NATIONAL UNIVERSITY OF LIFE AND ENVIRONMENTAL …

NATIONAL UNIVERSITY OF LIFE AND ENVIRONMENTAL SCIENCES

OF UKRAINE

CHAIR OF AGRICULTURAL BIOTECHNOLOGY

«PLANT BIOTECHNOLOGY BASIS OF PLANT PROTECTION»

Methodical instructions to implement laboratory work

for students of the specialty

6.090105 “Plant Protection”

Kyiv – 2014

2

UDC 606:632

The basic guidelines for laboratory works of the subject "Plant

Biotechnology Basis of Plant Protection".

Considered and approved at the Department of Agricultural Biotechnology

protocol # 5 of December 5, 2013.

Recommended for publication by the Scientific Council of Educational and

research institute of environment and biotechnology, protocol #4 of December 19,

2013.

Compiled by:

Lobova Oksana, candidate of biological sciences, associate professor

Pilipchuk Elena, postgraduate student

Reviewer:

Kolomiets Ulia, candidate of biological sciences, associate professor

Haychenko Vitalyi, doctor of biological sciences, professor

Educational publication

Recommendations for laboratory works for students of 6.090105 "Plant

Protection"

Responsible for the issue: Lobova Oksana, candidate of biological sciences,

associate professor

3

CONTENT

LABORATORY WORK №1 .......................................................................................................... 4

ORGANIZATION OF BIOTECHNOLOGICAL LABORATORY ............................................... 4

1.1. Features of laboratory equipment ......................................................................................... 4

1.2.Methods of ware, tools, materials sterilization ...................................................................... 4

1.3.Rules of work in laminar - box .............................................................................................. 6

1.4.Keep of lab - journal .............................................................................................................. 6

LABORATORY WORK №2 .......................................................................................................... 7

MOTHER LIQUOR MACROSALTS AND MICROSALTS FOR MS’S NUTRIENT MEDIUM

......................................................................................................................................................... 7

LABORATORY WORK №3 ........................................................................................................ 10

PREPARATION OF NON-HORMONAL NUTRIENT MEDIUM ............................................. 10

LABORATORY WORK №4 ........................................................................................................ 12

SEEDS STERILIZATION AND GROWING IN ASEPTIC CONDITIONS .............................. 12

LABORATORY WORK №5 ........................................................................................................ 15

STERILIZATION OF COURGETTES AND RECEIVING ASEPTIC CULTURE OF PLANTS

....................................................................................................................................................... 15

LABORATORY WORK №6 ........................................................................................................ 16

PREPARATION OF NUTRIENT MEDIUM FOR CALLUS ..................................................... 16

LABORATORY WORK №7 ........................................................................................................ 18

RECEIVING PRIMARY CALLUS FROM DIFFERENT EXPLANTS OF ASEPTIC PLANTS

....................................................................................................................................................... 18

LABORATORY WORK №8 ........................................................................................................ 19

SUBCULTIVATION OF PRIMARY CALLUS TO INCREASE ITS MASS ............................. 19

LABORATORY WORK №9 ........................................................................................................ 20

GETTING OF CALLUS CULTURE OF PLANT TISSUES ....................................................... 20

LABORATORY WORK №10 ...................................................................................................... 21

PREPARATION OF PLANT REGENERANTS THROUGH DIRECT AND INDIRECT

MORPHOGENESIS ...................................................................................................................... 21

4

Laboratory work № 1. Organization of biotechnological laboratory

Required space isolated rooms, modern equipment and high-quality reagents

of biotech laboratory.

1.1. Features of laboratory equipment

For the biotech research is necessary to have a specialized laboratory, which

is equipped with special facilities and equipment.

The biotechnological laboratory consists of several rooms:

1. The room for glassware wash, here are washer with deep with acid-

resistant sinks, distiller, shelvings for drying of glassware.

2. The room for preparation of nutrient mediums, here are technical,

analytical, torsion balance, рН-meter, a refrigerator, cupboard for keep of clean

glassware, reagents.

3. The room for sterilization of nutrient mediums, tooling, glassware, here

are autoclaves, drying oven with an operating mode 160 - 180°С.

4. Laminar-boxing. Sterility in laminar boxing provides the bacterial filter,

the size of pores 0,2 - 0,4 microns.

