Potato Research 40 (1997) 439 - 453 REVIEW

Innovative propagation methods in seed tuber

multiplication programmes
PAOLO RANALLI

Istituto Sperimentale per le Colture lndustriali, Via di Corticella 133, 40129 Bologna, Italy

Accepted for publication: 22 November 1997

Additional kevwords: vitroplantlets, microtubers, minitubers, genetic stability, storage and

physiological age" lield performance, seed production programmes, Solarium tuberosum L.

Summary
The production of large volumes of vitroplantlets and greenhouse tubers for increasing the rate
of multiplication at the start of seed programmes provides the opportunity of reducing the total
number of field generations grown before the seed moves into commerce. This implementation
is especially useful for countries where high quality potato seed tubers cannot be produced
because there are no vector-free production areas.
This review covers the lollowing steps: a) laboratory production of microplantlets and
microtubers: b) minituber production in the glasshouse: c) storage and dormancy of micro- and
minitubers: d) field performance of micro- and minitubers compared with conventiomd seed
tubers: e) incorporation of the mentioned propagules in seed production systems.
Many optimized protocols are already available for propagating plantlets, inducing
microtubers and obtaining minitubers in the glasshouse at all periods of the year. Advanced
molecular approaches techniques (RFLP and RAPD) to detect genetic variation in the
progeny of these propagules have been described. Investigations carried out in this field have
shown genetic stability, with the propagulcs usually reproducing plants true-to-type and tubers
without deviants. By contrast, variations were demonstrated in DNA extracted from old
suspension cell cultures. Field trials assessed a lower yield potential crops from in vitro
propagules compared with conventional seed tubers, mainly due to slow early crop
development and the failure of plants caused by early stress after emergence. This may cause
problems when the growing season is short because of the necessity for planting late to avoid
night frosts and the mandatory haulm killing dates, common in many seed producing areas.
Strategies for improving the lield performance of micro- and minitubers are discussed. The
most promising crop husbandry techniques appear to be: a) using tubers of a suitable
physiological age. properly presprouted and encapsulated: b) optimizing the time application
of fertilizer and irrigation, and c) using floating films.
Outside the classical seed tuber areas of Northern Europe where the length of the growing
period for prc-basic seed is usually not more than 80 days. the growing season is long enough to
obtain reasonable yields even from micro- and minitubers.

Introduction

C o n v e n t i o n a l s e e d p r o g r a m m e s take m o r e than 10 years and in each r o u n d of field

multiplication diseases b e c o m e increasingly c o m m o n and especially those
t r a n s m i t t e d t h r o u g h seed tubers. By contrast, the p r o d u c t i o n of large v o l u m e of
p r o p a g a t i o n m a t e r i a l in p r o t e c t e d e n v i r o n m e n t s requires only a few a d d i t i o n a l years
of t r a d i t i o n a l s e e d multiplication in the field to p r o d u c e the d e s i r e d seed lot with an
i m p r o v e d health status. This is useful especially in c o u n t r i e s where there are no
v e c t o r - f r e e p r o d u c t i o n areas for p r o d u c i n g high quality p o t a t o seed tubers.

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This review covers aspects concerning the laboratory and screenhouse production
of vitroplantlets, micro- and minitubers, their performance after planting and their
incorporation into seed production schemes.

Propaguleproduction
For many years, tissue culture has been used for improving potato production by
means of germplasm conservation (material is available all year round and is
protected from environmental and pathological risks), pathogen elimination (tissue
culture of the meristem tips of heat-treated plants and the use of antivirai chemicals)
and micropropagation (revolutionary impact in the seed potato industry by replacing
part of the system of clonal selection).
In the last few decades, alternative seed production programmes have been
developed in which the first multiplication steps are speeded up using in vitro
plantlets (Roca et al., 1978: Hussey & Stacey, 1981: Wattinema, 1983), microtubers
(Wang & Hu. 1982: Hussey & Stacey, 1984: Rosell et al., 1987: Forti et al., 1991) or
minitubers (Struik & Lommen, 1990: van der Zaag, 1990).

h7 vitro plantlets
Many techniques have been developed for producing potato plantlets on nutrient
medium in aseptic environments. The basic methods used are similar in most
laboratories and are based on the rapid growth of single node cuttings of stems with
multiple nodes on solid or liquid culture media.

