Plant Cell, l~ssue and Organ Culture 7." 3 -10 (1986] © Martinus Ni]hoffPublishers, Dordrecht - Printed in the Netherlands

I n d u c t i o n o f in v i t ro tube r s in a b r o a d range o f p o t a t o g e n o t y p e s

ROLANDO ESTRADA, PILAR TOVAR and JOHN H. DODDS*

International Potato Center, PO Box 5969, Lima, Peru

(Received I November 1985; in revised form 4 April 1986; accepted 6 April 1986)

Key words: in vitro tubers, potato, germplasm, seed tubers

Abstract. This paper describes a reproducible method for the induction of in vitro potato tubers in a wide range of genotypes. These in vitro tubers could be induced in all genotypes tested and show striking similarity to field produced tubers. In vitro tubers may prove most useful as material for International germplasm distribution.

Introduction

The potato is one of the world's most economically important crop plants. As a member of the Solanaceae family it is highly responsive to many tissue culture techniques. Subjecting the plant to a period of thermotherapy fol- lowed by meristem culture now routinely allows the production of pathogen- tested (virus free) stock cultures and in vitro cultures free from bacteria, fungi and viruses are used for micropropagation of large quantities of disease free plants [1, 2, 3]. These disease free plants form the nuclear stock of material in a potato seed tuber program. Disease free plants are high yielding and produce tubers of better marketable quality and resulting higher price.

International distribution of potato germplasm is routinely carried out for two principle reasons: (i) commercial export of seed tubers to those countries that lack the technical or physical facility to produce their own high quality seed, and (ii) the International Potato Center distributes germ- plasm for evaluation by national potato programs. There is an increasing tendency to export germplasm in vitro because of the ease of meeting plant quarantine regulations. The Potato Center exported over 7 500 in vitro plants in 1984 to over 40 different countries worldwide.

In recent years attention has focused on the induction of potato tubers in vitro in a limited number of varieties [4, 5, 6]. These tubers are small, light and are produced under aseptic conditions. In this paper we report a reproducible method for the induction of in vitro tubers in a range of geno- types from a wide genetic background and discuss the potential use of such tubers in International germplasm exchange.

* To whom correspondence should be addressed.

Materials and methods

Plant material

The plant materials used in this study are in vitro stock plants from the pathogen tested in vitro collection of the International Potato Center. These plants have previously been submitted to thermotherapy and meristem culture and have been rigorously tested for absence of viral and viroid patho- gens by electron microscopy, ELISA (Enzyme linked immunosorbant assay) and nucleic acid spot hybridisation. The in vitro plants were maintained on an in vitro conservation medium (see Table 2) at 8 °C with illumination (16 hour day) from fluorescent tubes at an intensity of 22 Wm -2 .

Table 1 shows a list of genotypes used in this study giving clone name and Potato Center accession number. The list contains varieties and advanced breeding lines and genetically it is composed of diploids, triploids, and tetra- ploids. The list of experimental material was specifically chosen to test this technology on the widest possible range of potato germplasm.

Methods

The general scheme used for routine induction of in vitro tubers is sum- marized in Figure 1. The growth and induction phases can be conveniently divided into three stages.

(A) Propagation o f Plantlets and Induction o f Tubers Stage 1 - Initial Propagation through the Use o f Single Node Cuttings. In vitro plants used in this study were taken from in vitro storage [7] and nodal cuttings were transferred to single node propagation medium (see Table 2), normally 4 nodal cuttings were inoculated into each test tube containing 4 cm a of propagation medium.

The plants were maintained at 22 °C under 16 hours day from fluorescent tubes at 22 Wm -2. Under these conditions, the young plantlets develop rapidly and provide sufficient material to start shaken liquid cultures.

Stage 2 - Shaken Liquid Cultures. Whole stem pieces of in vitro plantlets obtained in Stage 1 that have had the roots removed can be layered into a liquid propagation medium (see Table 2). Each stem se~aent was 4 cm long and normally had about 6 axillary buds along its length.

250 cm 3 erlenmeyer flasks containing 20cm 3 of shaken liquid propagation medium (see Table 2) were used as the culture system. Each flask was inocu- lated with a total of 30 nodal segments i.e. 5 stem pieces each with 6 nodes.