5. Light culture room, here are shelvings with glass shelfs, conditioner (the

photoperiod 14-16 hour).

6. Dark culture room.

1.2.Methods of ware, tools, materials sterilization

Sterilization methods of glassware, nutrient mediums, plant material

• Sterilization of laminar-boxing: work surface of laminar-boxing wipes alcohol.

• Sterilization of hands: hands wash soap solution, then wipe 96° alcohol.

• Sterilization of tooling: scissors, tweezers, scalpels sterilize 96º alcohol and roast

on fire of a alcohol-lamp. Then tooling put on support and use for one

manipulation.

5

• Sterilization of glassware: glassware washes by acid method, then wash flowing

water, rinse distilled water

• Sterilization of nutrient mediums: Plant tissue culture media are generally

sterilized by autoclaving at 121 °C, at 1 atmosphere. The time required for

sterilization depends upon the volume of medium in the vessel. The minimum

times required for sterilization of different volumes of medium are listed below.

Sterilization of plant material:

Table 1. Minimum autoclaving time for plant tissue culture medium

Volume of medium per vessel (ml) Minimum autoclaving (min)

25 20

50 25

100 28

250 31

500 35

1000 40

2000 48

4000 63

Preparation of nutrient mediums

The nutrient medium is a major factor of successful cultivation of isolated

bodies, tissues and cells of plants. Complete, plant cell culture medium is usually

made by combining several different components:

• Macroelements

• Microelements

• Amino acids, vitamins

• Regulators of growth

• Saccharose, glucose, fructose

• Agar - agar

Agar-agar is polysaccharide received from sea-weeds.

Macroelements: nitrogen, phosphorus, potassium, calcium, magnesium, iron.

Microelements: boron, zinc, copper, cobalt, manganese, iodine,

molybdenum.

6

1.3.Rules of work in laminar - box

While working in aseptic room observe the following rules:

1. Before the work necessary to seal the doors and windows.

2. Dress sterile gown, cap or bandage.

3. Perform disinfection of hands (rub them 80% ethanol).

4. Wipe 80% ethanol surfaces of tables, closely spaced outlet burner.

5. Place on the table the necessary tools to work.

6. Avoid unnecessary movements of hands with open Petri dish.

7. Through land plant material, utensils kept at an angle to avoid direct contact

with dust.

8. After landing, before closing, the edges of dishes and plugs burn in a flame

burner.

9. Planting is carried out as soon as plant material, reducing to a minimum the

time in which the culture dishes still open.

10. At the end of the tube culture dishes coated cellophane caps. The side faces

Petri dishes glued with adhesive tape.

1.4.Keep of lab - journal

In order to control compilation and analysis of the correctness of the

results should lead laboratory journal. Entries are made in a specific order:

1 - date research;

2 - the object of research;

3 - the conditions of the experiment;

4 - basic methods of research and analysis;

5 - the results (usually drawn in the form of tables, graphs, charts).

7

LABORATORY WORK №2

MOTHER LIQUOR MACROSALTS AND MICROSALTS FOR MS’S

NUTRIENT MEDIUM

Uterine solution by Macrosalts MS where their concentration is increased in

10 times. Salts g/500 ml of uterine solution:

NH4NO3 8,25

KNO3 9,5

CaCl2 x 2H2O 2,2

NaH2PO4 0,85

MgSO4 x 7H2O 1,85

For 1 liter of medium selected 100 ml of uterine solution.

Uterine solution of Fe-chelate for MS: salts, g/200ml of uterine solution:

Na2EDTA x 2H2O 1,492

FeSO4 x 7H2O 1,112


Initial solution of Fe-chelate (200 ml) prepares, dissolves of 1,492 g of

NA2EDTA * 2H2O and 1.112 g of FE2SO4 * 7H2O, and then brings it to a boil.

