Chapter 21 - Tissue Culture

Question 1

Are aseptic culture, in vitro culture and micropropagation equivalent terms?

Answer 1

nope

Question 2

What does totipotent mean?

Answer 2

The concept that a single cell has the genetic program to grow into an entire plant.

Question 3

in vitro

Answer 3

The culture of plant cells or organs in culture vessels (like test tubes) under controlled environment and nutritive growth medium

Question 4

How does the ratio of auxin and cytokinin influence root, shoot and callus formation?

Answer 4

high auxin to cytokinin ratio and roots are formed. high cytokinin to auxin ratio, shoots are formed.

Question 5

What are the 4 stages of micropropagation?

Answer 5

I. Establishment—placing tissue into cul-ture and having it initiate microshoots.

2. Shoot multiplication—inducing multiple shoot production. Stage
3. Root formation—initiating roots on microcuttings. Stage 4: Acclimatization—gradually moving plants to open-air conditions.

Question 6

the 3 origins of microshoots

Answer 6

Small shoots grown in vitro

Question 7

explant

Answer 7

The piece of the plant (propagule) used to initiate the micropropagation or tissue culture process

Question 8

What is the ‘5th Stage’ (Stage 0) of micropropagation?

Answer 8

Stage that includes preconditioning procedures applied to the source plants prior to taking explants for Stage I of micropropagation that can improve establishments of explants in culture.

Question 9

adventitious shoots

Answer 9

Shoots that arise from places where buds do not normally form, such as roots, leaves, flowers, and stem internodes

Question 10

How can stock plants be kept ‘cleaner’ before explants are taken for disinfestation?

Answer 10

greenhouse are “cleaner” sources of explants than are those growing out-of-doors. Overhead watering, sprinkling, or any activity that increases humidity around the plant should be avoided. Dormant plant parts can be taken and forced into growth under laboratory conditions to minimize contamination. insects and fungi should be controlled with chemicals if required.

Question 11

tissue culture, disinfestation

Answer 11

Chemical removal of surface contaminants that would otherwise grow in the tissue culture environment and kill the explant.

Question 12

Why do explants need disinfestation?

Answer 12

the explant is being placed on a tissue culture medium con-taining nutrients and sugar, the explant must be completely free of contaminants; otherwise these will actively overgrow and consume the explant within a few days.

Question 13

What are the usual disinfestants?

Answer 13

alcohol (ethyl, methyl, or isopropyl, usually about 80 percent) and bleach (calcium or sodium hypochlorite, usually with 5.25 to 6.0 percent active ingredient). Bleach is usually diluted at 10 to 20 percent before use.

Question 14

Why are surfactants or detergents are usually added to disinfestants?

Answer 14

A few drops of a surfactant or detergent should be added to the bleach to improve surface coverage.

Question 15

Why is ex-vitro rooting usually better in the longer term than in vitro root initiation?

Answer 15

an excellent transition from the culture environment to the open air, but also saves labor requirements for handling plants in micropropagation.

Question 16

How do in vitro leaves differ from normal leaves?

Answer 16

Leaf size and the number of cell layers that comprise the leaf are much reduced, compared to leaves developed outdoors. leaves do not develop epicuticular waxes on the leaf surface in normal amounts or with the same chemical properties

Question 17

How do in-vitro leaf differences make acclimatization difficult?

Answer 17

leaves on microshoots desiccate quickly after they are removed from in vitro culture.

Question 18

microcutting

Answer 18

The small shoots produced in tissue culture

Question 19

What treatments can be used to overcome acclimatisation difficulties?

Answer 19

gradually reduce humidity around the rooted microcuttings. Humidity may be maintained by using intermit-tent mist, fog, or enclosing plantlets in a polyethylene tent. Plantlets can also be “hardened” while they are still in vitro and prior to transplanting. Hardening involves various treatments to reduce humidity in the culture ves-sel. It may be as simple as removing the cap on the tissue culture vessel for 5 to 7 days prior to transplanting or using desiccants to reduce humidity.

Question 20

Meristen culture is usually used for __________?

Answer 20

eliminate diseases from plants, using a very small piece of tissue from the shoot tip as the initial explant.

Question 21

What is the tradeoff between meristem size and elimination of pathogens and culture survival?

Answer 21

smaller the explant the more likely the virus is not inside.

Question 22

Adventitious bud initiation can either be _______ or ______________ (via callus)

Answer 22

directly initiated or indirectly

Question 23

Compared to axillary shoot culture adventitious bud production can give _____ rates of multiplication, but also increased numbers of ______________?

Answer 23

high, aberrant off-type plants

Question 24

Why is it useful to germinate orchid seed in vitro?

Answer 24

germination because the seed is so small and contains no seed storage reserves. successful in a medium supplied with minerals and sucrose

Question 25

What is embryo rescue?

Answer 25

A procedure involving tissue culture of immature embryos that require controlled conditions to complete development. It is most commonly used by plant breeders for embryo rescue of genetic crosses that would not form seed on the plant.

Question 26

What are features of callus culture?