a) Growth of single node cuttings. Single nodes with leaves are excised from small in
vitro plantlets and inoculated onto the surface of agar-solidified medium (Espinoza et
al., 1984). The axillary bud quickly grows, and in 3-4 weeks a plantlet with six or
seven more nodes becomes available for subculture.

b) Growth of stem multiple nodes. In vitro plantlets are cut into stem cuttings each
with three or four nodes and the large leaves removed. Each stem piece is placed in
15 ml of liquid medium (Espinoza et al.. 1984) and the flasks are shaken (80 rpm).
After 2-3 weeks each flask contains 60 to 70 nodes and once a suitable number of
small plantlets has been produced, they can be transferred to non-sterile contitions,
i.e. transplanted into beds of pots for tuber production.

c) Basic media. In vitro culture of potato seed stocks is largely based on the
Murashige-Skoog medium originally used for tobacco tissue culture (Murashige &
Skoog, 1962). Growth regulators are usually omitted from media used for nodal
segment propagation and a few programmes use Geirite, a synthetic polysaccharide,
as a substituted for agar. Jones (1988) found that Gelrite improves rooting and
produces sturdier, darker green plantlets. Some fast growing cultivars do well with
the addition of succinic acid (Alar-85 of B9) at the rate of 2-5 mg/I (Jones, 1988).
Media pH shows a surprisingly wide range, with a minimum of 5.2 and maximum of

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 A D V A N C E S IN SEED TUBER P R O D U C T I O N

6.5. Numerous containers are used for in vitro culture. In North America glass test
tubes and Magenta polycarbonate boxes are used most frequently, while in Europe
glass test tubes predominate. Magenta caps and snap-on covers, all of translucent
polypropylene, are used most commonly in North America while in Europe
"'natural" plastic, glass, metal and cotton plugs are used.
In our laboratory (Ranalli et al., t988, 1990b), the routine production of in vitro
plantlets follows this protocol: initial explants derived from sprouts of virus-free
tubers are sterilized, cut into 5-10 mm nodal segments and planted in vessels, each
containing 100 ml of half-strength Murashige & Skoog (1962) medium, adjusted to
pH 5.7 and without growth regulators but with 3% sucrose and 0.7% agar. Cuttings
placed on nutrient agar produce upright shoots from the axillary bud, The resulting
plantlets are multiplied by cutting them into nodal segments, each including 4 mm of
stem, and subcultured on fresh medium every 4 weeks until there are sufficient
plantlets. The multiplication rate ranged from x 3 to x 5 per month, so that 3 to 5 new
cuttings can be obtained from one plantlet after 4 weeks growth. Cultured shoots
quickly develop roots and there is no need for a separate rooting stage. The plantlets
are lifted out, carefully separated and planted out in the greenhouse.

Microtubers
Many protocols have been developed to induce in vitro tubers. Important factors
during the tuberization period are: (a) the sugar concentration in the medium (8%
optimal): (b) the nitrogen content (there is a clear interaction between sugar
concentration and nitrogen concentration): (c) the temperature (incubation at
18-20 ~ is preferable): (d) the light conditions (incubation can occur in the dark or at
low light intensity (100-150 lux) with a photoperiod of 8 h).
A reliable microtuber production method on a medium free from any growth
regulating agent was reported by Garner & Blake (1989), Nodal cuttings were grown
on medium containing MS basal salts (Murashige & Skoog, 1962) with 80 g 1-l sucrose,
and incubated first under a 16 h and then an 8 h photoperiod. After 17 weeks of
incubation each cutting had produced approximately I microtuber weighing <200 mg.
In our laboratory (Forti et al., 1991: Ranalli et al., 1988), microtubers are routinely
produced by taking single node cuttings from in vitro plantlets and growing them in a
tuberizing medium containing half-strength Murashige & Skoog mineral salts and
vitamins, supplemented with 8% sucrose and 0.7% agar. Cultures including 12 nodes
are grown in 510 ml glass jars containing 50 ml of agar medium. The photoperiod is 8
hours with a light intensity of 400 lux provided by 1200 mm fluorescent tubes, and the
temperature is 22 ~
Microtubers begin to appear 3 weeks after transferring the cuttings to the medium:
some are apigeal and others hypogeal. Usually, epigeal microtubers grow at the end
of the shoots or are axillary, whereas microtubers developing within the culture
medium grow either from stolons or are produced from shoots that have grown
downwards into the medium. One or occasionally two tubers form on each plantlet
and tuberization (%), diameter and length of the tubers, and fresh weight are
assessed 8 weeks after the beginning of the induction. Microtubers vary in shape

