Procedures for Recovering Cassava and Sweet potato Germplasm

Distributed In vitro

Roca, W.M., Rodriguez. J.A and Roa. J

Genetic Resource Unit CIAT Apartado Aéreo 6713 Call, Colombia S.A

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 Table of Contents

Introduction 1

Recovery of the Cultures 1

Micropropagation

Potting

Field Transplantation

Green house transplantation

Appendix 1. Preparation of the Culture Medium

Appendix 1a. Preparation of Murashige and Skoog Stock Solutions

Appendix 1b. Preparation of Growth Regulator Stock Solutions

References

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1.0 Introduction
Invitro methods have been developed at CIAT for the international exchange of cassava (Manihot esculenta Crantz)
clones. Sterile cultures in artificial nutritive media are established from disease-free mother plants produced by
means of thermotherapy and meristem-tip culture and tested for known cassava viral, bacterial, and fungal
pathogens. The cultures for shipment consist of well-rooted plantlets in an agar medium contained in properly
capped1 6 x 125 mm test tubes. The test tubes, in turn are packed within polystyrene boxes.
The tubes are labeled with the clone’s name or number, a phytosanitary certificate, issued by the Colombian
authorities, and a phytosanitary statement which provides supplementary information regarding the health status of
the cultures, are included in the package.

Several countries have accepted the invitro system as a means to improve the phytosanitry aspects of the
international exchange of cassava germplasm. In the last few years, the technique has been used not only to
distribute selected germplasm from CIAT to national programs, but also to introduce into CIAT large numbers of
new germplasm collected in the crop’s major centers of variability. Furthermore, because of their small size and
their disease-free condition and high propagation potential, meristem-derived cultures have been used at CIAT to
develop an in vitro gene bank of cassava.

The in vitro system is only effective as a tool for germplasm exchange. If it is managed by well-trained personnel,
these guidelines are provided to acquaint the personnel of national programs and other collaborators with the
procedures to recover, propagate, and transplant materials from the cultures after arrival at the recipient institution.

1.1 Recovery of the Cultures

It is important to make the necessary arrangements to expedite clearance of the package through customs as soon as
it has arrived. Recipients will be advised on the approximate arrival date of the shipment.
1. Upon their arrival, unpack the test tubes carefully and place them on test tube supports, in an upright
position.
2. After short trips top to 1 week, the planlets can be potted directly (section 4) or can be micropropagated
prior to potting (section 3) if you have the facilities and personnel
3. If the shipment has taken 1-2 weeks to arrive at its destination, you should expose the cultures to an
Illumination of 2000 in two 40 W fluorescent lamps at 0.50 m above the cultures and a temperature of 26-
28°C for 1 week. This will allow the plants to recover from the detrimental effects of darkness. After this
recovery treatment, the cultures are ready for direct potting (section 3) or propagation and potting (section
2)
4. After longer trips (more than 3 weeks) and depending on genotype, etiolation and chlorosis, and sometimes
tissue necrosis due to phenolic oxidation, can occur with variable intensity due to the lack of light. You can
save extremely deteriorated cultures. If any available green bud (terminal or auxiliary) is aseptically
excised and cultured on a fresh medium (step B). Immediately upon unpacking, expose less damaged
cultures especially those with little or no shoot browning to temperatures of 22-24°C and 1000 lox of
illumination for 2 weeks. Even a slight growth of auxiliary buds, with or without leaf growth, is sufficient
to proceed to the micropropagation step.
1.2 Micropropagation
Micropropagation is carried out within a laminar flow cabinet or in a transfer room for aseptic work. Make sure to
have on hand:
• two No.10 scalpels, with their handles
• two forceps: one short (12cm) and one long (25 cm)
• several petri dishes containing 3-4 sterilized filter papers
• 2-3 flasks containing sterile distilled water
• an alcohol burner
• cotton wetted with 70% alcohol
• 2-3 flaps (10 x 20 cm each) of sterile filter paper

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 • a container with 95% ethanol
The culture medium includes the mineral salts of Murashige and Skoog + 2% sucrose + 1 mg/1 thiamine HCl = 100
mg/l Inositol + 0.05 mg/I benzyl aminopurine + 0.05 mg/I gibberellic acid + 0.02 mg/l naphathalene acetic acid; Ph
5.7-5.8; agar = 0.6%. The preparation of this medium is described in Appendix 1

Figure 1 micropropagation of cassava through nodal cuttings.

1.2.1 Procedures for micropropagation

1. Wash hands and arms with soap and running water and wipe with 70% alcohol. If possible, cover the hair
with a microbial cap and the mouth and nose with a microbial mask
2. Immerse the tools in 95% alcohol for 2-3 minutes; flame quickly, and place them under the sterile flaps of
paper.
3. Open one petri dish and wet the filter papers with the sterile water taking care to flame the flask. One at a
time, uncap 2-3 test tubes (Figure 1-a), and using short forceps, gently pull the plantlet from the tube. Place
all planlets within the petri dish (figure 1-b)
4. Using a scalped and the short forceps cut off all expanded leaves and roots. Then, cut the stem into
segments, each one comprising one node (Figure 1-e). Every segment or “nodal cutting” will then include
one auxiliary bud and a portion of stem (1-2 mm above and 4-5 mm below the bud, respectively). In
addition, cut the terminal bud from every shoot, leaving 4-5 mm of storm below the apex. Make sure to
make clean, horizontal cuts with the scalpel.
5. Using the larger forceps, gently pick up each “nodal cutting” and each terminal bud, and “ plant” each one
in agar solidified medium (Figure 1-d) in an 18-25 = X 150 MM TEST TUBE. Quickly flame the tube and
cap it immediately
During the operation, make sure to frequently wet the tools with 95% alcohol and flame them. Keep the
petri dish closed as much as possible.
6. Incubate the “node cutting’ cultures at 28°C under an illumination of 2000 lux until root initiation and,
thereafter, under 4000 lux, and maintain a 14 hour photoperiod throughout.
After about 3 weeks, every modal cutting will give rise to a complete planlet (Fig 1-e). Once, the plantlet
has grown to 6-8 cm tall and rooting is proportional to the amount of shoots, the cultures can be subjected
to a hardening treatment prior to potting. Hardening is not absolutely necessary, but it will improve the
potting.
7. For hardening, increase the illumination to 8,000-10,000 lux and, if possible, decrease the temperature to
24-25°C during 1-2 weeks
8. Three to four days prior to potting, take the paraffinated paper off the caps, whether or not you have
hardened the cultures.