Uterine solution of microsalts for MS, where their concentration is increased

in 100 times: salts, mg/200ml of uterine solution:

H3BO3 62

Na2MoO4 x 2H2O 2,5

KJ 8,3

MnSO4 x 4H2O 223

ZnSO4 x 7H2O 86

CuSO4 x 5H2O

CoCl2 x 6H2O

weigh up 10 mg of each salt and to

dissolve in 40 ml of water


Uterine solution of vitamins. Vitamins, mg/100 ml of uterine solution:

В1 (thiamine HCl) 10

В6 (pyrodoxine HCl) 50

РР(nicotinic acid) 50

For 1 liter of medium selected 1 ml of uterine solution. Solution with

vitamins (1.0 and 0.1 mg / ml) prepared and directly dissolved in bidistilled water.

8

Solutions with auxins 2,4-D, NAA, IAA, IMA and their analogues prepared by

dissolving 100 mg of the substance in 0.5 - 2 ml of ethanol, and add warm

water to 100 ml.

Kinetin, Zeatin, 6-BAP previously dissolved in a small amount of 0.5 N HCl

and heated at adding the right amount of water.

If you need to add to the nutrient medium abscisic acid (ABA), it dissolved in

70% of ethanol and adjusted to the desired volume.

Gibberellin acid dissolved directly in water and added to the culture media.

Table 2. Uterine solutions for preparates nutrient medium

Initial component Sample Necessary volume for

preparation of 1l of nutrient

medium

By MS By White

Macroelements, g/l

NH4NO3 16,5 –

100ml

KNO3 19.0 0,8

Ca(NO3)24H2O – 2,0

CaCl2 б/в 3,3 –

MgSO4 7H2O 3,7 3,6

KH2PO4 1,7 –

Na2EДТО 0,37 0,37

FeSO4 7H2O 0,28 0,28

KCl – 0,65

NaH2PO4 H2O – 0,165

Na2SO б/в – 2,00

Microelements, mg/100 ml

Н3BO3 620 150

1 ml

MnSO4 4H2O 2230 –

ZnSO4 7H2O – 150

ZnSO4 4H2O 860 –

KJ 83 –

Na2MoO4 2H2O 25 25

CuSO4 5H2O 2,5 4

CoCl2 6H2O 2,5 –

MnCl2 4H2O – 530

Вітаміни, мг/л

Тіамін HCl (В1) 10 10

1ml

Нікотинова к-та (РР) 50 50

Піридоксин HCl (В6) 50 10

9

Plant growth regulators

Plant growth regulators are the critical media components in determining

the developmental pathway of the plant cells. The plant growth regulators used

most commonly are plant hormones or their synthetic analogues.

There are five main classes of plant growth regulator used in plant cell

culture, namely:

• (1) auxins;

• (2) cytokinins;

• (3) gibberellins;

• (4) abscisic acid;

• (5) ethylene.

Table 3. Commonly used auxins, their abbreviation and chemical name

Dissolve 100 mg of matter in 0,5-2 ml of ethanol, warm up, add water to 100

ml (concentration of 1 mg/ml);

Table 4. Commonly used cytokinins, their abbreviation and chemical name

10

Dissolve 100 mg matter in 2 ml 0,5 НС1, warm up, add water to 100 ml

(concentration of 1 mg / ml);

― Gibberellins:

• GA - Gibberellin acid;

• Dissolve 100 mg matter in 100 ml water (concentration of 1 mg/ml);

― Abscisin:

• ABA - Abscisic acid;

• Dissolve in 3 ml of 70 % of ethanol, add water to 100 ml

• Regulators of growth keep in a refrigerator at temperature +4°С

To prepare solutions of vitamins

• Thiamine-HCl (В1), Pyridoxine-HCl (B6) nicotinic acid (РР), ascorbic acid

(С), dissolve in water (concentration of 1 mg/ml) Hydrocarbons and organic

supplements add in medium.

• The scheme of preparation of a nutrient medium: In a volumetric glass add

agar, saccharose, macroelements, microelements, vitamins, regulators of growth,

add distilled water up to the necessary volume and put on a magnetic mixer. The

agar to be dissolved, then add 1н КОН to finish рН medium with the necessary

value. Spill medium at a flask or test-tubes, close.

• Medium sterilize in an autoclave 20 - 25 mines at 1 atm.


PREPARATION OF NON-HORMONAL NUTRIENT MEDIUM

Nutrient medium - a major factor of successful cultivation of isolated organs,

tissues and cells of plants. Nutrient medium should include macrosalts, microsalts,

vitamins, carbohydrates, phytohormones. Some culture media include casein

hydrolyzate, certain amino acids.