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PAOLO RANALLI

(rounded or elongated), surface (smooth or rough), colour (yellowish to greenish).

weight (ranging from 24 to 273 mg), diameter (4-7 mm) and length (10-12 mm).

a) bnprovement of microtuber. In cvs Monalisa, Primura and Spunta activated

charcoal affected the induction and development of microtubers (Bizarri et al., 1995):
media containing activated charcoal for microtuber induction gave a higher rate of
tuberization and larger microtubers than hormone-free media.
The presence of activated charcoal in the nutrient media promotes cell and tissue
culture by absorbing inhibitory metabolites or other substances released during
growth, and probably affects nitrogen biochemistry and the ammonium:nitrate ratio
within the plant, the development of microtubers being strongly influenced by
nitrogen nutrition (Garner & Blake, 1989). Activated charcoal thus plays a role in
optimising conditions for rapid, mass tuberization of these cultivars, and produces
large microtubers for field planting.
Photoperiod plays an important role in the tuberization process (Gregory, 1956)
and should be optimized to enhance tuber size. The effects of different photoperiods
on in vitro tuberization of four potato cultivars (Red Pontiac, Shepody, Kennebec
and Yukon Gold) were investigated by Slimmon et ai. (1989). Microtubers from all
cultivars had a higher mean fresh weight when treated with an 8 h photoperiod
compared with total darkness; Red Pontiac and Shepody produced microtubers that
were well over twice the fresh weight of those produced in the total darkness
treatment. Delayed leaf senescence, microtuber greening and nodal rooting were
evident with 8 h photoperiod,
Most of the microtubers produced in total-darkness were positioned above the
medium. The majority of microtubers produced in 8 h photoperiod formed either
directly under the medium or were part-way into the medium, particularly with cvs Red
Pontiac and Shepody. Since these cultivars were the same as those that produced larger
microtubers, the positioning of the developing microtuber may have an effect on its
fresh weight. This effect could be a consequence of water and/or nutrient absorption
through the lenticel of microtubers in direct contact with the medium. Microtubers
which formed within the medium displayed very prominent lenticels compared with
those formed above the medium. Prominent lenticels may, however, cause the
microtubers to be more susceptible to desiccation immediately after harvesting.

b) Tuberization. As with in vivo tuberization, in vitro potato tuberization is a

developmental system that encompasses a number of important biological processes
regulated by differential expression of genes. The swelling or the axillary buds can be
detected 5 days after induction and the morphology of the microtuber is apparent after 6
days of culture (D6sir6 et al., 1995a,b). From the 12th day, the sessile microtuber becomes
round in shape with a diameter of 2-3 mm. Thereafter, with the growth of cortical cells
and the high accumulation of starch and protein, the final size is reached (4-5 mm).
Morphological, biochemical and molecular approaches have been made to
examine the mechanism associated with tuber initiation and development. Duncan &
Ewing (1984) used single-node cuttings to study anatomical changes associated with

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 ADVANCES IN SEED TUBER PRODUCTION

tuber formation from axillary buds. They showed that starch deposition and cell
mitosis were the earliest detectable changes in anatomy associated with tuber
initiation. Taylor et al. (1991) showed that there were a number of quantitative and
qualitative changes in protein composition and translatable mRNA occurring during
the very early stages of tuber development. The major qualitative changes appeared
after 11 days of culture and were characterized by the accumulation of polypeptides
with an apparent molecular mass of 42--43 kDa, which are likely to be part of the
patatin family (D6sir~ et al., 1995b). At molecular level, substantial increases in both
total protein and total RNA were observed at the onset of tuber morphology.
lmmunoblot analysis showed that the major tuber protein, patatin, could be detected
initially in day 4 buds and that a 22-kDa proteinase inhibitor could be detected
initially at day 8. Northern blot analysis corroborated this pattern of accumulation at
the RNA level for both protein types (Hannapel, 1991).