1.3 Potting
For potting, the following items are needed:

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 • Pots, Optimal potting is achieved using 3 to 4 inch jiffy-type pots, but clay or plastic bags 96.5 x 8
inches) can also be used
• Substratum, which consists of a mixture of one part [soil with three parts of fine sand. The sand must
be thoroughly washed with soft water.
• A fair amount of deionized water
• Long (15-20 cm) forceps
• Pot labels
• A water –soluble fertilizer rich in P; e.g. 10-52-10 (N-P-K)
• Plastic, clay or cement trays with sufficient draining holes, containing damped sterilized soil.
• A 2.0 X 10- X 0.3 –m transparent plastic frame to cover the trays
• A 2.0 X 1.0- X 0.8-m greenhouse table to hold the trays and the plastic frame
• The two or three tables of similar dimensions to carry out the potting and to hold the pots prior to field
transplanting
• Styrofoam cup
• You should do the potting in a fresh, but not too cold location that is protected from direct sun and
from insects.

1. A few hours prior to potting, damp with water the soil contained in the trays, place the trays on the
greenhouse table and cover them with the plastic frame (Fig 2). A high relative humidity environment
will form within the plastic chamber
2. Sterilize the substratum mixture using steam and fill in the pots. Wet the substratum to about one half
water saturation. Make a hole in the center of each pot (Fig 3)
3. Wash your hands thoroughly with soap and water. Uncap the test tubes, one at a time, and, with the aid
of the forceps, gently pull out the planlet (Fig 4). Using the fingers, pull the plant from the tube mouth.
4. Holding the plant on the palm of your hand, wash off as much agar as possible from the roots (Fig 5),
and place the roots and the lower part of the shoot within the hole in the pot (Fig 6). Press the
substratum and immediately apply deionized water around the plant (Fig 7)
5. After potting label the posts and water with the high P fertilizer 10-52-10. Use 2g fertilizer in 1 litre of
water and apply 80-100 ml solution per pot. Thereafter, when needed and until the time of field
transplanting, water with only the high P solution.
6. Place the pots in the high humidity chamber (Fig 8). Five or six days after potting and thereafter raise
the plastic frame slightly during the fresher hours of the day, close the frame at night. By day 10 or 12,
the plastic frame can be taken off completely.
7. After the 12th day, move the pots (still on the trays) to a warmer and much more illuminated part of the
greenhouse. In about 2 more weeks, the plants are ready for field transplanting.
The total time taken from receipt to this stage usually is 1 or 2 months depending on whether direct potting or
micropopagation and potting were carried out respectively.
1.4 Transplanting in the greenhouse
MATERIALS
• Black plastic bags for planting

• Sterile planting media sand/soil ratio of 3/1

• Transparent plastic cups

• Labels

• A greenhouse with temperature between 26 and 28 degrees for keeping only clean material

• In vitro plants in 17N media (enraizamiento) that are 6-8 cm long with maximum of 30 days after
sub-culturing

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 1. Prepare planting media a week before transplanting in the following way: - wash the sand 3 times
until it is totally clean, put to dry in the sun, sieve soil, mix and sterilize for 3 hours.

2. The plants should be in the screen-house for a minimum of 2 days before transplanting for them to
adopt.

3. Fill the bags for planting one day before

4. On planting day add to the planting media a solution of Banrot 0.75gr/lt, or a fungicide containing
active ingredients against mushrooms (Phytophthora, Phytium) bacteria. This should be prepared with
deionized or sterile distilled water.

5. A lot of care should be taken not to damage the roots, add little water to the flash to help loosen the
agar for the plants to be removed easily. Make a hole in the planting media and insert the plant.

6. A flask contains 5 plants, each is placed in a different bag, with its respective label, and a cup is
placed over it to increase humidity to protect it for the next 8 days. This cup should preferable be
transparent to allow in light.

7. After transplanting the material should be inspected daily, without removing the plastic cups to avoid
dehydration, and watered only when necessary.

8. Remove the cups after 8 days and water if necessary using deionized water and remove dead leaves
and initiate fertilizer application.

1.4.1 FERTILIZER APPLICATION

Fertilizer application is very important because it gives to the plants nutrients that it needs for its growth, due to this
the fertilizers are in two groups according to its functionality.
1. Fertilizers with micro nutrients that the plant needs in its initial stage: Plan/pro (plantex), cosmocel,
coljap development and coljap production each one of them is used in a concentration of 0.5%, The
major micro nutrients the fertilizers have to have are Calcium, Zinc, Magnesium, Sodium, Manganese
and Cobalt.

2. The fertilizers with macro nutrients that the plant needs at latter stage: N/P/K and the urea (little it is
used) one of them is used to a concentration of 1% with Nitrogen as a major component for foliar
growth

Fertilizer application is carried out every 8 days, alternating the fertilizer and precious amount of 50ml
should be done I the morning 8-10.00 am or later in the day 5-6.00 pm when it is cool so that the plants can
assimilate most of the fertilizer.

• First application should be done 15 days after transplanting and should be applied to the soil.

