Final exam

Question 1

Most common practices of in vivo clonal vegetative propagation

Answer 1

Cutting, budding and grafting

Question 2

Most common explants for tissue culture

Answer 2

Meristem, shoot tips and axillary buds

Question 3

Who and when were explants defined

Answer 3

Cutter 1965

Question 4

If you want to produce a disease free plant, what explant do you go for?

Answer 4

Meristem culture, taking off any sorrounding tissue

Question 5

Who developes meristem culture?

Answer 5

Morel and Martin 1952

Question 6

What medium do you use for meristem culture?

Answer 6

MS

Question 7

If you want to make a shoot tip culture what do you add to the media, and what for?

Answer 7

High quantities of cytokinins. They’ll avoid apical dominance, make it more branchy.

Question 8

Once you have shoot growth what goes next?

Answer 8

Rooting media with auxins

Question 9

What is the advantage of seed culture in the lab, rather than planting a seed?

Answer 9

You can get multiple shoots from one embryo, instead of one plantlet per seed

Question 10

What procedure do yu follow to produce plantlets starting from a single node?

Answer 10

Node with axillary buds -> Cytokinins -> Branches ->Rooting media -> Plantlet

Question 11

What are the main methods of plant tissue culture?

Answer 11

Shoot tip culture, meristem culture, axillary bud culture, single node culture

Question 12

What are the methods of plantlet production in micropropoagation?

Answer 12

Direc organogenesis, indirect organogenesis, axillary bud method

Question 13

When dou you go for direct organogenesis?

Answer 13

When axillary or apical meristem are difficult to obtain. Any tissue will produce shoots

Question 14

In what consists indirect organogenesis?

Answer 14

Formation of callus via phytohormones and then redifferentiation into organs.

Question 15

In what cosists the axillary bud method?

Answer 15

The axils of leaves produce axillary buds and then shoot tips. By adding cytokinins 10:1 auxins, apical dominance is suppressed and axillary buds develop.

Question 16

Best cytokinin for axillary bud method

Answer 16

6-benzyladenin

Question 17

If you go through axillary bud method and you get an apical meristem what do you do?

Answer 17

Kill it

Question 18

Stages of micropropagation

Answer 18

0- Selection of elite plant with desirable traits. Kept in controlled conditions, with llow humidity, irrigation, and without microbial infection for 3 months.

I. Aseptic culture establishment. Sterelization with 5 % socium hyplchlorite, 0.1 % mercuric chloride and/or 70% alcohol

II. Multiplication of explants. Oce can obtain nodal explants from shoots. Cytokinin rich emdia will give shoots

III. Germination of somatic embryo or rooting. Inoculation of shoots un rooting media with auxins.

IV. Hardening. Meks plants resistant to stress, moisture changes and disease. One must give protection form sunlight, adnd decrese humidity gradually over time.

Question 19

Hormone that stimulates callus formation

Answer 19

2,4-D

Question 20

What is vitrification?

Answer 20

Shoot is brittle, glossy and looks soaked, due to abnormal function of stomata, poorly developed vascular bundles and abnormal wax quantity.

Question 21

What can you do if you face vitrification?

Answer 21

Add growth retardants, bottom coolinf of culture tubes, increase the concentration of agar to 1 % or add hydrolysate compounds.

Question 22

What is a callus?

Answer 22

An unorganized mass of loosly areanged parenchymatous cells

Question 23

Why may a callus be hard?

Answer 23

Lignification of cells

Question 24

In what depends the darkness of a callus?

Answer 24

The quantity of plyphenols in the plant species

Question 25

Best explants for callus production

Answer 25

Juvenile tissue, seedlings, young shoots, buds, root tips, develpoing embryos; fruits, floral parts, tubers nad bulbs.

