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


Table of Contents

Introduction Recovery of the Cultures 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

1 1


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

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 2628°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


the cultures can be subjected to a hardening treatment prior to potting.05 mg/I benzyl aminopurine + 0. 2. decrease the temperature to 24-25°C during 1-2 weeks Three to four days prior to potting.1 Procedures for micropropagation 1. 4. 6. leaving 4-5 mm of storm below the apex. but it will improve the potting. Once. Hardening is not absolutely necessary. take the paraffinated paper off the caps. and maintain a 14 hour photoperiod throughout. 3. Keep the petri dish closed as much as possible. flame quickly. In addition. whether or not you have hardened the cultures. Using the larger forceps. For hardening. Incubate the “node cutting’ cultures at 28°C under an illumination of 2000 lux until root initiation and.05 mg/I gibberellic acid + 0. cut the stem into segments. if possible. uncap 2-3 test tubes (Figure 1-a). Ph 5. Wash hands and arms with soap and running water and wipe with 70% alcohol.000-10. the following items are needed: 4 . gently pick up each “nodal cutting” and each terminal bud. horizontal cuts with the scalpel.7-5. 1.02 mg/l naphathalene acetic acid. increase the illumination to 8. 5. gently pull the plantlet from the tube. Make sure to make clean.6%. and using short forceps. thereafter. under 4000 lux. 1. Open one petri dish and wet the filter papers with the sterile water taking care to flame the flask. every modal cutting will give rise to a complete planlet (Fig 1-e). Then. and place them under the sterile flaps of paper.2. Place all planlets within the petri dish (figure 1-b) Using a scalped and the short forceps cut off all expanded leaves and roots. Quickly flame the tube and cap it immediately During the operation. the plantlet has grown to 6-8 cm tall and rooting is proportional to the amount of shoots.• 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. After about 3 weeks.000 lux and. agar = 0. 8. The preparation of this medium is described in Appendix 1 Figure 1 micropropagation of cassava through nodal cuttings. and “ plant” each one in agar solidified medium (Figure 1-d) in an 18-25 = X 150 MM TEST TUBE.3 Potting For potting. 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. make sure to frequently wet the tools with 95% alcohol and flame them. If possible.8. cover the hair with a microbial cap and the mouth and nose with a microbial mask Immerse the tools in 95% alcohol for 2-3 minutes. each one comprising one node (Figure 1-e). respectively). 7. cut the terminal bud from every shoot. One at a time.

Thereafter. place the trays on the greenhouse table and cover them with the plastic frame (Fig 2). Holding the plant on the palm of your hand. A 2.5 x 8 inches) can also be used Substratum. close the frame at night. containing damped sterilized soil. Press the substratum and immediately apply deionized water around the plant (Fig 7) After potting label the posts and water with the high P fertilizer 10-52-10. A few hours prior to potting. 1. one at a time.3 –m transparent plastic frame to cover the trays A 2.0 X 10. clay or cement trays with sufficient draining holes. 2.0. e. Use 2g fertilizer in 1 litre of water and apply 80-100 ml solution per pot. Wet the substratum to about one half water saturation. In about 2 more weeks. but not too cold location that is protected from direct sun and from insects. Place the pots in the high humidity chamber (Fig 8). Using the fingers.g. which consists of a mixture of one part [soil with three parts of fine sand.0 X 1. but clay or plastic bags 96. 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. Optimal potting is achieved using 3 to 4 inch jiffy-type pots. Pots. and. pull the plant from the tube mouth. 3. wash off as much agar as possible from the roots (Fig 5). move the pots (still on the trays) to a warmer and much more illuminated part of the greenhouse. 6. 5. when needed and until the time of field transplanting. The sand must be thoroughly washed with soft water. gently pull out the planlet (Fig 4). water with only the high P solution. 4. damp with water the soil contained in the trays.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.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 5 . A high relative humidity environment will form within the plastic chamber Sterilize the substratum mixture using steam and fill in the pots.X 0. the plants are ready for field transplanting.• • • • • • • • • • • • 1. Uncap the test tubes. 10-52-10 (N-P-K) Plastic. 7. After the 12th day. Make a hole in the center of each pot (Fig 3) Wash your hands thoroughly with soap and water. A fair amount of deionized water Long (15-20 cm) forceps Pot labels A water –soluble fertilizer rich in P. and place the roots and the lower part of the shoot within the hole in the pot (Fig 6).X 0. By day 10 or 12. the plastic frame can be taken off completely. Five or six days after potting and thereafter raise the plastic frame slightly during the fresher hours of the day. with the aid of the forceps.