• After apply every 8 days. Plantex/Plan-pro (apply in the soil)

• Then coljap development (apply in the foliage)

• Coljap production (apply in the foliage)

• Cosmocel (apply in the soil)

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 • Triple 15 (N/P/K) (apply in the soil)

• Urea (apply in the soil)

NOTE;
Sometimes it is necessary to apply fungicides and pesticides to prevent fungal and/or pests. Apply 0.7 ml/l fungicide
or 1 ml/l of acaricide. These should not be done simultaneous with the fertilizations as the plant may get burn
because of excess chemical
1.5 Field Transplantation
Successful transplanting can be achieved under cloudy days or during the late afternoon. The soil should be in field
capacity and the plants 10-15 cm in height. Use jiffy-type pots or plastics bags and keep root disturbance minimum
1. Carry the plants in their pots, along with the trays, to the field and cut off the largest leaves
2. Remove the pots from the tray and place them within the hole large enough to hold the pot and up to
the loved two nodes of the shoot
3. Press the soil around the plant and water immediately
4. Maintain high soil humidity for 10-15 after transplanting, and watch out for ant, worm or cricket
attacks.
After planting, cover each plant with a Styrofoam cup (Make sure to make some openings on the upper side of the
cup). Leave the plants covered or 1 week, then uncover them once continue with stop
At the fourth week, the plants should be completely established in the field and can be handled conventionally.
However, it is recommended that you pay special attention to protecting the plants from insects or disease
throughout the crop. Keep in mind that these plants are elite stocks for the multiplication of clean planting material
and that their production shipment, and handling after arrival have been done with great care and effort
For news regarding the condition of the cultures upon arrival, any progress developments in handling or additional
information, please write to

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APPENDIX 1
Preparation of the Culture Medium
A) Basal Medium.
Can be prepared in either of two forms:

1. Using stock solutions of mineral salts, vitamin and growth regulators. For the preparations of the
Murashige and Skoog stock solutions see (Table 1)
To prepare 1 liter of medium, to 500 ml of double distilled water add:
20.0 ml of stock solution No.1
1.0 ml of stock solution No.2
1.0 ml of stock solution No.3
2.9 ml of stock solution No.4
5.0 ml of stock solution No.5

2. Using the pre-made Murashige and Skoog medium in powder form (without sucrose and without vitamins
and agar). Each bag contains 4.3g of powder, which serves to prepare 1 litre of basal medium. The powder
can be stored at 8-10°C or under desiccation, for up to 2 years. To prepare the basal medium dissolve the
entire contents of one bag in 500 ml double distilled water. Add the same volumes of stock solutions No.1
through No.5 as in Table 1.

B). Supplements.
Once either of the basal media is ready, proceed as follows:
• Add 5.0 ml of stock solution No.6 and 6.25 ml of stock solution No.7
• Dissolve 20.0 g of sucrose
• Add 5.0 ml of the benzyl aminopurine (BAP) stock solution and 2.0 ml of Naphthalene acetic acid
(NAA) stock solution
• Complete to 700ml with double distilled water
• Adjust the pΗ to 5.7-5.8
• Dissolve by heating 6.0 g of agar in 300ml of double distilled water
• Mix well the medium with the agar solution.
C. STERILIZATION
Quickly distribute the prepared medium in 18-X.150-mm test tubes (5ml/tubes); let cool slightly and cap the
tubes
Autoclave the tubes containing medium; 15 pounds (121°C) per square inch during 15 minutes; decompress
slowly
Place the tubes in a fresh place until the agar solidify, then store them in darkness at 6-8°C until used.

Appendix 1a. Preparation of Murashige and Skoog Stock Solutions

To prepare the stock solutions, dissolve one by one, all the ingredients presented in Table 1, in the volumes of
double distilled water shown.

Appendix 1b. Preparation of Growth Regulator Stock Solutions

Benzyl aminopurine (BAP)(10 ppm):
Dissolve 20 mg in a small volume of 1.0 N HCl; complete to 200 ml with double distilled water (This is a 100-
ppm solution of the growth regulator): Take 20 ml of the 100-ppm solution and complete to 200 ml (This is the
10-ppm stock solutions).

Gibberellic acid (GA3) (10 ppm):

Dissolve 22 mg (90% gibberellic acid) in a small volume of 1.0 N KOH; complete to 200 ml with water; take
20 ml of this solution and complete to 200 ml with water.

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Naphathalene acetic acid (NAA) (10 ppm):
Dissolve 20 mg in a small volume of 1.0 N KOH, complete to 200 ml with water; take 20 ml of this solution
and complete to 200 ml with water

Table 1. Murashige and Skoog Stock and Medium Preparation.

Stock Constituents Volume of stock per 1 liter basal

solution Substance Amount medium
no.a
1 NH₄NO₃ 82.5 g 20.0 ml
KNO₃ 95.0 g
MgSO₄. 7H₂O 18.5 g
KH₂PO₄ 8.5 g
Dissolve in 1000 ml water
2 H₂BO₃ 0.62 g 1.0 ml
MnSO₄ . H₂O 2.176g
ZnSO₄ . 7H₂O 0.86 g
Na₂MoO₄ . 2H₂O 0.025 g
CuSO₄ . 5H₂O 0.0025 g
CoCl₂ . 6H₂O 0.0021 g
Dissolve in 100 ml water
3 Kl 0.075 g 1.0 ml
Dissolve in 100 ml water
4 CaCl . 2H₂O 15 g 2.9 ml
Dissolve in 100 ml water
5b a) Na₂EDTA 1.492 mg 5.0 ml
b) FeSO₄ . 7H₂O 1.114 mg
Dissolve in 200 ml water
6 Thiamine-HCl 0.8 g 6.25 ml
Dissolve in 200 ml water
7 m-inositol 0.8 g 6.25 ml
Dissolve in 200 ml water

a. Stocks 2 and 6 should be kept frozen; all the others at 8-10°C. Keep stock 5 protected from light

b. Separately dissolve ‘a’ and ‘b’ in 50 ml with water; take 20 ml of this solution and complete to 200 ml with
water.