Minitubers
Minitubers are small tubers of 5-20 mm diameter (0.1-6 g) produced in vivo. They
combine the advantages of in vitro plantlets (disease-free, rapid and year-round
production) and tubers (easy storage and transport) and lack some of the
disadvantages of in vitro tubers (low multiplication rate, small size).
Minitubers can be obtained from in vitro plantlets or microtubers and plants raised
from both propagules can be subjected to a destructive harvest or to repeated
harvests.

a) Destructive harvest. In vitro plantlets of microtubers are planted in a screenhouse

or in the open field and harvesting occurs at the end of cycle. The propagules are
planted at low density and grown without special techniques to increase the number
of tubers per individual plant. This system produces a small number of tubers per m 2,
but the tubers will grow to a larger size (more than 15 g). Minituber production is
affected by genotype and crop husbandry techniques. Cultivars differ widely in their
capacity to produce minitubers, some being much more prolific than others. Cv.
Bintje produces an exceptionally high number of minitubers, and also gives the
highest yield. The type of greenhouse (glass, fibreglass, plastic), lighting, soil, time of
year and planting density also contribute to a wide range of tuber yields (Jones,
1988).

b) Repeated harvests. This approach, developed and studied by Lommen & Struik
(1992a,b,c), involves the planting of in vitro propagated plantlets in a glasshouse
under tuber-inducing conditions and removing tubers by repeated harvesting. With
this method it was possible to produce about 3500 minitubers >5 mm per m 2 when 350
plants were planted per m2 and tubers were harvested after 4, 7 and 10 weeks
(Lommen & Struik, 1992c).
The success of this method depends on three factors: (1) the state of the in vitro
propagated plantlets when they are planted in the glasshouse; (2) the climatic
conditions during minituber production in the glasshouse, and (3) the frequency and

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PAOLO RANALLI

timing of the consecutive harvests.

Experiments carried out by Ranalli et al. (1994c) showed that the bench technique
compared with screenhouse raising made it possible to increase the tuber production
per unit surface (+300-400%) and per plant (+200-300%), even if the size of tubers
obtained was smaller.

Genetic stabili O,
Two techniques can be employed in seed tuber production: meristem culture (culture
of dissected portions of the meristematic region of shoot tips) and nodal cuttings
(culture of single nodes with leaves excised from small in vitro plantlets). Techniques
that use existing buds for shoot formation provides more stability. For example,
starting from single explant cultures of cvs Kennebec, D6sir6e and Red Pontiac,
Ahloowalia (1994) produced more than 30,000 micropropagated plants in various
experiments during 1990 and 1991, and ca 25,000 minitubers were produced from
different cultivars: among in vitro plantlets, only a single variegated shoot was
observed, suggesting a high degree of genetic stability of the micropropagated
material. The minitubers also reproduced true-to-type plants and tubers with no
deviants for leaf shape, flower and tuber skin colour. This contrasts with the high
degree of somaclonal variation reported among plants produced through adventious
shoot formation (Roest & Bokeimann, 1980) and regeneration from callus
(Ahloowalia, 1982: Cassels et al., 1983).
A study to evaluate by molecular analysis the possible variation of plants of cvs
Monalisa and Spunta derived from conventional tubers, microtubers, cell cultures
and microplantlets was carried out by Mandolino et al. (1993, 1996). Analysis of the
genetic stability was performed by probe hybridization and genomic Southern blots
(RFLP-restriction fragment length polymorphism) and by randomly amplified
polymorphic DNA (RAPD) analysis. The result shows in both cultivars identity of
the DNA patterns of vitrotuber-deriving plants with the pattern of the conventional
tuber-deriving plant. By contrast, the pattern of identification of cv. Spunta was
greatly altered when DNA was extracted from two year old suspension cultures
growing in the presence of growth regulators is considered.

Storage or propagules and dormancy

Storage
Micro- and minitubers are dormant and must be stored before use. Difficulties in
storage and sprouting ability are greater in microtubers because they are very small
(average 90-120 mg). Investigations by Ranalli et al. (1994a) showed that the length
of dormancy is inversely correlated with tuber size, and that small tubers suffered
dehydration when stored for a long time, with reduced growth vigour when planted
directly in the field.
Release of dormancy
The mechanisms controlling potato tuber dormancy maintenance and release are not
well understood. Water content of the microtubers decreases during the first weeks of