Answer 26

Callus is produced on explants in vitro as a response to wounding and growth substances, either within the tissue or supplied in the medium.

Question 27

What is somaclonal variation?

Answer 27

Somaclonal variability results because (a) variation is inherently present within the source plant, (b) the callusing system results in variation, or (c) it can be induced by mutagenic agents

Question 28

What is the main hormone needed for callus production?

Answer 28

Auxin at a moderate to high concentration is the primary growth substance used to produce callus. The principal auxins include indoleacetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D). Cytokinin (such as kinetin, or benzyladenine) is supplied in a lesser amount.

Question 29

What is the most effective auxin for callus production?

Answer 29

(IAA)

Question 30

Is exogenous cytokinin needed for callus production?

Answer 30

if not adequate levels remain in the explant.

Question 31

is a callus culture a homogenous group of cells.

Answer 31

no

Question 32

A high cytokinin/auxin ratio leads to ________ ?

Answer 32

adventitious shoot growth

Question 33

A high auxin/cytokinin ratio leads to ___________ ?

Answer 33

adventitious root growth

Question 34

An intermediate ratio of auxin and cytokinin leads to _____________ ?

Answer 34

a combination of root and shoot growth

Question 35

How are cell suspensions formed?

Answer 35

Callus cells grown in liquid culture that is constantly agitated. Agitation breaks cells apart, preventing them from forming large callus clumps.

Question 36

How are protoplast cultures initiated?

Answer 36

Plant cells without a cell wall. The cell wall is removed by microbial enzymes.

Question 37

What are some uses of protoplants culture?

Answer 37

Protoplast culture is particularly important in genetic engineering, since protoplasts can absorb DNA, proteins, and other large macromolecules. fusion of protoplasts from two different genotypes, such as two species,

Question 38

What is somatic embryogenesis?

Answer 38

The development of embryos from vegetative cells rather than from the union of male and female gametes to produce a zygote. In this process, a bipolar structure is produced with a root-shoot axis and a closed independent vascular system.

Question 39

Organogenesis is _____________?

Answer 39

is the process by which the three germ tissue layers of the embryo, which are the ectoderm, endoderm, and mesoderm, develop into the internal organs of the organism

Question 40

What are some differences between zygotic and somatic embryos?

Answer 40

Zygotic embryos of a given species are adapted to grow in very species-specific and complex nutrient environments. Whereas, with somatic embryos, there is no endosperm tissue to feed the developing embryo and modulate nutrient flow.

Question 41

synthetic seeds

Answer 41

Somatic embryos enclosed in an artificial seed coat, which may be a way of sowing somatic embryos for mass propagation.

Question 42

How is somatic embryogenesis used in plant propagation?

3

Answer 42

1. still in the research stages but to create synthetic seeds.
2. used to recover plants that have unique genetic properties,

Question 43

What hormone is most effective for induction of somatic embryogenesis?

Answer 43

auxin 2, 4-d induction medium.

Question 44

What is the difference between direct and indirect somatic embryogenesis?

Answer 44

direct somatic embryos can develop directly from explants without an intervening callus stage (already contains potential). indirect requires a callus and filter stage.

Question 45

Once formed somatic embryos must be moved to a _______________ medium to allow continued development of the embryo?

Answer 45

growth-regulator-free medium without 2,4-D

Question 46

What hormone is used to help embryos to mature?

Answer 46

Abscisic acid (ABA) is added to the maturation medium to promote normal somatic embryo development and inhibit precocious germination

Question 47

Why are relatively low light intensities used for the tissue culture environment?

Answer 47

because sucrose is provided in the medium of heterotrophic cultures rather than relying on photosynthesis for energy. Higher light irradiance in heterotrophic cultures can result in loss of chlorophyll and leaf necrosis.

Question 48

What is a heterotrophic culture?

Answer 48

adding an organic carbon. (use of sugars) in a culture.

Question 49

Elevated levels of ________ and low levels of __________ gases are useful in culture vessels

Answer 49

increased CO2,

reduced ethylene levels.

Question 50

What is hyperhydricity?

Answer 50

(originally called vitrification) is characterized by a translucent, water-soaked, succulent appearance that can result in cultures that deteriorate and fail to proliferate

Question 51

What can be added to culture media to combat excessive exudation?

Answer 51

treating the explant with an antioxidant (citric or ascorbic acid), including an adsorbent material in the medium (polyvinylpyrrolidone or activated charcoal), and frequent transfers to a new medium.

Question 52

What is habituation?

Answer 52

is the autotrophic growth in cultures that had previously required auxin or cytokinin for growth. For example, shoot cultures (Fig. 43) may become habituated to cytokinin and continue to proliferate even after the culture has been transferred to a medium without any growth regulator

Question 53

When is somaclonal variation useful?

Answer 53

Genetic mutations that occur in tissue culture are referred to as somaclonal variation. for developing mutants that may show desirable plant form, flower color, or resistance to disease.

Question 54

When is somaclonal variation undesirable?

Answer 54

In most cases

Question 55

Which is generally more genetically stable – axillary or adventitious bud production?

Answer 55

axillary bud production.