Addition of Growth Regulator Stocks to the Medium: To determine the volume (Appendix 1) of each growth
regulator stock solution necessary to obtain the prescribed concentrations (step B), apply the following
formulation:

C₁V₁ = C₂V₂
C₁ = Concentration of stock – 10 mg/l
C₂ = Final concentration of growth regulator in the medium
Benzyl aminopurine = 0.05 mg/l
Gibberellic acid = 0.05 mg/l
Naphthalene acetic acid = 0.02 mg/l
V₁ = Volume (in ml) of stick solutions needed = X
V₂ = Final volume of medium = 1000 ml

X= 0.05mg/l x 1000ml = 5.0 ml of either BAP/GA3

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 10mg/l

References

1. CIAT (Centro International de Agricultura Tropical). 1983. Elite Cassava germplasm from CIAT. Cali,
Colombia.20 p

2. Hewitt, W.B and Chiarappa, L (eds) 1977. Plant health and quarantine in the international transfer of
genetic resources. CRS Press, Cleveland, Ohio.

3. IBPGR (I international Board for Plant Genetic Resources) 1983a. Genetic resources of cassava and
wild relatives. IBPGR Secretariat, Rome, 56 p.

4. 1983b. Practical constraints affecting the collection and exchange of wild species and primitive
cultivars. IBPGR Secretariat, Rome, 11 p.

5. Lozano, J.C; Belloti, A. Reyes, J.A; Howeler, R; Leihner, D; and Doll, J. 1981. Field problems in
cassava. Centro Internacional de Agricultura Tropical, Cali, Colombia. 192 p.

6. Murashige, T and Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue
cultures. Physiol. Plant. 15:473-497.

7. Roca, W.M. In press. Cassava. In: D.A Evans; W.R. Sharp; P.V. Ammirato; and Y. Yamada (eds),
Handbook of plant cell culture. Vol. 2: Crop species. MacMillan, New York

8. Schilde, L and Roca, W.M.In Press. Pathogen elimination in potato and cassava. In: J. Cock (ed),
Propagation of tuner root crops. Centro Internacional de Agricultura Tropical, Cali, Colombia.

9. Tery, E.R 1982. A review of cassava and sweet potato diseases and their relation to germplasm
exchange. Research Briefs, Vol.3 International Institue of Tropical AGRICULTURE, Ibadan, Nigeria,
4p

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 Micropropagation and Maintenance

Rolando Lizárraga, Ana Panta, Nelson Espinoza,John Dodds

(Reproduced from CIP Research Guide 32)

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 Table of Contents

Introduction 1

Advantages of tissue culture 1

Introduction of in vivo material into Invitro
Meristem isolation cultures

Thermotherapy

Field Transplantation

Green house transplantation

Appendix 1. Preparation of the Culture Medium

Appendix 1a. Preparation of Murashige and Skoog Stock Solutions

Appendix 1b. Preparation of Growth Regulator Stock Solutions

References

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Tissue culture allows the rapid clonal propagation of a large number of plantlets over a short
period, as well as the maintenance of germplasm under controlled conditions in small
spaces and with reduced labor requirements.

This document complies the advances, methodologies and materials used for tissue culture
at the various institution including International Potato Center (CIP), Mikocheni Agricultural
research Institute etc. It analyzes isolation, micropropagation, and long term storage
techniques.

Advantages of tissue culture

CIP maintains a sweet potato germplasm collection of over 5,000 accessions. Their clonal
maintenance on the field is expensive and involves the risk of loss due to infectious diseases
or unfavorable climatic conditions. Thus, in vitro maintenance presents the following
advantages:

• Lower labor costs,

• Absence of field infections,
• Protection against unfavorable climatic conditions,
• Timely access to material under maintenance,
• Timely access to material for pathogen clean-up,
• Permanent availability of (when pathogen tested) material for propagation and
exportation.

Introduction of in vivo material into in vitro

The explants from the mother plant must come from the greenhouse. Alternatively mature
plants directly from the fields normally are very dirty and are difficulty to thoroughly cleaned
them, read for initiation. They can be re-established in the green house for 2-3 month with
frequent spraying with insecticides and acaricide to eliminate the ectoparacites such as
aphides white flies or mites. Plantlets of 2-3 months old, which are in excellent health
conditions, and free of lateral buds that are, too sprout are best for initiation. The stems are
excised from the mother plant and the leaves removed. Part of the petiole should be left to
cover the buds. Stem cuttings 2 to 3 cm long each must include an axillar bud and a portion
of the internodes behind the bud for ease of manipulation.

Presurface sterilization

Before sending them to the in vitro laboratory, the stem cuttings are treated with a wide
spectrum acaricide to destroy the mites at any stage of development. This can be achieved
by any commercial acaricide for few minutes. Then acaricide is rinsed away by washing the
stems in running water for 30-45 minutes. They are then placed in a clean bottle/beaker
covered with a Petri dish/aluminum foil until starting surface disinfection.

To start surface disinfection of the stem cuttings, remove the water from the container, add
96% ethanol and let stand for two seconds. Discard the alcohol and immediately add a 2.5%
solution of calcium hypochlorite (brought to pH 8 by addition of HCl). Sodium hypochlorite or
chlorine (bleach) may also be used. If possible, add a few drops of a dispersing-adhering
agent such as Tween 20 or 80 (4 drops/l of solution). The bottle is then placed in the laminar
flow transfer chamber. After 15 minutes under sterile conditions, the hypochlorite is
eliminated, by washing three times with sterile water. To reduce phenolization of the

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explants, after the last rinse they are left in a sterile 100 ppm solution of ascorbic acid
before proceeding to excision. Blot the explants into a filter paper to dry off the hypochlorite
solution.

Under these conditions, proceed to excise the buds by eliminating the largest possible
number of leaflets and leaf primordia. The excised portions should be as small as possible.
The optimum size is 0.6 mm but the explant may be bigger if no adequate excision
instruments, such as a stereoscopic microscope, are available.

The buds/shoot tips are initiate in initiation media and sealed with parafilm and incubate in
the growth room with required environment for 4 weeks. In the first propagation stage, 16 x
125 mm culture vessels are used. In the second stage, either 18 x 150 mm or 25 x 150 mm
tubes may be utilized.

In a situation where cultures are initiated using meristem, Due to the fact that meristematic
portions larger than 0.6 mm are used, saprophytic bacteria mainly may contaminate some
material. If so, proceed in either of two ways.