4. 1. 5. After transplanting the material should be inspected daily. Phytium) bacteria. Fill the bags for planting one day before On planting day add to the planting media a solution of Banrot 0. 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) 6 . and a cup is placed over it to increase humidity to protect it for the next 8 days. or a fungicide containing active ingredients against mushrooms (Phytophthora. due to this the fertilizers are in two groups according to its functionality. The major micro nutrients the fertilizers have to have are Calcium. without removing the plastic cups to avoid dehydration.1 FERTILIZER APPLICATION Fertilizer application is very important because it gives to the plants nutrients that it needs for its growth. 4. and watered only when necessary. This should be prepared with deionized or sterile distilled water. 1. Manganese and Cobalt. Zinc.75gr/lt. Fertilizer application is carried out every 8 days. 3. alternating the fertilizer and precious amount of 50ml should be done I the morning 8-10. Magnesium. Remove the cups after 8 days and water if necessary using deionized water and remove dead leaves and initiate fertilizer application. 2. The plants should be in the screen-house for a minimum of 2 days before transplanting for them to adopt. add little water to the flash to help loosen the agar for the plants to be removed easily. mix and sterilize for 3 hours.00 am or later in the day 5-6. 7. • • • • • First application should be done 15 days after transplanting and should be applied to the soil.5%. Prepare planting media a week before transplanting in the following way: . Make a hole in the planting media and insert the plant.wash the sand 3 times until it is totally clean.1. put to dry in the sun. A lot of care should be taken not to damage the roots. A flask contains 5 plants. cosmocel. 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 2. coljap development and coljap production each one of them is used in a concentration of 0. Fertilizers with micro nutrients that the plant needs in its initial stage: Plan/pro (plantex). After apply every 8 days. sieve soil. 6.00 pm when it is cool so that the plants can assimilate most of the fertilizer. with its respective label. 8. Sodium. This cup should preferable be transparent to allow in light. each is placed in a different bag.

Apply 0. worm or cricket attacks. to the field and cut off the largest leaves 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. the plants should be completely established in the field and can be handled conventionally. and watch out for ant. Keep in mind that these plants are elite stocks for the multiplication of clean planting material and that their production shipment.5 Field Transplantation Successful transplanting can be achieved under cloudy days or during the late afternoon. 2. Press the soil around the plant and water immediately 4. After planting.• • NOTE. Leave the plants covered or 1 week. The soil should be in field capacity and the plants 10-15 cm in height. cover each plant with a Styrofoam cup (Make sure to make some openings on the upper side of the cup). it is recommended that you pay special attention to protecting the plants from insects or disease throughout the crop. 7 . These should not be done simultaneous with the fertilizations as the plant may get burn because of excess chemical 1. along with the trays. Maintain high soil humidity for 10-15 after transplanting. Triple 15 (N/P/K) (apply in the soil) Urea (apply in the soil) Sometimes it is necessary to apply fungicides and pesticides to prevent fungal and/or pests. please write to 1. Use jiffy-type pots or plastics bags and keep root disturbance minimum Carry the plants in their pots. and handling after arrival have been done with great care and effort For news regarding the condition of the cultures upon arrival. then uncover them once continue with stop At the fourth week. any progress developments in handling or additional information. However.7 ml/l fungicide or 1 ml/l of acaricide.