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 ADVANCES IN SEED TUBER PRODUCTION

storage whereas the total soluble protein is more variable. Two-dimensional

polyacrilamide gel electrophoresis of proteins extracted from microtubers re,~,eals
changes during storage. The major changes appear after 3 weeks and are
characterized by the accumulation of three polypeptides, with an apparent molecular
mass of 3l kDa. A protein set with molecular mass of 40 kDa, which is likely so be
part of the patatin family, and one polypeptide with a molecular mass of 29.8 kDa
increase strongly between 4 and 9 weeks. When first germination is possible after 9
weeks, these polypeptides decrease in amount or are no longer detected. It can be
speculated that their synthesis and accumulation during the dormancy-breaking
treatment are necessary to obtain germination, especially as their function may be
related to first events leading to dormancy break.
A comprehensive study of this subject was carried out by Ranalli et al. (1994a).
Induction. dormancy length, advancing of physiological age and related field
performance of microtubers were investigated. After storage in the dark at 3 ~ for
different periods (25, 56.84. and 105 days) microtubers were transferred to 20 ~ for
between 4 and 17 weeks. Following removal to room temperature, sprouting and
dormancy were recorded. The dormancy duration was linearly and inversely
correlated with the length of storage and decreased from 28.1 to 19.9, ll.1 and 7.8
days, respectively for 4 to 16 weeks storage. Cvs Arsy, Nicola and Jaerla took
consistently more time to break dormancy.
After sprouting, tubers were kept at 20 ~ for various intervals and a range of
physiological ages (0, 368, 720, and 1008 degree days above 4 ~ were. accumulated.
The field comparison of microtubers showed a plant growth response, and tuber
yield/plant was influenced by the cultivar and physiological age. In early cultivars
(Jaerla). a better performance was shown by younger mother tuber,,: the opposite
trend was noted in Alpha (a late cultivar) with an increase in stems/plant,
tubers/plant and tuber yield/plant for tubers with greater physiological age.
In another experiment (Leclerc et al., 1995), microtuber size, media growth
regulators, incubation period and endogenous abscissic acid content of microtubers
were evaluated for their effects on length of dormant period of cvs Kennebec, Russet
Burbank and Superior microtubers. The dormant period of microtubers was found to
be cultivar-specific and significant correlation was observed between in vitro and in
vivo dormant periods. Smaller microtubers (<250 mg) had longer dormant periods
than did those >250 mg. Dormant periods were unaffected by addition of coumarin
or 2-(chloroethyl)-trimethylammoniumchloride and 6-benzylaminopurine to the
culture media or by the incubation period (28 and 56 days). Developmental age had
no effect on the ability of individual buds to break dormancy and elongate. A positive
correlation was observed between tissue levels of abscissic acid and microtuber
dormant periods.

Per]brmance of potato minitubers after d(fferent storage periods

After planting, the performance of minitubers may be affected by physiological age,
as observed with "normal" seed tubers (Madec & Perennec, 1955). The physiological
age of tubers increases with increasing storage duration (Krijthe, 1962: Bodlaender &

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PAOLO RANALLI

Marinus, 1987) and is affected by conditions and treatments during tuber growth
(Krijthe, 1962; van Ittersum & Struik, 1992) and during storage and presprouting
(O'Brien et al., 1983: van Ittersum et ai., 1993).
An experiment was conducted in growth chamber, using 15 mm minitubers of two
clones (B-41189 and B-42199) subjected to two temperature regimes of storage
(18/14 ~ and 23/18 ~ and five physiological ages (2, 4, 6, 8 and 10 weeks)
(Rykaczewska. 1992). The two temperature regimes gave plants characterized by
different rates of emergence, morphology, yield and numbers of tubers. The higher
temperature resulted in earlier emergence and smaller and deformed leaves in both
clones. The yield of tubers per plant decreased from the higher temperatures (212 g)
to lower temperature (283 g), while the average number of tubers per plant increased
(6.8 and 9.3 respectively at higher and lower temperatures). The plants grown from
minitubers were generally one-stemmed, however the yield and number of tubers
increased systematically with increased physiological age of the seed tubers. The
averages across the two clones were 200 g and 6.9 tubers/plant from 2 weeks of
presprouting and 288 g and 9.3 tubers/plant from 10 weeks of presprouting.

Utilization of propagules

In vitro plantlets
These are delicate planting stocks requiring special growing conditions and usually
cannot be multipled in the field. Planted in nursery beds and compared with
microtubers, these propagules produce significantly more smaller tubers than plants
grown from microtubers (Wiersema et al., 1987). The in vitro plantlets are a suitable
material to produce minitubers when transferred outside into an aphid-proof
screenhouse or into a growth chamber and subjected to destructive of repeated
harvests. Generally they are transplanted into growth chambers at high plant density
and minitubers can be harvested at intervals of three weeks from the same plantlets
(Lommen & Struik, 1992c). The period from planting to the last harvest may extend
to 10 weeks, and with several plantings minitubers can thus be produced throughout
the year.