1. If a stereoscopic microscope is available, excise meristems between 0.4 and 0.6 mm

long, plant them in the introduction medium (MMB-I) and transfer them weekly to the
MMB-II medium.
2. Eradication of bacteria or yeasts may be attempted by adding antibiotics to the
medium. For bacteria it is recommended to use Rifampicine (Rimactan 300 CIBA) at
400 ppm.

A concentrated (12,000 ppm), filter-sterilized solution of Rifampicine is used to soak small (5

x 5 mm) squares of filter paper, which are allowed to dry in the laminar flow chamber.
Approximately 0.03 cm3 of concentrate solution is used for every square of filter paper. They
are best if used before seven days for they progressively lose they effectiveness. This
process must be carried out under aseptic conditions. The paper squares are introduced into
the culture medium with the planted bud, which must be transferred to a fresh medium with
another antibiotic paper every 3 to 5 days. Other antibiotics such as Cefotixine (Mefoxin
Merck) in doses of 500 ppm may also be used.

For contamination by yeast it is advisable to use 0.25 to 0.5 ppm doses of Amphotericine B.
The filter paper procedure described above will be used in all instances.

1. Meristem isolation and culture

The meristem is a tissue made of cells under division and is the active growth point of the
bud. The dome of the bud contains the meristematic cells and is surrounded by foliar
primordia and primary leaves. The meristematic cells divide and form new tissue. Nutrition
of the dissected section is provided by the artificial medium. The isolation of the
meristematic zone under aseptic conditions and its culture in an adequate nutritive medium
allow plantlet development with a differentiation pattern similar to that of a normal plant.
The aseptic dissection of the meristem for virus eradication is a delicate process requiring
skill.

Photo 1 shows a photographic sequence of the dissection procedure.

14
 QuickTimeª and a
decompressor
are needed to see this picture.

1.
2.
3. The plant stems are cut in segments including a node and the corresponding axillar
bud. The material is deeped into a disinfectant and sodium hypochlorite as for the
initiation of invitro cultures.

15
 4. After rinsing in distilled sterile water, place the material under the dissection
microscope and use a needle or surgical knife to remove the leaves around the
growth point until only the cupule and two or three foliar primordia are left.
5. The cupule and foliar primordia are dissected with the surgical blade and transferred
to culture medium MMB-I. The dissected meristem is transferred weekly to a fresh
MMB-II medium. After 6 to 8 weeks the meristem will develop into a plantlet. The
plantlets are now ready for subculture in the propagation medium (see section on
Culture Media).

2. Thermotherapy

At CIP before excision of the meristems, the plants undergo a month thermotherapy at 38
C for 16 hours and at 32 C for eight hours under constant light conditions. This high
temperature treatment has increased the efficiency of the production process of virus free
material. After thermotherapy either axillar or apical meristems may be used indistinctly.

QuickTimeª and a
decompressor
are needed to see this picture.

Photo 1. Photographic sequence of meristem dissection:

A: Isolated and disinfected apical bud

B: Dissection after removal of primary leaves
C: Meristem with two foliar primordia

Micropropagation

The purpose of micropropagation is to obtain a large number of clonal plants in a short time.
The following methods are used at CIP:

Propagation by nodes

This is based on the principle that the node of an in vitro plantlet placed in an appropriate
culture medium will induce the development of the axillar bud resulting in a new in vitro
plantlet. It must be noted that this type of propagation is based on the development of a
pre-existing morphological structure. The nutritional-hormonal condition of the medium
plays a simple role in breaking the dormancy of the axillar bud and promoting its rapid
development. The propagation medium described in the section on Culture Media is used.

Callus formation and plant regeneration must be carefully avoided because they tend to
affect the genetic stability of the genotype.

16
The plantlets grow under long day conditions (16 hours of light at 45 m E/m2/seg2 or 3,000
lux) and at temperatures ranging from 25° C to 28° C. Under these conditions
micropropagation is fast. Each node will develop into a plantlet occupying the full length of
the test tube. The plantlets will be ready for subculture after six weeks (Photos 2 and 3).

The resulting sweetpotato in vitro plantlets may be easily transplanted to in vivo conditions
either in small pots or directly to field beds (Photo 4).

Propagation by stem cuttings in liquid medium

As with the potato, it is possible to micropropagate sweetpotato in bottles containing a liquid

medium (Photo 5). Stem cuttings with 5 to 8 nodes are prepared by removing both the apex
and root of the plantlet to be propagated. The cuttings are placed in a liquid medium
containing gibberellic acid to break the dormancy of the stem cutting’s axillar buds. The
nodes will sprout and new plantlets develop over a period of 3 to 4 weeks. The plantlets may
be used as initial material for simple node propagation or once again for propagation with
stem cuttings in liquid medium, depending on program needs. Shaking of cultures may
accelerate and promote the development of new plantlets. However, this is not essential.

Photo 2. Plantlet growing from a node

Photo 3. Sequence of in vitro development

QuickTimeª and a
decompressor
are needed to see this picture.

Photo 4. Plantlets after transfer to peat-moss pot

QuickTimeª and a
decompressor
are needed to see this picture.

17
a: Homemade peat-moss pot (newsprint)

b: Commercial peat-moss pot (jiffy 7)

Photo 5. Liquid medium culture for rapid propagation

QuickTimeª and a
decompressor
are needed to see this picture.

Long term maintenance

Long term maintenance is important for propagation and conservation itself. For clonally
propagated cultures it is important that every propagule be free of even the slightest
genetic alterations that may build up from one generation to the next and result in major
changes affecting uniformity and production.

18
For the maintenance of germplasm clones, it is crucial to conduct a detailed analysis of the
culture’s genetic stability. The clonal storage of germplasm consists on the maintenance of
specific gene combinations (genotypes). If a plantlet’s genetic combination changes during
storage, the validity of the storage techniques must be examined. The capacity to detect
genetic changes during propagation and maintenance depends on the methods used.