8 .0 ml of stock solution No.8 • Dissolve by heating 6.25 ml of stock solution No. Supplements. Using stock solutions of mineral salts. For the preparations of the Murashige and Skoog stock solutions see (Table 1) To prepare 1 liter of medium.1 through No. Add the same volumes of stock solutions No. Preparation of Growth Regulator Stock Solutions Benzyl aminopurine (BAP)(10 ppm): Dissolve 20 mg in a small volume of 1. complete to 200 ml with water. Gibberellic acid (GA3) (10 ppm): Dissolve 22 mg (90% gibberellic acid) in a small volume of 1. The powder can be stored at 8-10°C or under desiccation.0 N HCl. Appendix 1a.4 5.0 ml of stock solution No. To prepare the basal medium dissolve the entire contents of one bag in 500 ml double distilled water. in the volumes of double distilled water shown. Using the pre-made Murashige and Skoog medium in powder form (without sucrose and without vitamins and agar).0 ml of Naphthalene acetic acid (NAA) stock solution • Complete to 700ml with double distilled water • Adjust the pΗ to 5.0 N KOH. proceed as follows: • Add 5. B). for up to 2 years. all the ingredients presented in Table 1.3 2. Each bag contains 4.6 and 6.150-mm test tubes (5ml/tubes). Can be prepared in either of two forms: 1.APPENDIX 1 Preparation of the Culture Medium A) Basal Medium.7 • Dissolve 20. 15 pounds (121°C) per square inch during 15 minutes. vitamin and growth regulators. complete to 200 ml with double distilled water (This is a 100ppm 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). Appendix 1b. Preparation of Murashige and Skoog Stock Solutions To prepare the stock solutions.0 ml of stock solution No. which serves to prepare 1 litre of basal medium. dissolve one by one.0 ml of stock solution No. STERILIZATION Quickly distribute the prepared medium in 18-X.5 2.7-5. C.3g of powder. let cool slightly and cap the tubes Autoclave the tubes containing medium. then store them in darkness at 6-8°C until used.0 ml of the benzyl aminopurine (BAP) stock solution and 2.0 g of sucrose • Add 5. to 500 ml of double distilled water add: 20.5 as in Table 1.0 g of agar in 300ml of double distilled water • Mix well the medium with the agar solution. decompress slowly Place the tubes in a fresh place until the agar solidify. Once either of the basal media is ready.1 1. take 20 ml of this solution and complete to 200 ml with water.0 ml of stock solution No.2 1.9 ml of stock solution No.

6H₂O Dissolve in 100 ml water Kl Dissolve in 100 ml water CaCl .075 g 15 g 1. Separately dissolve ‘a’ and ‘b’ in 50 ml with water.025 g 0.0 ml of either BAP/GA3 9 .0025 g 0. Murashige and Skoog Stock and Medium Preparation. 7H₂O KH₂PO₄ Dissolve in 1000 ml water H₂BO₃ MnSO₄ .62 g 2.0021 g 0. Stocks 2 and 6 should be kept frozen.0 N KOH. 2H₂O CuSO₄ .0 ml 2.25 ml a.492 mg 1. take 20 ml of this solution and complete to 200 ml with water Table 1.05mg/l x 1000ml = 5.8 g Volume of stock per 1 liter basal medium 20.05 mg/l Gibberellic acid = 0.0 ml 6 7 6.05 mg/l Naphthalene acetic acid = 0. H₂O ZnSO₄ .0 g 18. 5H₂O CoCl₂ .9 ml 5.02 mg/l V₁ = Volume (in ml) of stick solutions needed = X V₂ = Final volume of medium = 1000 ml X= 0.114 mg 0.0 ml 3 4 5b 1. Stock solution no. 2H₂O Dissolve in 100 ml water a) Na₂EDTA b) FeSO₄ .Naphathalene acetic acid (NAA) (10 ppm): Dissolve 20 mg in a small volume of 1. Keep stock 5 protected from light b.25 ml 6. 7H₂O Na₂MoO₄ .5 g 8.5 g 95.5 g 0. all the others at 8-10°C. 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). complete to 200 ml with water. 7H₂O Dissolve in 200 ml water Thiamine-HCl Dissolve in 200 ml water m-inositol Dissolve in 200 ml water Amount 82. apply the following formulation: C₁V₁ = C₂V₂ C₁ = Concentration of stock – 10 mg/l C₂ = Final concentration of growth regulator in the medium Benzyl aminopurine = 0.8 g 0.86 g 0.a 1 Constituents Substance NH₄NO₃ KNO₃ MgSO₄. take 20 ml of this solution and complete to 200 ml with water.0 ml 2 1.176g 0.

Tery. Cassava. In: D. In: J. In press. Elite Cassava germplasm from CIAT.10mg/l References 1. Belloti. 1983b.3 International Institue of Tropical AGRICULTURE. Cleveland. Physiol. MacMillan. 11 p. J. P. Colombia. Cali. Schilde. W.B and Chiarappa. Vol. A. 6. Propagation of tuner root crops. Centro Internacional de Agricultura Tropical. Reyes. and Doll. Research Briefs. 56 p. J. 3.R. L and Roca. Lozano. Centro Internacional de Agricultura Tropical. Murashige. 5. Ohio.V. W. Cali. Colombia. Roca. L (eds) 1977. Sharp. 192 p. Rome. 4p 7. Howeler. Nigeria. Genetic resources of cassava and wild relatives. D.20 p 2. Colombia. Ammirato. Plant. A revised medium for rapid growth and bioassays with tobacco tissue cultures. 10 . Handbook of plant cell culture. Yamada (eds). Plant health and quarantine in the international transfer of genetic resources. W. E. Rome. CIAT (Centro International de Agricultura Tropical). J. Ibadan. 1981. T and Skoog F. Leihner.C. W. Cali. A review of cassava and sweet potato diseases and their relation to germplasm exchange. Pathogen elimination in potato and cassava. and Y. Vol. Hewitt. Practical constraints affecting the collection and exchange of wild species and primitive cultivars. R.M.1962. New York 8.M. IBPGR (I international Board for Plant Genetic Resources) 1983a.In Press. 15:473-497.R 1982.A. 4. IBPGR Secretariat. 9. IBPGR Secretariat. 2: Crop species. Field problems in cassava. 1983. CRS Press. Cock (ed).A Evans.