Objective of work: To prepare the culture medium of Murasige-Skoog for

further culturing on them isolated tissues, cells and organs of plants.

11

Materials and equipment: macrosalts and microsalts, vitamins, growth

regulators, alcohol, scales, spatulas, tile, laboratory glassware, magnetic mixer,

HCl, KOH.

1. Preparation of nutrient medium - 0.5 liters:

1. In the flask: pour 50 ml of distilled water and add exactly measure of each

solution of macro- and microelements, Fe-chelate and vitamins.

• Weigh the required amount of mesoinozyt, add to the solution.

• Weigh the required amount of sucrose, add to the solution.

• Add distilled water to the required volume - 500 ml.

• Weigh agar-agar and add to the solution.

• pH is set on the pH meter, bring it to the desired value and add (dropwise)

0.1n KOH, 0.1 N NaOH or 0.1 N HCl.

2. Heat the culture medium to dissolve agar within 25-30 minutes.

3. Pour the medium in tubes, cover with foil and carefully wrapped around

the neck of the glass.

4. Sterilization of nutrient medium – in an autoclave under pressure 0.08 -

0.1 MPA (1 atm.), T = 115 - 120 °C for 15-20 minutes.

Table 5. The composition of the nutrient medium:

Elements of

nutrient medium 1 l 0,5 l 0,250 l 0,100 l

Macro MS 100 ml 50 ml 25 ml 10 ml

Micro MS 1 ml 0,5 ml 0,25 ml 0,1 ml

Fe-chelate 5 ml 2,5 ml 1,25 ml 0,5 ml

Mesoinosyte 0,1 g 0,05 g 0,025 g 0,01 g

Vitamins МS 1 ml 0,5 ml 0,25 ml 0,1 ml

Sacharose 30 g 15 g 7,5 g 3 g

Agar-agar 6,8 g 3,4 g 1,7 g 0,68 g

КОН 10-16 drops 8 drops 4 drops 2 drops

12


SEEDS STERILIZATION AND GROWING IN ASEPTIC CONDITIONS

Seeds sterilization and aseptic cultivation of plants

The surfaces of plants are usually contaminated with spores of various

microorganisms and fungi (endogenous and epiphytic microflora, viruses).

However, the internal tissues are sterile, though they may be in non-pathogenic

bacteria that are not always found.

The basic requirement for obtaining callus and suspension cultures is a

sterilization plant facilities. Type of sterilizing agents, concentration and duration

depend on the density and sensitivity of tissue that sterilized.

Standard requirements for sterilizing agents

Before sterilization tissue is pre-cleaned. For this storing organs (roots,

tubers), thick stems of plants, some seeds of woody species:

• thoroughly wash with soap under running water, while pre-flushing by

brush;

• remove the upper epidermis (in roots and tubers);

• washed with distilled water, laminar-box dipped for a few seconds in

absolute alcohol.

The surface of the shoots and leaves of trees rubbed with cotton wool soaked

with ethanol (alcohol), and seed and fruit rinsed with ethanol. Treatment of 70%

ethanol improves performance sterilizing solutions.

In order to more fully wetting the surface pubescent surface sterilizing

solution is added in recent detergents - surfactants, detergents (soap, detergent,

etc.) 4 - 5 drops to 1 liter of water.

Seed sterilization and aseptic cultivation of plants

Depending on the degree of seeds contamination divided into three groups:

- With minor surface contamination by microorganisms (seed wheat, sorghum,

cabbage);

13

- Infection of the outer surface of a seed (lettuce, spinach, radishes, tomatoes,

corn, carrots);

- The presence of microorganisms on the surface and in the middle of seeds (rice,

sunflower, soybean, pine).

Knowing which group belongs seed processing mode chosen seed, but

sterilizing substance, its concentration and the time of sterilization are selected for

each type and grade empirically.

Purpose of work. Select concentration of sterilizing solution and time of

sterilization of seeds that maximize the overall effectiveness of the process.

Receive sterile seeds and grow from it aseptic plants.