Microtubers
Microtubers may prove most useful as material for international germplasm
distribution and exchange. The export of sterile in vitro tubers would allow the
benefits of in vitro export without the technical problems of shipping green plants. A
major problem with export of in vitro plantlets is that if they are kept in the dark for
longer than 3 weeks during transit they will die. In seed tuber production schemes,
microtubers, like in vitro plantlets, can be planted into the field. However, this is
hazardous because the plants grown from them are susceptible to frost which can lead
to complete loss. Although the use of plastic film reduces this risk, lower emergence
and survival rates and increased risks of infection with Rhizoctonia solani and virus
can occur (Struik & Lommen, 1990).
The field performance of microtubers has been investigated by many workers.

446 Potato Research 40 (1997)

 ADVANCES IN SEED TUBER PRODUCTION

Haverkort & Marinus (1990) used microtubers weighing less than 0,5 g planted
directly in the field or allowed to grow in small plots in the greenhouse at higher
temperatures than outside until about 15 cm high, when they were transplanted to the
field. Directly planted microtubers yielded less than half of a conventional crop (342
vs 870 g/plant in cv. Gloria and 452 vs 978 in cv. Morene). Transplanting of
microtuber plants led to an increase with cv. Morene only from 452 to 697 g/plant and
from 5.57 to 9.73 tubers/plant. The lower yields of crops from microtubers was mainly
due to slow early crop development, and full ground cover was reached 5 weeks later
than with crops from seed tubers. It is clear that crops from microtubers yield less
than crops from normal seed tubers, particularly when they have to be harvested
early to maintain health.
Bus et al. (1990) investigated the possibility of using microtubers (cv. Bintje) for
seed potato production in the field at four locations in The Netherlands. Field
planting of microtubers with and without the use of plastic film to increase
temperature and to preserve soil moisture was compared with transplanting plantlets
grown to 8-10 cm from microtubers in a greenhouse and with normal seed. The
microtubers emerged 2-3 weeks later and developed very slowly compared with
normal seed. The effect of plastic film was limited because of warm weather, and
transplanting led to a more regular crop. Earthing up more than once did not increase
the number of tubers and larger microtubers led to a more rapid crop development
than smaller ones. The efficiency of light use of crops from microtubers was equal to
the efficiencies of crops from normal seed tubers.
The role of husbandry techniques on the performance of microtubers was also
investigated by Struik & Lommen (1990). A crop grown from microtubers without
plastic film reached 50% ground cover approximately 6 weeks later than a crop
grown from normal seed. When the crops must be killed at an early stage because of a
short growing season or mandatory haulm killing dates, the slow rate of development
will result in a much lower yield. This effect depends on the size of the mother tubers
and is affected by their age.

Minitubers
Of the two methods tested, growing micropropagated plants in soil seems to have
several advantages over microtubers for obtaining minitubers. Not only are the
durations of in vitro microtuberization, storage, breaking microtuber dormancy and
growing all avoided by direct transfer of micropropagated plants to soil, but the
minitubers produced are relatively large, allow better handling, and conform to the
morphology of the parental cultivars. Whereas the microtubers of all cultivars look
similar in shape and skin colour, minitubers maintain their tuber identity in tuber
shape, skin colour and texture. Hence, minitubers allow a better control of varietal
purity and detection of off-types and somac[onai variants for skin colour and texture
than microtubers. In addition, the direct transfer of plants to soil allows a rapid
through-put of plants in the culture rooms.
The performance of minitubers was investigated taking into account different
aspects:

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a) Sprout growth. In general, micro- and minitubers have a tendency to produce

single stems which later produce several lateral shoots. By contrast, the standard
sized tubers produce multiple shoots on sprouting, and very few lateral shoots. The
primary shoots in the micro- and minitubers appear to have apical dominance, and
inhibit sprouting of the shoots from the remaining eyes.

b) Emergence. Slow emergence from minitubers is due to small amounts of tuber

reserves available for growth and to slower haulm development caused by the
relatively small root system of plants from the small tubers. In addition, both faster
emergence from conventional tubers compared with minitubers and quicker
development of ground cover after emergence led to a higher amount of radiation
being intercepted during the growing season. Because of the increased quantity of
radiation intercepted, dry-matter production from conventional tubers is also higher.