Ana Panta.John Dodds (Reproduced from CIP Research Guide 32) 11 .Micropropagation and Maintenance Rolando Lizárraga. Nelson Espinoza.

Preparation of the Culture Medium Appendix 1a. Preparation of Growth Regulator Stock Solutions References 12 . Preparation of Murashige and Skoog Stock Solutions Appendix 1b.Table of Contents Introduction Advantages of tissue culture Introduction of in vivo material into Invitro 1 1 Meristem isolation cultures Thermotherapy Field Transplantation Green house transplantation Appendix 1.

They are then placed in a clean bottle/beaker covered with a Petri dish/aluminum foil until starting surface disinfection. Permanent availability of (when pathogen tested) material for propagation and exportation. To start surface disinfection of the stem cuttings. To reduce phenolization of the 13 . add 96% ethanol and let stand for two seconds.5% solution of calcium hypochlorite (brought to pH 8 by addition of HCl). The bottle is then placed in the laminar flow transfer chamber. This can be achieved by any commercial acaricide for few minutes. remove the water from the container. as well as the maintenance of germplasm under controlled conditions in small spaces and with reduced labor requirements. Then acaricide is rinsed away by washing the stems in running water for 30-45 minutes. Timely access to material for pathogen clean-up. Timely access to material under maintenance. Alternatively mature plants directly from the fields normally are very dirty and are difficulty to thoroughly cleaned them. the stem cuttings are treated with a wide spectrum acaricide to destroy the mites at any stage of development. too sprout are best for initiation. micropropagation. which are in excellent health conditions. Plantlets of 2-3 months old. Advantages of tissue culture CIP maintains a sweet potato germplasm collection of over 5. Introduction of in vivo material into in vitro The explants from the mother plant must come from the greenhouse. Presurface sterilization Before sending them to the in vitro laboratory. 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. and long term storage techniques. The stems are excised from the mother plant and the leaves removed. 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. read for initiation. Discard the alcohol and immediately add a 2.Tissue culture allows the rapid clonal propagation of a large number of plantlets over a short period. Mikocheni Agricultural research Institute etc. the hypochlorite is eliminated. After 15 minutes under sterile conditions. Part of the petiole should be left to cover the buds. Protection against unfavorable climatic conditions. Thus. This document complies the advances. Their clonal maintenance on the field is expensive and involves the risk of loss due to infectious diseases or unfavorable climatic conditions. by washing three times with sterile water. Absence of field infections. add a few drops of a dispersing-adhering agent such as Tween 20 or 80 (4 drops/l of solution). and free of lateral buds that are. methodologies and materials used for tissue culture at the various institution including International Potato Center (CIP). If possible. in vitro maintenance presents the following advantages: • • • • • • Lower labor costs.000 accessions. It analyzes isolation. Sodium hypochlorite or chlorine (bleach) may also be used.