Question 26

Conditions for callus culture

Answer 26

``` MS media Auxin 1:1 Cytokinin Sucrose 3% w/v pH 5.6-6 optimal 5.8 T 25 +- 2°C Light: 5000-10000 lux/m 16 hrs light/8 hrs dark ```

Question 27

How a callus produce?

Answer 27

In the outer layer of cortical cells of stem. Cells start dividing, increasing presure in the epidermins until rupturing

Question 28

How long a callus takes to form?

Answer 28

2-3 weeks, may take up to 4 weeks.

Question 29

Disadvantages of callus

Answer 29

Can cause chromosomal aberrations, such as polyploidy (caused by endoreplication) or aneuploidy (anaplase irregularity)

Question 30

Morphological stages of somatic embryogenesis

Answer 30

Globular - Heart - Torpedo - Germinated s. embryo

Question 31

How to create a somatic embryo?

Answer 31

Start with a media with high concentrations of auxins (mainly 2,4-D) and embryo develops when concentrations is reduced

Question 32

What is the most successful explant for direct embryogenesis?

Answer 32

female gametophytes

Question 33

Explants for direct embryogenesis

Answer 33

Ovules, nucellar embryos, nucellus tissue

Question 34

What to do to get embryogenesis from microspores?

Answer 34

Although it has been possible to achive embryogenesis from microspores, the more accepted method is through anther culture that results in the formation of callus and therefore indirect embryogenesis

Question 35

What is a protocorm?

Answer 35

Orchids cells have a small embryo with no endosperm. In germination the embryo enlarges to form a corm-like structure with quiscient shoot and root maristem at opposite poles

Question 36

What is basal media in animal cell culture?

Answer 36

Media with vitamins, inorganic cells, carbone source like glucose and serum

Question 37

What is reduced-serum media?

Answer 37

Basal media enriched with nutrients and animal-derived factors, to reduce the use of serum

Question 38

What is serum-free media?

Answer 38

Replace sera with nutrients and hormonal formulations. | One can make the media slective for a specific cell type

Question 39

pH for most mammalian cells, transformed ones and insect

Answer 39

Mammalian cells 7.4 Transformed 7 - 7.4 Insect cells (sf9)(sf21) 6.2

Question 40

What is the CO2 for?

Answer 40

Helps to buffer pH

Question 41

Temperatures for human and mammal cell cullture

Answer 41

36-37°C

Question 42

Temperature for insect cell culture

Answer 42

27°C

Question 43

Temp for avian cell culture

Answer 43

38.5°C, can be a 37°C but grow slower

Question 44

Temp for cold blooded animal tissue culture

Answer 44

15°C-26°C

Question 45

Sigs of deterioration of animal cell culture

Answer 45

Granularity around the nucleus, detachment from substrate, cytoplasmic vacuolation

Question 46

Categories of mammalian cells

Answer 46

Fibroblast cells: Bipolar or multipolar, elongated, grow attached to substrate Epithelial-like cells: Polygonal with more regular dimensions. Grow attached to substrate Lymphoblas-like cells: Spherical, grown is suspension culture. They don't attache to substrate

Question 47

Whats trasfection?

Answer 47

Artificial introduction of nucleic acids to a cell, via non-viral means

Question 48

Transcient transfection

Answer 48

Limited time duration, doesn't integrates to genome. Doesn't replicate

Question 49

Stable transfection

Answer 49

Permanent, integrates to genome, replicates whern host replicates

Question 50

What is cathionic lipid mediated transfection

Answer 50

Introduction of genetic materia through endocytosis, provoqued by a difference between the negative charge of cell membrane and the positive charge of liposome-nucleic acid complex

Question 51

What does a cathionic lipid cosists of?

Answer 51

A positively charged head and 1 or 2 hydrocarbon chains. The head interacts with the phosphate backbone of nucleic acid

Question 52

Famous Cathionic lipid

Answer 52

Lipofectamine

Question 53

What is the single structural unit of antibodies

Answer 53

immunoglobulin domain

Question 54

What does IgG

Answer 54

Highest opsonization and neutralization activities. There are 4: IgG1, IgG2, IgG3, IgG4

Question 55

What does IgM

Answer 55

Produces firts upon antigen invasion, increases transciently

Question 56

What does IgA

Answer 56

Expressed in mucosal tissue, forms dimers after secretion

Question 57

What does IgD

Answer 57

Unknown

Question 58

What does IgE

Answer 58

Allergies

Question 59

The combination of what is a hybridoma?

Answer 59

Antibody-producing lymphocyte B cell and myeloma cell

Question 60

What is produced with hybridomas?

Answer 60

Monoclonar antibodies

Question 61

Who first produced hybridomas?

Answer 61

Gerge Kohler, Cesal Milstein & Niels Jerne 1975

Question 62

What gene myeloma cell lack?

Answer 62

HGPRT

Question 63

Chemical that indices fusion?

Answer 63

Polyethylen glycol (PEG)

Question 64

In what media are hybridomas cultured?

Answer 64

Hypoxanthine aminopterine thymidine (HAT)

Question 65

What plathway inhibits aminopterin?

Answer 65

De novo

Question 66

What genes the salvage pathway needs?

Answer 66

HGPRT and TK

Question 67

Myeloma can go through the salvage pathway? T/F

Answer 67

F

Question 68

How long B or T cells live in media?

Answer 68

7 - 10 days

Question 69

Why we can't just culture B cells?

Answer 69

Because they don't have tumorogeninc property of immortaluty

Question 70

Why myeloma cells die in hybridoma media?

Answer 70

The media has aminopterin, wich blocks de novo pathway, so it has to go through the salvage pathways, that requires of the HGPRT gene, that myeloma cells, so it cannot survive if it is not fused with a B cell that has HGPRT