In many germplasm collections the stored genotypes are routinely evaluated for the
morphological characteristics of the plantlets growing under controlled conditions. If the
plants show new morphological characteristics (e.g. leaf shape, or storage root color) some
genetic changes are obvious. However, genetic changes such as virus resistance may not be
detected through the observation of morphological changes.

Biochemical methods are now used to study the genetic stability of both potato and
sweetpotato. They are the analysis of soluble protein and isoenzyme patterns. Although
these methods are highly effective in determining changes in genetic products, they do not
allow directly to determine changes in the genes.

New methods such as Restricted Fragment Length Polimorphism (RFLP) are considered to be
more sensitive ways of determining genetic changes. It is important that germplasm banks
and seed programs use the most effective methods in determining the genetic fidelity of the
propagation and maintenance systems.

Growth restriction media

After many years of research, propagation media for sweetpotato have been developed that
optimize rapid in vitro growth. However, maintenance requires limiting growth to a minimum
while maintaining culture viability. Use of growth restriction media allows to maximize the
interval between transfers (subcultures) of in vitro plantlets. At CIP, transfer of most
sweetpotato material under maintenance is carried out once a year, and in some cases once
every year and a half.

Laboratory experiments aimed at limiting in vitro growth of sweetpotato include the use of
hormonal growth retardants such as abscisic acid (ABA), growth inhibitors such as B995 or
chloride chloride (CCC), as well as osmotic regulators with addition of low assimilation
sugars such as manitol or sorbitol.

The difficulty involved in this type of study is that under these conditions genotypes react
differently. Studies of germplasm collections in vitro maintenance should aim at developing
maintenance media broad enough for a large variety of genotypes.

Also, the storage medium should not allow callus induction that may result in genetic
alterations.

Many storage methods have been reported for sweetpotato. At present, the method
described in the next section is used at Cip.

Restriction of storage temperature the growth of in vitro plantlets may be restricted

reducing incubation temperature. Adequate in vitro growth of sweetpotato can be obtained
with temperatures between 28° C and 30° C. At 8° C, survival time is less than one month.
For genotypes studied to date, 15° C seems to be the optimum temperature. However, this
has to be confirmed yet.

19
As with other in vitro cultures such as cassava and potato, low temperature and growth
retardants may be used simultaneously. For now, the combined use of osmotic stress and
low temperature (15° C) appears as the best and least costly way of maintaining
sweetpotato germplasm collections.

Culture media used in the work reported here are based on Murashige-Skoog (1962) and
Gamborg B-5 (1968) salts.

Medium for initiation

• Calcium pantothenate 2 ppm

• Gibberellic acid 20 ppm
• Ascorbic acid 100 ppm
• Calcium nitrate 100 ppm
• Putrescine HCl 20 ppm
• L-Arginine 100 ppm
• Coconut milk 1%
• Sucrose 5%
• Agar or 0.7%
• Phytagel/Gelrite 0.25%

Medium for transfer of meristems or buds (MMB-II)

• Calcium pantothenate 2ppm

• Gibberelic acid 15 ppm
• Ascorbic acid 100 ppm
• Calcium nitrate 100 ppm
• Putrescine HCl 20 ppm
• L-Arginine 100 ppm
• Saccharose 5%
• Agar or 0.7%
• Phytagel/Gelrite 0.25%

Propagation medium (MPB)

• Calcium pantothenate 2 ppm

• Gibberelic acid 10 ppm
• L-Arginine 100 ppm
• Ascorbic acid 200 ppm
• Putrescine HCl 20 ppm
• Sucrose 3%
• Agar or 0.8%
• Phytagel/Gelrite 0.3%

Maintenance medium (MCB)

• Glucose 2%

20
 • Sorbitol 2%
• Putrescine HCl 20 ppm
• Phytagel/Gelrite 0.4%

A pH 5.8 is used in all media.

Note: These culture media were prepared to attain maximum uniformity in a collection including a
large number of varieties. When few varieties are involved, it is advisable to use simple culture media
such as Murashige and Skoog salts with the addition of gibberellic acid and sucrose.

21
Cryotherapy protocol for pathogen elimination from Sweet
potato and Cassava

Introduction

Although several protocols for cryotherapy of shoot tips of sweet potato have
been published (Towill and Jarret, 1992; Hirai and Sakai, 2003; Wang and
Valkonen, 2008a), encapsulation-vitrification described by Hirai and Sakai
(2003) and Wang and Valkonen (2008a) proved to be efficient for sweet
potato. Therefore, encapsulation-vitrification is described in detail as follows:

Procedures for Cryopreservation of Sweet potato shoot tip by

encapsulation vitrification

1. Initiate stock cultures of sweet potato on MS medium containing 30 g/l

sucrose, 2.6 g/l gelrite (pH, 5.8) and grow them in the growth room
with 24-280C and 16 hrs light for 4 weeks to get good shoots
2. Excise shoot tips (1-1.5 mm) from 4-weeks old stock cultures and
encapsulate them with 3% Na-alginate solution in 0.1 M CaCl2 solution
for 20 min to form beads (4 mm in diameter), each bead should
containing one shoot tip
3. Preculture beads in liquid MS medium containing 0.3 M sucrose for 16
h on a shaker (90 rpm)
4. Transfer the beads into liquid MS medium containing 1.6 M sucrose
and place the cultures for 3 h on a shaker (90 rpm). After shaking of 3
h surface-dry the beads by place them on sterilized filter paper inside
laminar flow;
5. Transfer the beads into PVS2 solution and vitrify the beads for 60-90
min by shaking on orbital shaker and surface dry them after 90 min.
6. Transfer the beads into 1.8-ml cryovials, each cryotube containing 10
beads, and directly immerse the cryovials into liquid nitrogen for 1 h
7. Quickly transfer the cryovials in a water bath at 40oC for 3 min;
8. Empty the beads into liquid MS containing 1.2 M sucrose for 20 min at

22
 room temperature and surface-dry the beads as in step 4
9. Post-culture the beads on solid NH4-free MS containing 0.5 mg/l BAP at
24-28o C in the dark for 7-10 days
10. Transfer the beads onto solid MS medium containing 10 mg/l GA3 and
cultured under light at 24-28 o
C until the shoot tips develop shoot.
Subculture is done once every 4 weeks.