either 18 x 150 mm or 25 x 150 mm tubes may be utilized. Blot the explants into a filter paper to dry off the hypochlorite solution. In a situation where cultures are initiated using meristem. saprophytic bacteria mainly may contaminate some material. Under these conditions. Other antibiotics such as Cefotixine (Mefoxin Merck) in doses of 500 ppm may also be used. are available. For bacteria it is recommended to use Rifampicine (Rimactan 300 CIBA) at 400 ppm. plant them in the introduction medium (MMB-I) and transfer them weekly to the MMB-II medium. which must be transferred to a fresh medium with another antibiotic paper every 3 to 5 days. after the last rinse they are left in a sterile 100 ppm solution of ascorbic acid before proceeding to excision. proceed in either of two ways. For contamination by yeast it is advisable to use 0. The excised portions should be as small as possible.000 ppm). A concentrated (12.explants. Photo 1 shows a photographic sequence of the dissection procedure. In the first propagation stage. 14 . 1. The optimum size is 0. The paper squares are introduced into the culture medium with the planted bud. Nutrition of the dissected section is provided by the artificial medium. Approximately 0. 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 second stage.03 cm3 of concentrate solution is used for every square of filter paper. which are allowed to dry in the laminar flow chamber.4 and 0. Eradication of bacteria or yeasts may be attempted by adding antibiotics to the 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. If a stereoscopic microscope is available. They are best if used before seven days for they progressively lose they effectiveness. The filter paper procedure described above will be used in all instances. 16 x 125 mm culture vessels are used. The aseptic dissection of the meristem for virus eradication is a delicate process requiring skill.6 mm but the explant may be bigger if no adequate excision instruments.25 to 0. filter-sterilized solution of Rifampicine is used to soak small (5 x 5 mm) squares of filter paper. excise meristems between 0.6 mm are used. 2. If so. The meristematic cells divide and form new tissue. This process must be carried out under aseptic conditions.6 mm long. such as a stereoscopic microscope.5 ppm doses of Amphotericine B. proceed to excise the buds by eliminating the largest possible number of leaflets and leaf primordia. Meristem isolation and culture The meristem is a tissue made of cells under division and is the active growth point of the bud. Due to the fact that meristematic portions larger than 0. 1. The dome of the bud contains the meristematic cells and is surrounded by foliar primordia and primary leaves.

The material is deeped into a disinfectant and sodium hypochlorite as for the initiation of invitro cultures. 3. 2. 1. 15 . The plant stems are cut in segments including a node and the corresponding axillar bud.QuickTimeª and a decompressor are needed to see this picture.

After rinsing in distilled sterile water. After 6 to 8 weeks the meristem will develop into a plantlet. 5. 16 . 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. Callus formation and plant regeneration must be carefully avoided because they tend to affect the genetic stability of the genotype. The plantlets are now ready for subculture in the propagation medium (see section on Culture Media). The propagation medium described in the section on Culture Media is used. 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 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. 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. The dissected meristem is transferred weekly to a fresh MMB-II medium. It must be noted that this type of propagation is based on the development of a pre-existing morphological structure. After thermotherapy either axillar or apical meristems may be used indistinctly. QuickTimeª and a decompressor are needed to see this picture. Photo 1.4. This high temperature treatment has increased the efficiency of the production process of virus free material. 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. The cupule and foliar primordia are dissected with the surgical blade and transferred to culture medium MMB-I. 2.

Under these conditions micropropagation is fast. Photo 4. Plantlets after transfer to peat-moss pot QuickTimeª and a decompressor are needed to see this picture. it is possible to micropropagate sweetpotato in bottles containing a liquid medium (Photo 5). However. Each node will develop into a plantlet occupying the full length of the test tube. Sequence of in vitro development QuickTimeª and a decompressor are needed to see this picture. Photo 2.The plantlets grow under long day conditions (16 hours of light at 45 m E/m2/seg2 or 3. this is not essential. The plantlets will be ready for subculture after six weeks (Photos 2 and 3). The plantlets may be used as initial material for simple node propagation or once again for propagation with stem cuttings in liquid medium. Propagation by stem cuttings in liquid medium As with the potato. depending on program needs. The nodes will sprout and new plantlets develop over a period of 3 to 4 weeks. Plantlet growing from a node Photo 3. The cuttings are placed in a liquid medium containing gibberellic acid to break the dormancy of the stem cutting’s axillar buds. 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). Shaking of cultures may accelerate and promote the development of new plantlets. Stem cuttings with 5 to 8 nodes are prepared by removing both the apex and root of the plantlet to be propagated.000 lux) and at temperatures ranging from 25° C to 28° C. 17 .

Long term maintenance Long term maintenance is important for propagation and conservation itself. Liquid medium culture for rapid propagation QuickTimeª and a decompressor are needed to see this picture.a: Homemade peat-moss pot (newsprint) b: Commercial peat-moss pot (jiffy 7) Photo 5. 18 . 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.

