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Final exam Flashcards

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1
Q

Most common practices of in vivo clonal vegetative propagation

A

Cutting, budding and grafting

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2
Q

Most common explants for tissue culture

A

Meristem, shoot tips and axillary buds

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3
Q

Who and when were explants defined

A

Cutter 1965

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4
Q

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

A

Meristem culture, taking off any sorrounding tissue

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5
Q

Who developes meristem culture?

A

Morel and Martin 1952

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6
Q

What medium do you use for meristem culture?

A

MS

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7
Q

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

A

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

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8
Q

Once you have shoot growth what goes next?

A

Rooting media with auxins

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9
Q

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

A

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

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10
Q

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

A

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

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11
Q

What are the main methods of plant tissue culture?

A

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

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12
Q

What are the methods of plantlet production in micropropoagation?

A

Direc organogenesis, indirect organogenesis, axillary bud method

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13
Q

When dou you go for direct organogenesis?

A

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

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14
Q

In what consists indirect organogenesis?

A

Formation of callus via phytohormones and then redifferentiation into organs.

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15
Q

In what cosists the axillary bud method?

A

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.

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16
Q

Best cytokinin for axillary bud method

A

6-benzyladenin

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17
Q

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

A

Kill it

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18
Q

Stages of micropropagation

A

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.

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19
Q

Hormone that stimulates callus formation

A

2,4-D

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20
Q

What is vitrification?

A

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

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21
Q

What can you do if you face vitrification?

A

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

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22
Q

What is a callus?

A

An unorganized mass of loosly areanged parenchymatous cells

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23
Q

Why may a callus be hard?

A

Lignification of cells

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24
Q

In what depends the darkness of a callus?

A

The quantity of plyphenols in the plant species

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25
Q

Best explants for callus production

A

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

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26
Q

Conditions for callus culture

A 
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
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27
Q

How a callus produce?

A

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

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28
Q

How long a callus takes to form?

A

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

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29
Q

Disadvantages of callus

A

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

30
Q

Morphological stages of somatic embryogenesis

A

Globular - Heart - Torpedo - Germinated s. embryo

31
Q

How to create a somatic embryo?

A

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

32
Q

What is the most successful explant for direct embryogenesis?

A

female gametophytes

33
Q

Explants for direct embryogenesis

A

Ovules, nucellar embryos, nucellus tissue

34
Q

What to do to get embryogenesis from microspores?

A

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

35
Q

What is a protocorm?

A

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

36
Q

What is basal media in animal cell culture?

A

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

37
Q

What is reduced-serum media?

A

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

38
Q

What is serum-free media?

A

Replace sera with nutrients and hormonal formulations.

One can make the media slective for a specific cell type

39
Q

pH for most mammalian cells, transformed ones and insect

A

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

40
Q

What is the CO2 for?

A

Helps to buffer pH

41
Q

Temperatures for human and mammal cell cullture

A

36-37°C

42
Q

Temperature for insect cell culture

A

27°C

43
Q

Temp for avian cell culture

A

38.5°C, can be a 37°C but grow slower

44
Q

Temp for cold blooded animal tissue culture

A

15°C-26°C

45
Q

Sigs of deterioration of animal cell culture

A

Granularity around the nucleus, detachment from substrate, cytoplasmic vacuolation

46
Q

Categories of mammalian cells

A

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

47
Q

Whats trasfection?

A

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

48
Q

Transcient transfection

A

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

49
Q

Stable transfection

A

Permanent, integrates to genome, replicates whern host replicates

50
Q

What is cathionic lipid mediated transfection

A

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

51
Q

What does a cathionic lipid cosists of?

A

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

52
Q

Famous Cathionic lipid

A

Lipofectamine

53
Q

What is the single structural unit of antibodies

A

immunoglobulin domain

54
Q

What does IgG

A

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

55
Q

What does IgM

A

Produces firts upon antigen invasion, increases transciently

56
Q

What does IgA

A

Expressed in mucosal tissue, forms dimers after secretion

57
Q

What does IgD

A

Unknown

58
Q

What does IgE

A

Allergies

59
Q

The combination of what is a hybridoma?

A

Antibody-producing lymphocyte B cell and myeloma cell

60
Q

What is produced with hybridomas?

A

Monoclonar antibodies

61
Q

Who first produced hybridomas?

A

Gerge Kohler, Cesal Milstein & Niels Jerne 1975

62
Q

What gene myeloma cell lack?

A

HGPRT

63
Q

Chemical that indices fusion?

A

Polyethylen glycol (PEG)

64
Q

In what media are hybridomas cultured?

A

Hypoxanthine aminopterine thymidine (HAT)

65
Q

What plathway inhibits aminopterin?

A

De novo

66
Q

What genes the salvage pathway needs?

A

HGPRT and TK

67
Q

Myeloma can go through the salvage pathway? T/F

A

F

68
Q

How long B or T cells live in media?

A

7 - 10 days

69
Q

Why we can’t just culture B cells?

A

Because they don’t have tumorogeninc property of immortaluty

70
Q

Why myeloma cells die in hybridoma media?

A

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

Tissue culture (3 decks)

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