous propagation year round
• potential for disease-free propagules

Question 23

micropropagation disadvantages

Answer 23

• equipment/facility intensive operation
• requires trained technicians
• protocols not optimized for all species
• propagules may be too expensive

Question 24

transient transformation

Answer 24

• does not depend on integration into plant genome
• fast
• expression often disappears after less than a week

Question 25

stable transformation

Answer 25

- integrated into plant genome - take longer - transgene passed to daughter cells -expression throughout plant

Question 26

transformation methods

Answer 26

- agrobacterium - viruses - rhizobium

Question 27

agrobacterium transformation method

Answer 27

- infects plant by inserting its DNA into plant DNA - host cells grow and divide rapidly - produce opine (carbon source and nitrogen source for bacteria)

Question 28

Agrobacterium advantages and disadvantages

Answer 28

advantages: - more efficient than biolistics - low # of insertion events/cell disadvantages: - doesnt work for all species - insertion of bacterial DNA with GOI - IP issues

Question 29

agrobacterium transformation steps

Answer 29

1) co-cultivation - bacteria infect plant tissue (plasmid DNA inserted into plant cells) 2) Kill phase - bacteria must be killed off, transferred to antibiotic media with high cytokinin conc. 3) regeneration phase - high BA content, cells with gene divide and become meristematic, must select for cells containing gene with antibiotic

Question 30

biolistics

Answer 30

- gold particles coated with DNA - projected into plant cells - selection of transgenic cells - regeneration

Question 31

biolistics advantages vs disadvantages

Answer 31

advantages - any tissue that can be regenerated through tissue culture can be transformed - good for transient expression studies disadvantages - several copies of transgene insert into a chromosome (high copy events silenced) - not as efficient as agrobacterium - large DNA fragments may be damaged bc shearing

Question 32

CRISPR delivery

Answer 32

agrobacterium - inserts into plant genome - remove crispr genes by breeding with unaltered plants protoplast - deliver crispr enzymes and RNA guide molecules that dont insert into the genome biolistics - insert DNA, crispr enzymes and RNA - if DNA is inserted into genome it must be removed

Question 33

events

Answer 33

insertion of a particular transgene into a specific location on a chromosome

Question 34

event: inserts into gene critical for survival or development

Answer 34

effect: knocks out gene and cell dies or cant divide

Question 35

event:inserts into locus that does not allow for expression

Answer 35

effect: never expressed transgene

Question 36

event: expressed in original transgenic plant but is silenced in later generations

Answer 36

effect: offspring doesnt express transgene

Question 37

event: inserts into gene critical for reproduction

Answer 37

effect: cannot pass event to offspring

Question 38

event:inserts into region genes not normally found or has an alteration in sequence

Answer 38

effect: expression occurs but not optimal level

Question 39

event: inserts in a gene that influences agronomic traits and yield

Answer 39

effect: yield drag

Question 40

why are desriable events rare?

Answer 40

- transgene must insert in chromosome part that allows gene expression - cant disrupt other plant genes - most events never leave lab or greenhouse - others rejected in field - must produce many events to find a desirable one