the method described in the next section is used at Cip. At present. At CIP. transfer of most sweetpotato material under maintenance is carried out once a year. If the plants show new morphological characteristics (e. New methods such as Restricted Fragment Length Polimorphism (RFLP) are considered to be more sensitive ways of determining genetic changes. or storage root color) some genetic changes are obvious. The clonal storage of germplasm consists on the maintenance of specific gene combinations (genotypes). Studies of germplasm collections in vitro maintenance should aim at developing maintenance media broad enough for a large variety of genotypes. The difficulty involved in this type of study is that under these conditions genotypes react differently. 15° C seems to be the optimum temperature. survival time is less than one month.For the maintenance of germplasm clones. this has to be confirmed yet. the storage medium should not allow callus induction that may result in genetic alterations. The capacity to detect genetic changes during propagation and maintenance depends on the methods used. genetic changes such as virus resistance may not be detected through the observation of morphological changes. They are the analysis of soluble protein and isoenzyme patterns. Use of growth restriction media allows to maximize the interval between transfers (subcultures) of in vitro plantlets. At 8° C. Adequate in vitro growth of sweetpotato can be obtained with temperatures between 28° C and 30° C. Growth restriction media After many years of research. However. maintenance requires limiting growth to a minimum while maintaining culture viability. the validity of the storage techniques must be examined. leaf shape. In many germplasm collections the stored genotypes are routinely evaluated for the morphological characteristics of the plantlets growing under controlled conditions. Biochemical methods are now used to study the genetic stability of both potato and sweetpotato. growth inhibitors such as B995 or chloride chloride (CCC). Also. it is crucial to conduct a detailed analysis of the culture’s genetic stability. 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. If a plantlet’s genetic combination changes during storage. Many storage methods have been reported for sweetpotato. Restriction of storage temperature the growth of in vitro plantlets may be restricted reducing incubation temperature. However. Laboratory experiments aimed at limiting in vitro growth of sweetpotato include the use of hormonal growth retardants such as abscisic acid (ABA). For genotypes studied to date. propagation media for sweetpotato have been developed that optimize rapid in vitro growth. 19 . and in some cases once every year and a half. However. Although these methods are highly effective in determining changes in genetic products. they do not allow directly to determine changes in the genes.g. as well as osmotic regulators with addition of low assimilation sugars such as manitol or sorbitol.

As with other in vitro cultures such as cassava and potato. low temperature and growth retardants may be used simultaneously.8% Phytagel/Gelrite 0.3% Maintenance medium (MCB) • Glucose 2% 20 . the combined use of osmotic stress and low temperature (15° C) appears as the best and least costly way of maintaining sweetpotato germplasm collections. 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. Culture media used in the work reported here are based on Murashige-Skoog (1962) and Gamborg B-5 (1968) salts.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. For now.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.

4% A pH 5.8 is used in all media. 21 .• • • Sorbitol 2% Putrescine HCl 20 ppm Phytagel/Gelrite 0. it is advisable to use simple culture media such as Murashige and Skoog salts with the addition of gibberellic acid and sucrose. Note: These culture media were prepared to attain maximum uniformity in a collection including a large number of varieties. When few varieties are involved.

encapsulation-vitrification is described in detail as follows: Procedures for Cryopreservation of Sweet potato shoot tip by encapsulation vitrification 1. Therefore.8-ml cryovials. Hirai and Sakai. 6. Excise shoot tips (1-1.1 M CaCl2 solution for 20 min to form beads (4 mm in diameter). Preculture beads in liquid MS medium containing 0. 2.6 g/l gelrite (pH. After shaking of 3 h surface-dry the beads by place them on sterilized filter paper inside laminar flow. and directly immerse the cryovials into liquid nitrogen for 1 h 7.6 M sucrose and place the cultures for 3 h on a shaker (90 rpm). Quickly transfer the cryovials in a water bath at 40oC for 3 min. Transfer the beads into liquid MS medium containing 1. 5. 2008a).8) and grow them in the growth room with 24-280C and 16 hrs light for 4 weeks to get good shoots 2. each bead should containing one shoot tip 3.5 mm) from 4-weeks old stock cultures and encapsulate them with 3% Na-alginate solution in 0. 8. Empty the beads into liquid MS containing 1.3 M sucrose for 16 h on a shaker (90 rpm) 4. each cryotube containing 10 beads. encapsulation-vitrification described by Hirai and Sakai (2003) and Wang and Valkonen (2008a) proved to be efficient for sweet potato. 5. 1992. Initiate stock cultures of sweet potato on MS medium containing 30 g/l sucrose. Transfer the beads into 1.2 M sucrose for 20 min at 22 . 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.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. Wang and Valkonen. 2003.

5 mg/l BAP at 24-28o C in the dark for 7-10 days 10. 2006) 23 . Subculture is done once every 4 weeks. 3. Flow chart of cryotherapy of shoot tips by encapsulation-vitrification (Wang and Perl. Post-culture the beads on solid NH4-free MS containing temperature and surface-dry the beads as in step 4 9. In vitro stock shoots shoot tip Encapsulat ion Precultu reee Loading Vitrification ding Regeneratio n Survival Unloading Thawin g Freezing Fig. 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.