The flasks were shaken on an orbital shaker 60rpm, under 16 hour day from fluorescent tubes (22Wm-2). When cultured in this medium, the axillary buds grow rapidly and in two or three weeks the flasks are fiUed

Table 1. Potato genotypes used in this study, their name and accession number

Name CIP Code No. Genotype

R-128.6 376999.6 Tetraploid LT-1 377257.1 Tetraploid LT-2 377258.1 Tetraploid Maria Tropical 377260.1 Tetraploid LT-4 377319.7 Tetraploid LT-5 377957.5 Tetraploid LT-6 377939.5 Tetraploid LT-7 378017.2 Tetraploid ASN-69.1 573275 Tetraploid 1-1062 575010 Tetraploid N-503.31 573255 Tetraploid AGG-69.1 676026 Tetraploid AND-69.1 676028 Tetraploid BL-1.10 678009 Tetraploid BL-2.9 678011 Tetraploid BL-2.2 678019 Tetraploid Chiquillo 701022 Tetraploid Pinaza 702445 Triploid Atacama 702698 Tetraploid - 702867 Tetraploid Chaucha Roja 703269 Diploid Maman Pecke 703276 Diploid - 703294 Diploid Mariva 720025 Tetraploid Rosita 720044 Tetraploid Anita 720047 Tetraploid Montsama 720049 Tetraploid CGN-69.1 720050 Tetraploid CFE-69.1 720053 Tetraploid, Tollocan 720054 Tetraploid 65-Za-5 720055 Tetraploid CEX-69.1 720057 Tetraploid ARX-69.1 720060 Tetraploid Monserrate 720071 Tetraploid CFK-69.1 720084 Tetraploid Serrana Inta 720087 Tetraploid Huinkul 720090 Tetraploid 69-56-52 720093 Tetraploid Mutca 720097 Tetraploid CEX-69.1 720121 Tetraploid Mex 750815 720122 Tetraptoid Mex 750826 720124 Tetraploid Mex 750847 720126 Tetraploid Cipa Virus 720128 Tetraploid Pentland Crown 800034 Tetraploid DTO-2 800144 Tetraploid DTO-28 800169 Tetraploid DTO-33 800174 Tetraploid BR-63.74 800223 Tetraptoid N-565.1 800301 Tetraploid Atlantic 800827 Tetraploid Spunta 800923 Tetraploid Saturna 800943 Tetraploid San Gema 800949 Tetraploid Amapola 800950 Tetraploid

Table 2. Composition of different culture media used for conservation, propagation and tuberisation of in vitro potato piantlets. All media are based on the Murashige and Skoog (MS) [8] salts mixture with the following additions. All media were sterilised by auto- claving at 121 °C for 15 minutes

Single node Shaken liquid In vitro In vitro tuber Compound (mg/1) propagation propagation conservation induction

Thiamine HCL Ca-Pantothenic acid Gibberellic acid Benzyl amino purine Naphthalene acetic acid Chlorochlorine chloride Inositol Sucrose Mannitol ~ a r

MS + MS + MS + MS + 0.4 0.4 0.4 0.4 2.0 2.0 0.25 0.4

0.5 5.0 0.01

500 100 100 100 100

3% 2% 3% 8% 4%

0.8% 0.8%

I Propagate nodal cuttings

cut nodal cuttings

in vitro ~. ':t:}. 7"L plantlet ~ change media for tuber

~ induction media ~r

in vitro /q~ ~,~ shoot culture tuber formation ~ I

T ~ add CCC to existing media

l

1 ~*~k Liquid shaken

culture ~ propatlat ion

Figure 1. Schematic representation of methods used for in vitro tuber induction.

with plantlets. It is from these rapidly propagating plantlets in a liquid

media system that in vitro tubers are induced.

Stage 3 - Induction o f Tubers. In vitro tubers are induced as the result of an addition of Chlorocholine chloride (CCC) to the medium together with

Figure 2. In vitro tubers induced on cultivar Mariva.

an increase in the concentration of sucrose and benzyl amino purine (see Table 2).These additions can be achieved in two ways, firstly, by removingthe existing propagation medium and replacing it with medium containing CCC, BAP and 8% sucrose or, secondly, by adding stock CCC, BAP and sucrose solu- tions to the flask containing the original propagation medium. The flasks after addition of CCC stimulus were transferred to total darkness without agitation at 22 °C, in vitro tubers formation was analysed 40 days later, Figure 2.

(B) Harvest of ln Vitro Tubers

Flasks containing in vitro tubers for collection were moved to a sterile trans- fer bench and tubers harvested aseptically and kept in sterile petri dishes.

Table 3. Number of in vitro tubers per flask produced and tuber fresh weight for selected genotypes. 250 cm 3 Erlenmeyer flask were inoculated with 30 nodes and cultured under conditions indicated in materials and methods sections. Means and standard deviations for a minimum of 11 flasks of each genotype

Clone Mean No. tubers/flask Mean weight (mg)/tuber

Mariva 9.9 ± 1.8 163.3 ± 23.3 LT-2 12.1 ± 1.9 230.3 ± 30.1 DTO-2 9.7 ± 3.5 108.6 +- 16.9 LT-7 19.4 ± 3.1 64.5 ± 10.5 DTO-33 8.2 -+ 3.9 80.9 ± 26.2 LT-1 15.3 ± 3.5 54.9 ± 11.3 Pifiaza 20.8 ± 3.3 39.0 ± 7.0 Atacama 8.9 ± 1.6 59.0 ± 9.7 702867 13.4 ± 1.4 93.1 ± 19.0 Atzimba 9.8 ± 2.1 77.7 ± 11.2

Figure 3. Comparison of color and shape of in vitro and field produced tubers.