c) Field pelformance. (1) Effect of physiological ageing. This topic was investigated
by Lommen & Struik (1993) with minitubers of cvs Agria and Liseta. Their
performance was assessed 65. 128, 191,254, 317, 380, 443, 506 and 569 days after
harvest. Minitubers (1-2 g) were planted in pots and grown for 8 weeks in a
controlled environment. Tallest plants and largest leaf areas per plant were observed
in plants from tubers that had been stored 317 days. (2) Effect of weight of
minitubers. This parameter affects sprout growth, emergence, crop establishment
and yield formation. The behaviour of minitubers in five weight classes, ranging
between 0.19 and 3.00 g, was studied with respect to sprouting and emergence under
controlled conditions (Lommen & Struik, 1994). Lighter tubers took longer to
produce sprouts of 2 mm than heavier tubers and when tubers with sprouts of the
same length were planted in pots, sprouts from lighter tubers took longer to emerge.
Emergence was later and differences between weight classes were larger when tubers
were planted deeper (6 or 9 cm) or when they had shorter sprouts at planting (2 or 4
mm) (Lommen, 1994). Conventional tubers appeared superior to minitubers in all
characteristics mentioned except radiation conversion coefficient which was similar.
Other experiments were aimed at enhancing the performance of minitubers by
proper handling and planting techniques. Encapsulation of the minitubers in peat-lite
mix, closer seed spacing and planting two minitubers per hill enhanced performance
and increased yields compared with minitubers without mix, wider seed spacing and
one minituber per hill (Melching et al., 1993). Minituber comparisons showed that
peat-lite mix enhanced emergence, produced larger, more vigorous vines and higher
yields (44% average increase). Reasons for enhanced production may be attributed
to a warmer initial tuber environment, a more uniform moisture supply or better
nutritional conditions regardless of encapsulation and closer seed spacing with two
minitubers/hill increased yields by an average of 35% and 20%, respectively.
compared with wider spacing and one minituber/hill. Finally, yields of encapsulated
minitubers compared favorably with cut (50 g) and whole (80 g) seed, averaging
103 % of the cut and 87 % of the whole seed yields respectively.

448 Potato Research 40 (1997)

 ADVANCES IN SEED TUBER PRODUCTION

Fiehl pe#~f'ormance o f micro - arid minitubers compared with normal tubers

Investigations by Ranalli et al. (1994b) compared normal tubers, minitubers and
microtubers of cv. Monalisa planted at similar densities but at two distances between
rows (60 and 90 cm). The field performance of propagules was assessed by recording
plant emergence, ground cover and tuber yield.

Plant emergence aml ground cover. Plants from microtubers emerged 8-9 days later
than those from minitubers and 14.2 days later than those from normal tubers. The
micro- and minitubers had only one emerging stem and after one month differences
in haulm growth were evident: the stems of micro- and minitubers branched
profusely to give short, bushy plants. The number of days to 50% emergence was not
significantly affected by distance between rows but varied from 24.2 for microtubers
to 13.9 for minitubers and 1(I.5 for normal tubers (mean of two spacings). Also, the
percentage ground cover 51 days after planting was not influenced by spacing
between rows and average values were 10.5, 37.8 and 72.4% respectively for micro-
mini-, and normal tubers. These differences persisted during the later developmental
stages of the plants so that final ground cover was almost complete only in the crops
derived from normal tubers, and decreased with the size of the mother tubers.

Tuber viehl. The distance between rows significantly affected the performance of
micro- and minitubers. At close row spacing, microtubers and minitubers vielded
more than at wide spacing. With microtubers, total tuber yields were 27.3 and 6.7 t/ha
respectively for close and wide spacings and with minitubers they ranged from 38.9 to
24.4 t/ha. Normal seed yielded 47.5 and 54.2 t/ha, respectively.
Tuber size distribution was also influenced by the source of the material, and the
proportion of tubers in the ware fraction (>45 ram) increased from microtubers.
minitubers to normal tubers. Progenies of microtubers were about 74% in tuber class
>36 ram, 25% in class 35-55 mm and 1.0% in class 55-80 mm. By contrast, plants
originating from normal tubers produced most tubers in the 36-55 mm class (57.9%).
The size of mother tubers affected the number of tubers per m 2 and microtubers
produced the highest values at close spacing between rows. The occasional
phenomenon of a higher number of tubers in micro-propagated material than in
conventional planted crops has been reported (Levy, 1985: Leclerc & Donellv. 1990)
but its cause is not fully understood. In plants produced from microtubers, tuber
initiation probably occurs over a longer period and resorption is lower than with
certified seed-produced plants. In plants produced from normal seed, some
researchers (Doncaster & Gregory, 1948: Krijthe. 1955) found that all the tubers are
initiated over a very short period, while others (Milthorpe. 1963: Moorbv & Milthorpe.
1975) believe that tubers continue to form late in season, but many are resorbed.
Microtubers yield less than minitubers and normal tubers because of the large
number of small tubers in their progeny. This could be improved by preplanting the
microtubers in pots in the greenhouse before transplanting to the field and might lead
to earliest and larger foliage ground cover and perhaps to more tubers per plant
(Haverkort & Marinus. 1990). The performance of minitubers is superior to