Remove the cryovial from LN and quickly deep them into water bath set at 45 o C to thaw them for 1 min 8. Initiate invitro stock of cassava onto MS + 0. Excise shoot tips of (1 mm with 2 leaf primordia) under the microscope and preculture in MS + 0..02 mg/l BA + 0. 2003) is presented as the followings 1.01 mg/ NAA + 30 g/l sucrose (pH=5.6) solid media and grow them into a growth room for 8 weeks 2.1 mg/l GA3 + 0.6) for 12 days 3.3 M sucrose for 16 h 4.01 mg/ NAA + 30 g/l sucrose solid media for recovery and regeneration 24 .02 mg/l BA + 0. Culture shoot tips on MS +0.01 mg/ NAA + 30 g/l sucrose solid media overlaid with filter paper for 4 days 10.01 mg/ NAA + 30 g/l sucrose (pH=5. Decant the remaining PVS2 solution and wash the ST by incubating them into unloading solution containing MS + 1.02 mg/l BA + 0. 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 7.2 M sucrose for 20 min inside laminar flow 9.Protocol for cryotherapy of cassava shoot tips by vitrification (Charoensub et al.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. Transfer 10 shoot tips each into 1.02 mg/l BA + 0.Transfer to fresh MS +0. 0. Vitrify (Osmoprotection stage) the shoot tips in 1.5 cm long) and culture on MS + 0.8 ml cryotubes by adding 1 ml of PVS2 solution and incubate at RT for 45 min.1 mg/l GA3 + 0.1 mg/l GA3 + 0. 6. Excise a nodal segments (one lateral bud.8 ml cryovials containing MS + 0.1 mg/l GA3 + 0.

shoot In vitro stock tip shoots Precultu re Loading Vitrificatio n Regenerati on Survival Unloadi ng Thawing Freezing 25 .

Flow chart of cryotherapy of shoot tips by droplet-vitrification (Wang and Perl. 2006) 26 .shoot In vitro tip stock shoots Precultu re Loading Vitrificatio n Droplet s Regenerat ion Survival Unloadin g Thawin g Freezin g Fig. 5.

6. 2006) 27 .shoot In vitro tip stock shoots Encapsulati on Precultu re Air-drying Regenerat ion Survival Thawing Freezin g Fig. Flow chart for cryotherapy of shoot tips by encapsulation-dehydration (Wang and Perl.

4M sucrose pH5.4M sucrose 2M glycerol balance the pH to 5.8-2 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 =5.5% Na-alginate solution: BM 25g/l Na-alginate 0.7 2.1M CaCl2 solution MS 11.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 28 .1g/l CaCl2 0.7 and autoclave 0.

7 29 . pH =5.5mg/l BA + 1mg/l GA3. pH =5.7 Plant vitrification solution 2 (PVS2): BM 30% glycerol 15% ethylene glycol 15% DMSO 0. pH =5.25M sucrose.7 Rooting media MS 30g/l sucrose 2.75M sucrose.6g/l gelrite pH 5.2M sucrose. BM + 0. pH =5.7 Osmoprotection solution: liquid BM + 1. BM + 0.7 Survival medium: BM + 0.5M sucrose.6M sucrose + BM + 2M glycerol.7 Washing solution: BM + 1.Preculture medium: BM + 0.4M sucrose Balance pH to 5.

2 M sucrose 2M glycerol 30 .5mg/l BA 30g/l sucrose 2.6g/l gelrite pH 5.7 Unloading solution NH4-freeMS 1.2 M sucrose 30g/l sucrose 2.7 survival media NH4-freeMS 0.6g/l gelrite pH 5.Regrowth medium MS 10mg/l GA3 30g/l sucrose 2.7 Unloading medum NH4-freeMS 1.6g/l gelrite pH 5.

but virus-indexing using indicator plant is cost-effective and also reliable (Wang and Valkonen.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. 2008b). setosa (Wang and Valkonen. this simple. for example. 31 . 2008b). efficient method is presented here: A silicon tube is used to hold grafting point Cultivars to be tested A “V” cut at basal part of scion In vitro grafting A “vertical cut” at top of rootstock Virus indexing Indicator Fig. ELISA and RT-PCR. 7. Various detection methods for viruses have been developed. Virus indexing by micro-grafting of cultivars tested upon the indicator plant. Therefore. I.