Materials and equipment. Soapy solution, 70% ethanol solution, cups with

sterile water, concentrated solution "bleaching powder“, cups for sterilizing

solutions, graduated cylinder, seeds, tubes with non-hormonal nutrient medium for

plants, sterile Petri dishes with a filter paper, sterile Petri dishes, gauze, forceps,

scalpels, alcohol lamp, ethyl alcohol for sterilization of instruments, laminar-box.

Progress of work:

1. Prepare a solution of "whiteness" of various concentrations.

I - 1 part of preparation and 4 of water (1:4)

II - 1 part of preparation and 3 of water (1:3)

III - 1 part of preparation and 2 of water (1:2)

IV - 1 part of preparation and 1 of water (1:1)

Solutions used once immediately after preparation.

2. Wash the seeds in soapy water.

3. In gauze bags puts 10 seeds.

4. Seeds 1 min placed in a beaker with 70% ethanol.

5. By sterile forceps carry bags of seeds in cups with the appropriate concentration

of sterilizing solution and maintain the appropriate time.

6. Sterilized seeds were washed three times for 10 min in sterile water. This sterile

tweezers carry bags of glass with sterilizing solution into a glass with sterile

distilled water. Withstand 10 min. Washing is repeated 3 times, using a new batch

14

of water.

7. Wash bags with seeds by sterile tweezers transferred to a sterile Petri dish with

filter paper.

8. By sterile forceps carry one of the bags to another Petri dish, roll him with

tweezers and roll seeds in a Petri dish.

9. By sterile forceps carry seeds on a nutrient medium in a test tube.

10. Test tubes with seeds placed in a thermostat at 25 ± 1 ° C.

11. After 4 - 6 days check the purity of seed germination.

12. After germination of seeds is transferred to a thermostat with lighting 400 lux

and a temperature of 25 ° ± 1 ° C.

Table 6. For checking the purity of seed germination

Concentration of

"whiteness" in

volume

Duration of

sterilization, min.

Total

quantity of

seeds,

pieces

Quantity of infected

seeds after 7 days

Germination of

seeds Effectiveness

of

sterilization

(%) pieces % pieces %

1:4

10

15

30

1:3

10

15

30

1:2

10

15

30

1:1

10

15

30

15


STERILIZATION OF COURGETTES AND RECEIVING ASEPTIC

CULTURE OF PLANTS

Purpose of work. Select concentration of sterilizing solution and time of

sterilization of seeds that maximize the overall effectiveness of the process.

Receive sterile seeds and grow from it aseptic plants.

Materials and equipment. Soapy solution, 70% ethanol solution, cups with

sterile water, concentrated solution "bleaching powder“, cups for sterilizing

solutions, graduated cylinder, seeds, tubes with non-hormonal nutrient medium for

plants, sterile Petri dishes with a filter paper, sterile Petri dishes, gauze, forceps,

scalpels, alcohol lamp, ethyl alcohol for sterilization of instruments, laminar-box.

Progress of work:

1. Prepare a solution of "whiteness" of various concentrations.

1 part of preparation and 2 of water (1:2)

Solutions used once immediately after preparation.

2. Wash the seeds in soapy water.

3. In gauze bags puts 10 seeds.

4. Seeds 1 min placed in a beaker with 70% ethanol.

5. By sterile forceps carry bags of seeds in cups with the appropriate concentration

of sterilizing solution and maintain the appropriate time.

6. Sterilized seeds were washed three times for 10 min in sterile water. This sterile

tweezers carry bags of glass with sterilizing solution into a glass with sterile

distilled water. Withstand 10 min. Washing is repeated 3 times, using a new batch

of water.

7. Wash bags with seeds by sterile tweezers transferred to a sterile Petri dish with

filter paper.

8. By sterile forceps carry one of the bags to another Petri dish, roll him with

tweezers and roll seeds in a Petri dish.

9. By sterile forceps carry seeds on a nutrient medium in a test tube.

10. Test tubes with seeds placed in a thermostat at 25 ± 1 ° C.

16

11. After 4 - 6 days check the purity of seed germination.

12. After germination of seeds is transferred to a thermostat with lighting 400 lux

and a temperature of 25 ± 1 ° C.

Table 7. For checking the purity of seed germination

Concentration

of "whiteness"

in volume

Duration of

sterilization,

min.