Potato Research 40 (1997) 449

PAOLO R A N A L L I

microtubers but inferior to normal tubers. The distance between rows significantly
affects the performance of micro- and minitubers and closer spacing gives a
significant increase in tuber yield per ha in plants produced from both propagules.
Ground cover produced by plants derived from micro- and minitubers is not
complete, showing that part of the field does not contribute to the nutrient supply of
the crops and thus could explain lower yield potential with respect to normal seed,
Yields from small seed tubers might be improved by increasing sprout or stem
density, or decreasing the rectangular arrangement of plants by reducing the spacing
between rows. Closer spacing between rows is probably more effective in increasing
yield than planting at greater sprout density.

Concluding remarks

According to Struik & Lommen (1990). field experiments with micro- and minitubers
have revealed the following problems:
a.the lack of tolerance to early heat and drought stress due to small size of mother
tubers:
b.the need for special cultivation techniques, including the preparation of a good
seed bed and timely irrigation and fertilization:
c. poor early vigour, requiring more attention to weed control.
These problems might be solved by producing larger-sized microtubers using
modified in vitro procedures. Hussey & Stacey (1984) tested the effect of liquid
media on enhancing both tuber number and weight and they produced tubers up to
200 mg, possibly by allowing more efficient nutrient absorption. The density of
cuttings in the culture vessels during the tuberization period also influenced tuber
size and weight. Forti et al. (1991) used densities of 25 and 8 nodes per vessel and
obtained microtubers with diameter ranging from 3-5 mm to 5-8 mm, and weight
ranging from 80 mg to 450 mg for high and low densities respectively.
Increase in vigour during the early development is provided by covering the plants
with floating plastic film. proper pre-treatment of the tubers (chitting, pre-sprouting,
encapsulation), pre-growth in a greenhouse followed by transplanting of the young
plant material and by timely irrigation and fertilization.
In this way it is possible to reduce the time of emergence, to increase the proportion
of emerging and surviving plants, to enhance haulm growth after emergence and to
increase the harvest index.

Incorporating propagules in seed production system

In Northern European countries micropropagation is combined with traditional

clonal selection procedures and so a proportion of the basic plants used as starting
material for clonal selection has already been replaced by in vitro plantlets or by
minitubers. In some countries all basic seed is also now derived from
micropropagated plants. It is assumed that this replacement has improved the
standard of health of the seed (van der Zaag, 1990).

450 Potato Research 40 (1997)

 A D V A N C E S IN S E E D TUBER P R O D U C T I O N

In Southern Europe, the use of propagules raised in vitro (microplantlets or

microtubers) or in the greenhouse and in aphid-proof screen (minitubers) has
produced a revolutionary impact on the production of pre-basic seed where high
quality potato seed tubers cannot be produced because of the lack of vector-free
production areas (Ranalli et al., 1990a). The availability of a large quantity of initial
health material propagated in protected environments requires only a few additional
years of conventional seed multiplication to reach the desired seed lot of health seed
tubers.
Outside the classical seed tuber areas of Northern Europe where the length of the
growing period for pre-basic seed is usually not more than 80 days, the growing
season is long enough to obtain reasonable yields even from micro- and minitubers
(Ranalli et al., 1990b). In this area, the growing season of seed potato crops is not
limited by night frost in the spring nor by cold in the winter, but. by contrast, heat and
water stresses can occur in the summer. However. by precise choice of the sites, it is
possible to design more cycles each year and so improve the integration with
minituber production. For these environments investigation of some physiological
aspects of minitubers will help improve the performance and use of the propagules.

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