In vitro plantlets can survive two to three weeks without light. Extra agar media is added to prevent damage from movement during shipment (Photo 1). Do not remove the plantlets. each with three well-developed plantlets. Do not open the tubes. 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. receipt. place the test tubes under diffused light for about 1 week in a clean room. Transport of tissue culture material 1. When this is not feasible. Each package contains several small glass test tubes. The cultures should be cleared from customs as quickly as possible. Packing the material is packed in polystyrene in a cardboard container. tissue cultures are hand-carried to ensure rapid transport. 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. and propagation of invitro sweet potato plantlets Tissue culture materials consist of small. Use of tissue culture material. Shipment. Whenever possible. If the plantlets have become yellow. the fastest possible method is usually airfreight. When advance notice of the shipment is known.Transport.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. Handling after arrival. Carefully remove the test tubes from the package in a laboratory or clean room. 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. aseptic plantlets growing on a synthetic nutrient medium. The plantlets can be used in two different ways: • • Transfer to planting mix Micropropagated 32 . alert the customs officials of its expected arrival. Invitro shoot cultures are free from diseases.

14. Keep the pots in a clean location. Work with one tube at a time. Mix peat moss and sand (1:2 volume). 13. Place the plant into a humid chamber during 48 h. trying not to wet the rest of the plantlet. fungicides. Wash the agar from the roots by gently immersing them several times in sterilized water. cover them with aluminum foil. and sterilize the planting mix and some additional sand separately by any other means (heat. insects. rinse them well with running water. Irrigate the pots with a small amount of water. Take the pots and the in vitro culture to a clean bench that is protected from air currents. Using clean fingers. Remove the humid chamber and wait until the roots are established (about 10 days). and sterilize for 1 hour. 15. etc. If an autoclave is available. Then rinse hands in 70% alcohol. Gently pull the plantlets with the agar out of the test tube using sterilized forceps (flamed to red heat and cooled) (Photo 2).). Plant each plant individually in the holes in the potting mix with the roots plus one or two nodes below the surface (Photo 3). Before removing the plantlets. or other contaminants. 12. disinfect the outside of the test tube using a piece of cotton or cloth moistened with 70% alcohol to reduce the risk of contamination. Place the sterilized sand around the plantlet and press lightly to keep the plantlet straight in the pot. remove the parafilm and the plastic cover from the test tube. If an autoclave is not available wash the clay pots (jiffy pots are already sterile) with detergent. Wash your hands with strong soap and 1% calcium-hypochlorite solution. 3.Transfer to planting mix 1. 33 . fill the pots with peat moss/sand mix. 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. Photo 2 11. at 25 to 27° C with 14-16 hours illumination. steam. 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. dirt. dust.

Apply 50-100 cm3 per 20 cm diameter pot. Dispense 4 cm3 of the medium in each test tube. 5. Again. Working under sterile conditions (sterile area or "microvoid") follow steps 8 to 10 of previous section. you may dissolve supplementary nutrient in the irrigation water. 3. Micropropagation 1. Cap the tubes with plastic caps or cotton wool plugs and autoclave them for 15 minutes. do not overwater. 19.5 cm stem segment with an axillary bud (Photo 4). Do not overwater.g. Transfer the plantlets from the test tuber to a sterile petri dish and make nodal cuttings using sterile scalpel and forceps. 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. Commercial peat moss often contains fertilizer.16. thus less additional nutrient may be required. Be careful not to break the roots. 18. Until the plants are well rooted irrigate lightly with tap water if it has a low salt content. transfer to larger (e. otherwise use demineralized or rain water. 20 cm diameter) pots.2-0. Once the plants are established. Each nodal cutting consists of a 0. When the plants are well rooted. Photo 3 17. Keep the test tubes vertical while the agar sets. 4. Photo 4 34 . When roots are established. Gradually expose the plants to the normal atmosphere by removing the beakers for short periods each day. 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. Prepare the nutritive growth medium according to the procedure given in the section on Medium to Sweet potato Micropropagation.

35 . label and place in a clean area where the room temperature is 25-27° C. Photo 5 7. Ensure that each cutting lies on the agar surface with its axillary bud pointing upwards (Photo 5). Place each nodal cutting in a test tube. Place 1 or 2 cuttings per test tube.6. or for further micropropagation (Photo 6).000 lux illumination for 14-16 hours each day. Give 45m E/m2/seg2 or 3. seal with parafilm. Close the test tubes. 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.

36 .

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