shoot Encapsulat Precultu

Loading
In vitro stock tip ion reee Vitrification
shoots ding

Thawin
Survival Unloading
Regeneratio g Freezing
n

Fig. 3. Flow chart of cryotherapy of shoot tips by encapsulation-vitrification (Wang and Perl, 2006)

23
 Protocol for cryotherapy of cassava shoot tips by vitrification

(Charoensub et al., 2003) is presented as the followings

1. Initiate invitro stock of cassava onto MS + 0.02 mg/l BA + 0.1 mg/l GA3 + 0.01
mg/ NAA + 30 g/l sucrose (pH=5.6) solid media and grow them into a growth
room for 8 weeks
2. Excise a nodal segments (one lateral bud, 0.5 cm long) and culture on MS +
0.02 mg/l BA + 0.1 mg/l GA3 + 0.01 mg/ NAA + 30 g/l sucrose (pH=5.6) for 12
days
3. Excise shoot tips of (1 mm with 2 leaf primordia) under the microscope and
preculture in MS + 0.3 M sucrose for 16 h
4. Transfer 10 shoot tips each into 1.8 ml cryovials containing MS + 0.4 M
sucrose + 2 M glycerol for 20 min (You can pick up the ST with sterile tweezers
and drop them into cryovials together with loading buffer into vials)
NOTE: From these stage to last stage is critical important you prepare all the
required matariaal for eash step just before the end of each step)
5. Vitrify (Osmoprotection stage) the shoot tips in 1.8 ml cryotubes by adding
1 ml of PVS2 solution and incubate at RT for 45 min.
6. After 45 min decant half on the PVS2 solution and immerse cryotubes in LN
for 1h each of cryotubes contains remaining 0.5 ml PVS2
7. Remove the cryovial from LN and quickly deep them into water bath set at 45
o
C to thaw them for 1 min
8. Decant the remaining PVS2 solution and wash the ST by incubating them into
unloading solution containing MS + 1.2 M sucrose for 20 min inside laminar
flow
9. Culture shoot tips on MS +0.02 mg/l BA + 0.1 mg/l GA3 + 0.01 mg/ NAA + 30
g/l sucrose solid media overlaid with filter paper for 4 days
10.Transfer to fresh MS +0.02 mg/l BA + 0.1 mg/l GA3 + 0.01 mg/ NAA + 30 g/l
sucrose solid media for recovery and regeneration

24
 shoot Precultu Vitrificatio
In vitro stock Loading
tip re n
shoots

Regenerati Unloadi
Survival Thawing Freezing
on ng

25
 shoot Precultu Vitrificatio Droplet
In vitro Loading
tip re n s
stock shoots

Regenerat Unloadin Thawin Freezin

Survival
ion g g g

Fig. 5. Flow chart of cryotherapy of shoot tips by droplet-vitrification (Wang

and Perl, 2006)

26
 shoot Encapsulati Precultu
In vitro Air-drying
tip on re
stock shoots

Regenerat Freezin
Survival Thawing
ion g

Fig. 6. Flow chart for cryotherapy of shoot tips by encapsulation-dehydration (Wang and Perl, 2006)

27
Appendix
Facilities and equipment required
Tissue culture facilities (Laminar flow cabinet and growth room with 24-280C 16hr light)
Liquid nitrogen (LN)
Water bath (400C)
Petri dishes (90 mm diameter)
Erlenmeyer’s conical flask (100ml)
Filter papers
Cryovials (1.8-2 ml capacity)
Pipettes 1 & 5 ml
Pipettes tips 1& 5 ml
Orbital shaker (90rpm)

Buffer and solutions

Basal medium:

MS (Commercial available premix without vitamins)

1g/l casamino acids

30g/l sucrose +

2g/l geltrite,

pH =5.7

2.5% Na-alginate solution:

BM

25g/l Na-alginate

0.4M sucrose

2M glycerol

balance the pH to 5.7 and autoclave

0.1M CaCl2 solution

MS

11.1g/l CaCl2

0.4M sucrose

pH5.8

28
 Preculture medium:

BM +

0.25M sucrose, BM + 0.5M sucrose, BM + 0.75M sucrose; pH =5.7

Osmoprotection solution: liquid BM + 1.6M sucrose + BM + 2M glycerol, pH =5.7

Plant vitrification solution 2 (PVS2):

BM

30% glycerol

15% ethylene glycol

15% DMSO

0.4M sucrose

Balance pH to 5.7

Washing solution:

BM +

1.2M sucrose,

pH =5.7

Survival medium:

BM +

0.5mg/l BA +

1mg/l GA3,

pH =5.7

Rooting media
MS
30g/l sucrose
2.6g/l gelrite
pH 5.7

29
Regrowth medium
MS
10mg/l GA3
30g/l sucrose
2.6g/l gelrite
pH 5.7

survival media
NH4-freeMS
0.5mg/l BA
30g/l sucrose
2.6g/l gelrite
pH 5.7

Unloading medum
NH4-freeMS
1.2 M sucrose
30g/l sucrose
2.6g/l gelrite
pH 5.7

Unloading solution
NH4-freeMS
1.2 M sucrose
2M glycerol

30
Virus indexing

Virus detection in plants regenerated from cryo-treated shoot tips is a critical

step because viral conditions can be confirmed only through this step.
Various detection methods for viruses have been developed, for example,
ELISA and RT-PCR, but virus-indexing using indicator plant is cost-effective
and also reliable (Wang and Valkonen, 2008b). Therefore, this simple,
efficient method is presented here:

A silicon tube
is used to
hold grafting
point

Cultivars to be A “V” cut at

tested basal part of
scion

In vitro Virus
grafting indexing
A “vertical
cut” at top of
rootstock

Indicator

Fig. 7. Virus indexing by micro-grafting of cultivars tested upon the indicator plant, I. setosa (Wang
and Valkonen, 2008b).