Total

quantity of

seeds,

pieces

Quantity of

infected seeds

after 7 days

Germination

of seeds

Effectivenes

s of

sterilization

(%) pieces % pieces %

1:2

10

15

30


PREPARATION OF NUTRIENT MEDIUM FOR CALLUS

Objective of work: To prepare the culture medium of Murasige-Skoog for

further culturing on them isolated tissues, cells and organs of plants for receiving

primary callus.

Materials and equipment: macrosalts and microsalts, vitamins, growth

regulators, alcohol, scales, spatulas, tile, laboratory glassware, magnetic mixer,

HCl, KOH.

Preparation of nutrient medium

Progress of work:

1. In the flask: pour 50 ml of distilled water and add exactly measure of each

solution of macro- and microelements, Fe-chelate and vitamins.

• Weigh the required amount of mesoinozyt, add to the solution.

• Weigh the required amount of sucrose, add to the solution.

• Add distilled water to the required volume - 500 ml.

• Weigh agar-agar and add to the solution.

• pH is set on the pH meter, bring it to the desired value and add (dropwise)

0.1n KOH, 0.1 N NaOH or 0.1 N HCl.

2. Heat the culture medium to dissolve agar within 25-30 minutes.

17

3. Pour the medium in tubes, cover with foil and carefully wrapped around

the neck of the glass.

4. Sterilization of nutrient medium – in an autoclave under pressure 0.08 -

0.1 MPA (1 atm.), T = 115 - 120 °C for 15-20 minutes.

Table 8. The composition of the nutrient medium

Elements 1 l

Macro MC 100 ml

Micro MC 1 ml

Fe-chelate 5 ml

Inositol 0,1 g

White’s vitamins

(nicotinic acid

0,05 g, tiamine

0,01 g for 100 ml)

1 ml

2,4 D 3 ml

Saccharose 30 g

Agar 6,8 g

КОН 10-16 drops

Callus cells differ by:

• intensity of growth,

• consistency

• color

• the ability to green at light and other properties.

Cell colonies in agar medium can be compact and firm, and plump that takes

in callus and break into separate pieces.

The last type of callus in liquid medium easily separated single cells and

gives rise to suspension culture. It is noted that the compact callus may give rise to

loose tissue, but not conversely.

Consistency of callus depends largely on the composition of the medium.

Anatomical structure is characterized by loose of many organized centers of

meristematic activities separated by large undifferentiated cells.

18

Thick callus less differentiated and contain many large vacuolised cells.

There is a difference between these types of callus and cell packing: thick callus

composed of tightly adjacent to each cell.


RECEIVING PRIMARY CALLUS FROM DIFFERENT EXPLANTS OF

ASEPTIC PLANTS

Receiving primary callus from different explants of aseptic plants

Purpose of work. Learn to chosen tissue explants and culturing conditions to

induce dedifferentiation and formation of callus.

Materials and equipment. Plants of different species, soap solution,

concentrated solution "whiteness", glasses with sterilizing solutions, graduated

cylinder, sterile distilled water, test tubes with nutrient medium, Petri dishes with

sterile filter paper, sterile Petri dishes, nylon, forceps, scalpels, alcohol, laminar

box.

Progress of work:

Explants are aseptic plants derived from seeds under in vitro (tobacco, pea,

carnation, sunflower, tomato).

1. Sterile forceps removed the plant from the tube and placed in a sterile Petri

dish.

2. Hold the tweezers, by sterile scalpel cutting the plant explants: cuttings, leaf

plates, fragments of stems, roots. The leaves make additional cuts in the

central vein.

3. Explants cultivated on callus medium in a bottle that sign with the name of

the type explants (petiole, leaf, stem, root).

4. Cultured in the dark at +25 - 26,5 °C. They spend subcultivation every 3 - 4

weeks and determine the frequency of callusgenesis as the number of

explants with callus to the total planted explants as a percentage. The data

recorded in the table.