F o r each flask, n u m b e r , size and we igh t o f in vi t ro t ube r s was d e t e r m i n e d .

Table 3 shows yield da ta f rom a l imi ted n u m b e r o f geno types .

(C) Storage and Sprouting

In v i t ro t ube r s in Sterile 9 cm pe t r i p la tes sealed w i t h paraff irn were s to red in

the dark at 4 ° C for b e t w e e n 6 weeks an d 10 m o n t h s . These dishes did n o t

c o n t a i n any med ia or m o i s t e n i n g agent . Once r emo v ed to r o o m t e m p e r a t u r e

sp rou t ing o f the t ube r s qu ick ly occurred.

(D) Packaging for In ternational Dis tn'bu tion

Sprouted in vitro tubers were placed carefully into sterile petri dishes con- raining sterile cotton wool moistened with a few millilitres of sterile distilled water. The packaged tubers are distributed by air freight, each package certified with a government phytosanitary certificate. Sending sprouted tubers has the advantage that material can be used immediately upon arrival.

Remits and discussion

Using the propagation and induction methods shown in Figure 1, and an induction medium known to be optimal for tuberization (9) in vitro tuberi- zation was attempted in over 50 different genotypes (Table 1). In all cases it was possible to induce the formation of in vitro tubers. Table 3 shows yield characteristics of a selected number of genotypes.

The tubers are small, the mean tuber diameter is about 5 mm. In vitro tubers appear to be morphologically similar to field produced

tubers (Figure 3). It has also been shown that these tubers can be used to produce seed tubers with no notable differences to using in vitro plantlets. In vitro tuber dormancy can be influenced by daylength conditions during their induction phase, when induced under short days they have a dormancy period of 60 days when stored at 4 °C while induction in complete darkness causes a dormancy period of 210 days under similar storage conditions.

It would appear, therefore, that plants ctenved from these tubers are normal and they should, therefore, be able to play a useful role in the pro- duction of seed tubers. This is already the case in some national potato programs.

The export of germplasm by the International Potato Center is an im- portant part of its mandate to encourage potato production for use as a basic food. A major problem with export of in vitro plantlets is that if the plants are kept in the dark for longer than 3 weeks during transit, they will die. The export of sterile in vitro tubers would allow the phytosanitary benefits of in vitro export without the technical problems of shipping green plants. Upon receipt in vitro tubers can be maintained in vitro for further micro- propagation or can be transplanted into pots to provide donor plants for production of conventional cuttings. Using these methods over 3000 in vitro tubers have already been exported to over 7 different countries.

It is preferable to ship the in vitro tubers in the sprouted state rather than in the dormant state because often the national programs need to plant directly. Sending dormant tubers would require reproducible methods for dormancy break of in vitro tubers.

Further studies will be necessary to understand more fully how these in vitro tubers can fit into a commercial seed production program, however, in the near future it can be anticipated that their use for International germplasm will rapidly expand.

10

References

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2. Roca WM, Bryan J, Roca MR (1979) Tissue culture for International transfer of potato genetic resources. Amer Pot Journal 56:1-11

3. Espinoza N, Estrada R, Tovar P, Bryan J, Dodds JH (1984) Tissue culture micro- propagation, conservation and export of potato germplasm. CIP Specialized Tech- nology Document. Lima, Peru

4. Wang P, Hu C (1982) In vitro mass tuberisation and virus-free seed potato pro- duction in Taiwan. Amer Pot Journal 59:33-37

5. Hussey G, Stacey NJ (1981) In vitro propagation of potato (Solanum tuberosum) Ann Bot 48:787-796

6. Koda Y, Okasawa Y (1983) Influences of environmental, hormonal and nutritional factors on potato tuberisation in vitro. Jap J Crop Sci 52:589-591

7. Schilde-Rentschler L, Espinoza NO, Estrada R, Lizarraga R (1982) In vitro storage and distribution of potato germplasm. 5th International Plant Tissue Culture Con- gress. Japan. Ed Fujiwara

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

9. Tovax P, Estrada R, Schilde-Rentsehler L, Dodds JH (1985) Induction of in vitro potato tubers. CIP Circular 13 (4): 1-4. International Potato Center, Lima, Peru