31
Transport, receipt, and propagation of invitro sweet potato
plantlets

Tissue culture materials consist of small, aseptic plantlets growing on a synthetic nutrient medium. The
aseptic nature of this material makes it an ideal method for international exchange of germplasm as it
minimizes the risk of transmitting fungal and bacterial diseases. This document contains information for
the recipient of invitro plantlets on the handling procedures to be followed for further micro-propagation or
transfer to non-sterile growing conditions.

Transport of tissue culture material

1. Packing the material is packed in polystyrene in a cardboard container. Each package contains several
small glass test tubes, each with three well-developed plantlets. Extra agar media is added to prevent
damage from movement during shipment (Photo 1).Photo 1
The test tubes are capped with plastic covers and sealed with parafilm to prevent entry of contaminants
into the cultures and loss of water from the medium.

Shipment.
Whenever possible, tissue cultures are hand-carried to ensure rapid transport. When this is not feasible,
the fastest possible method is usually airfreight. In vitro plantlets can survive two to three weeks without
light.

Handling after arrival.

The cultures should be cleared from customs as quickly as possible. When advance notice of the
shipment is known, alert the customs officials of its expected arrival. Carefully remove the test tubes from
the package in a laboratory or clean room. Do not open the tubes. Do not remove the plantlets. If the
plantlets have become yellow, place the test tubes under diffused light for about 1 week in a clean room.
Invitro shoot cultures are free from diseases. Work under clean conditions according to the following
description (steps 4 and 5 of the following section on "Transfer to Planting Materials") to prevent
contamination during and after unpacking.

Use of tissue culture material.

The plantlets can be used in two different ways:
• Transfer to planting mix

• Micropropagated

32
Transfer to planting mix
1. Materials
• Peat moss Fine sand (1 mm diameter) Aluminum foil Clay pots (8 to 10 cm diameter) or jiffy pots
Larger pots (20 cm diameter) Distilled water1% calcium-hypochlorite solution70% alcohol solution
Strong soap

• Fertilizer with high content of phosphorus (5-50-17) or (12-12-12)

2. Mix peat moss and sand (1:2 volume).

If an autoclave is available, fill the pots with peat moss/sand mix, cover them with aluminum foil,
and sterilize for 1 hour. If an autoclave is not available wash the clay pots (jiffy pots are already
sterile) with detergent, rinse them well with running water, and sterilize the planting mix and some
additional sand separately by any other means (heat, steam, fungicides, etc.).
Take the pots and the in vitro culture to a clean bench that is protected from air currents, dust,
dirt, insects, or other contaminants.
Wash your hands with strong soap and 1% calcium-hypochlorite solution. Then rinse hands in
70% alcohol.
Irrigate the pots with a small amount of water.
Prepare the pot that is to receive the plantlet by making a hole in the center of the peat
moss/sand mix with a clean stick or pencil.
Before removing the plantlets, disinfect the outside of the test tube using a piece of cotton or cloth
moistened with 70% alcohol to reduce the risk of contamination.
Using clean fingers, remove the parafilm and the plastic cover from the test tube. Work with one
tube at a time.
3. Gently pull the plantlets with the agar out of the test tube using sterilized forceps (flamed to red
heat and cooled) (Photo 2).
Photo 2

11. Wash the agar from the roots by gently immersing them several times in sterilized water, trying
not to wet the rest of the plantlet.
12. Plant each plant individually in the holes in the potting mix with the roots plus one or two nodes
below the surface (Photo 3).
13. Place the sterilized sand around the plantlet and press lightly to keep the plantlet straight in the
pot.
14. Place the plant into a humid chamber during 48 h. Remove the humid chamber and wait until the
roots are established (about 10 days).
15. Keep the pots in a clean location, at 25 to 27° C with 14-16 hours illumination.

33
 16. Until the plants are well rooted irrigate lightly with tap water if it has a low salt content; otherwise
use demineralized or rain water. Do not overwater.
Photo 3
17. When roots are established, you may dissolve supplementary nutrient in the irrigation water.
Commercial peat moss often contains fertilizer, thus less additional nutrient may be required.
18. Gradually expose the plants to the normal atmosphere by removing the beakers for short periods
each day.
19. Once the plants are established, transfer to larger (e.g. 20 cm diameter) pots. Be careful not to
break the roots. When the plants are well rooted, normal fertilizer can be dissolved in the
irrigation water. At CIP we use 5 g N:P:K at 12-12-12 per liter water. Apply 50-100 cm3 per 20 cm
diameter pot. Again, do not overwater.

Micropropagation
1. Materials and equipment
• Culture medium (see next two following sections)

• Test tubes

• Plastic caps or cotton wool

• Forceps

• Scalpel

• Parafilm

• Alcohol lamp

• Alcohol 70%

• Autoclave

• Sterile work area ("microvoid")

2. Prepare the nutritive growth medium according to the procedure given in the section on Medium
to Sweet potato Micropropagation.
3. Dispense 4 cm3 of the medium in each test tube. Cap the tubes with plastic caps or cotton wool
plugs and autoclave them for 15 minutes. Keep the test tubes vertical while the agar sets.
4. Working under sterile conditions (sterile area or "microvoid") follow steps 8 to 10 of previous
section.
5. Transfer the plantlets from the test tuber to a sterile petri dish and make nodal cuttings using
sterile scalpel and forceps. Each nodal cutting consists of a 0.2-0.5 cm stem segment with an
axillary bud (Photo 4).
Photo 4

34
 6. Place each nodal cutting in a test tube. Ensure that each cutting lies on the agar surface with its
axillary bud pointing upwards (Photo 5). Place 1 or 2 cuttings per test tube.
Photo 5
7. Close the test tubes, seal with parafilm, label and place in a clean area where the room
temperature is 25-27° C. Give 45m E/m2/seg2 or 3,000 lux illumination for 14-16 hours each day.
8. The axillary bud of each nodal cutting grows into a new plantlet within 2-4 weeks and is ready for
transplanting to pots as previously described, or for further micropropagation (Photo 6).

35
36