+

19

Table 9. Testing of environments for induction of leaf callus explants

Nu

trie

nt

med

ia

Ty

pes

of

exp

lan

tats

Qu

anti

ty o

f

exp

lan

ts,

pie

ces

Dat

e of

pla

nti

ng

Dat

e of

ind

uct

ion

Per

cen

tag

e of

call

usg

enes

is

Co

nd

itio

n o

f

call

us*

* State of callus estimate as follows:

+ + + – white viable callus, growing;

+ + - – callus in good condition, yellow;

+ - - – flabby, dark yellow callus;

- - - – dark brown, viable callus.


SUBCULTIVATION OF PRIMARY CALLUS TO INCREASE ITS MASS

After the formation of callus aseptically separated and placed it on the

surface of fresh culture medium. As a result of receiving a sterile culture of callus

tissue, which can be maintained for a long time, occasionally dividing it into

fragments. Callus subcultivated on medium of the same composition as for

dedifferentiation or in medium with a reduced concentration of auxin to stimulate

the growth of callus mass.

Progress of work:

1. Explants with primary callus transferred to a sterile Petri dish.

2. Hold explants by tweezers, cut callus by scalpel.

3. By scalpel share callus tissue level fragments and transferred to fresh

culture medium.

4. Signs Petri dish and close by parafilm.

5. Petri dish cultured in an incubator at 25 ± 1 ° C.

6. After 4 - 6 weeks callus tissue is divided into equal pieces and then

transferred to fresh culture medium.

20


GETTING OF CALLUS CULTURE OF PLANT TISSUES

Purpose of work. Get callus culture of plant tissues.

Progress of work:

1. Conduct preliminary training of laminar-box to work, which involves

sterilizing by ultraviolet light for 30 min., followed by wiping work surfaces by

sterilizing solution (70 - 96% ethanol).

2. The basic requirement for operations is adherence to sterility, as in an oven

pre maintain tools and utensils for 2 - 2.5 hours at a temperature 180 - 210 °C or

sterilized by autoclaving for 25 min. at 0.1 - 0.12 MPa.

3. Immediately before working tools dipped in ethanol and burn surfaces in

alcohol lamp.

4. The starting material (any parts of plants) thoroughly washed in soapy water,

then rinse lightly pink solution of KMnO4 (potassium permanganate) and

transferred to a sterile working box.

5. By sterile instruments cut raw material into pieces of 2 - 3 cm and placed in

a pre- sterilized gauze bags for further immersion in the capacity of sterilizing

solution (a solution of mercuric chloride, bleach, ethanol).

Table 10. Exposure to sterilizing solutions

Object Solution

70% ethanol Diacyd

Seeds 1 – 2 min 15 – 30 min

Leaf 20 s 6 min

6. The starting material was washed several times with sterile distilled water to

wash off sterilizing solution.

7. Sterile plant material is divided into segments and by sterile instruments

cutting into pieces of 2 x 2 x 2 mm or 4 x 4 x 4 mm.

8. Used instruments lowered into a glass with alcohol and burn in a flame of

alcohol lamp, then placed between the pages of sterile thick paper.

21

9. Source plant material (explants) are exposed on the surface of agar medium

in test tubes, flasks or Petri dish and placed in a growth chamber (in an

incubator). Grown in the dark at a temperature of 23 - 28 °C.

10. After 3 - 4 weeks on the cut surface marked the formation of primary callus

tissue. Preparation of callus tissue depends on the type of plant and tissue. The

cells of plants that are in the final stages of differentiation under the influence

of inducers of cell division - cytokinins and auxins, moving in dedifferentional

status and reduced meristematic activity. After the formation of primary callus

under aseptic conditions it is separated from the explants and transplanted to a

new agar nutrient medium.


PREPARATION OF PLANT REGENERANTS THROUGH DIRECT AND

INDIRECT MORPHOGENESIS

Explants are aseptic tobacco plants.

Progress of work:

1. Puff plate damaged by cutting central vein cultured on MC medium with the

addition of 2,4-D at a concentration of 4 mg / L in the dark at a temperature of 25 -

27 ° C.

2. After 7 - 10 days cell explants to form callus.

3. Clip morphogenic callus and cultivated it on MC medium with the addition

of 6-BAP at a concentration of 1 mg / l.

4. After 10 days produced optimally formed shoots.

5. For rhizogenesis - shoots transferred to culture medium without growth

regulators and inositol.

NATIONAL UNIVERSITY OF LIFE AND ENVIRONMENTAL …

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