Residual Effects of Sucrose and Hormonal Treatments of the Tuberization Medium on <i>in Vitro</i> Germination of Potato (<i>Solanum tuberosum</i> L.) Microtubers

doi:10.4236/ajps.2013.49230

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American Journal of Plant Sciences, 2013, 4, 1872-1878

http://dx.doi.org/10.4236/ajps.2013.49230 Published Online September 2013 (http://www.scirp.org/journal/ajps)

Residual Effects of Sucrose and Hormonal Treatments of

the Tuberization Medium on in Vitro Germination of

Potato (Solanum tuberosum L.) Microtubers

Abraham Diémé1, Mame Abdou Nahr Sambe1, Emile Codjo Agbangba2, Mame Ourèye Sy1

1Laboratoire Campus de Biotechnologies Végétales, Département de Biologie Végétale, Faculté des Sciences & Techniques,

Université Cheikh Anta Diop, Dakar, Sénégal; 2Laboratoire d’Ecologie Végétale et d’Ecohydrologie, Département de Biologie

Végétale, Faculté des Sciences & Techniques, Université Cheikh Anta Diop, Dakar, Sénégal.

Email: [email protected], [email protected]

Received July 9th, 2013; revised August 9th, 2013; accepted August 27th, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The residual effects of sucrose concentrations (80 or 100 g·L−1) and hormonal treatments (BAP + Kinetin or Coumarin)

of tuberization medium on in vitro microtubers germination of three potato varieties (Solanum tuberosum L.) so called

Aida, Atlas and Odessa, are described. After 3 weeks of incubation at 28˚C ± 1˚C, 70% of Aida microtubers variety,

previously formed in the MT2 medium [MS/2 + 80 g·L−1 Sucrose], germinated. The best germination rate for varieties

Atlas (100%) and Odessa (66.66%) was obtained on microtubers previously formed in the medium MT2 [MS/2 + 100

g·L−1 Sucrose]. The addition of hormones in the tuberization medium allowed optimizing the microtubers germination

of the Aida variety unlike the other varieties. Indeed, for the Aida variety, the combination M5 [Kin 2.5 mg·L−1 + Coum

0.025 mg·L−1 + Sucrose 80 g·L−1] increased the germination rate from 70% up to 93.33%. The best germination rate

(90%), noticed with microtubers of Atlas variety, initially formed in M2 medium [Kin 1 mg·L−1 + BAP 1 mg·L−1 + Su-

crose 100 g·L−1], was lower than that one (100%) obtained on medium without hormones. For Odessa variety, the

maximum germination rate (53.33%) of microtubers, from the medium M4 [Kin 2.5 mg·L−1 + BAP 1 mg·L−1 + Sucrose

100 g·L−1], was also lower than that one (66.66%) observed in the medium without hormones. Aida and Atlas varieties

thus offer a better germination rate than Odessa after their cold storage.

Keywords: Solanum tuberosum; Microcuttings; Microtubers; In Vitro Germination; Sucrose; Cytokinins; Coumarin

1. Introduction

The potato is the fourth largest food crop production in

the world, after the three major cereals such as wheat,

rice and maize. Potato culture is a strategic sector in Se-

negal. This production occurs from November to March

in the Niayes zone, during which the North Atlantic

fringe of the country benefits from winds relenting tem-

peratures and thus allow the tuberization of this specula-

tion. The potato culture represents 10% of the national

production of vegetables and roughly 50% of trade im-

portation. Analysis of the national potato production cost

illustrates that potato seeds represent one third of impor-

tation, which heavily influences the trade balance in Se-

negal. Indeed, conventional seed production systems are

currently completely disorganized so that the needs of

quality seeds are important. Local certified pest-free

seeds (5170 tons in 2010) do not cover all the national

requirements, consequently, a massive import of seeds is

practiced (24,000 tons per year).

Most varieties of potatoes grown in the world are va-

rieties with tetraploid true-seeds giving heterogeneous

progeny. Thus, the use of the potato tuber as basic seed is

a current practice and can easily meet the requirement of

genetic conformity [1,2]. But the conventional produc-

tion of potato plants is a speculation that requires signifi-

cant technical expertise as produced tubers must include

a minimum rate of plant pathogenic infections and any

viral amount respecting the original genetic traits of the

variety. The techniques developed have always strived to

bring in cropping systems plant material with a mini-

mum of infective load. For this, the traditional culture

techniques obey a clonal selection model that includes a

number of measures favorable to the preservation of

good health status over multiplication and thus the intrin-

Residual Effects of Sucrose and Hormonal Treatments of the Tuberization Medium on in Vitro

Germination of Potato (Solanum tuberosum L.) Microtubers

1873

sic qualities of varieties. However, this method of mass

selection has severe limitations especially for latent viral

infections that may be transmitted to the next generations.

The advent of in vitro culture techniques enabled us

through micropropagation to accelerate the number of

seedlings produced and improve their health status while

providing virus-free seeds with the development of sero-

logical detection methods such as meristem culture cou-

pled with thermotherapy. The discovery of new chemical

molecules also contributed to improve the possibilities of

fight against the vectors of viral diseases such as aphids

and their transmission to plants. The techniques of vege-

tative propagation permit getting four types of seeds:

plantlets, microtubers, minitubers and tubers. Indeed, the

acclimatization step of plants can be replaced by in vitro

microtuberization i.e. vitrotubers production which is one

of the practical ways, the most effective for the propaga-

tion of basic material, the transport of germplasm and the

conservation of potatoes varieties grown after sanitation

[3,4].

The in vitro method of vegetative multiplication al-

lows respecting easily the imperatives of conformity such

as permanency and genetic stability for different studied

potato varieties [5]. Microtuberization of explants re-

quires an important provision of sucrose and growth re-

gulators. It was more precocious when the culture me-

dium was enriched with cytokinins. Harmey et al. [6]

stated that adding growth regulators increases the devel-

opment of microtubers if an amount of 8% sucrose is

supplied. Ebadi et al. [7] found healthy microtubers with

3 to 4 months of dormancy by cultivating isolated cul-

tures of two to three nodes in bio semi-continuous biore-

actors of attained microtubers in concentration above 10

mg· L −1 BAP and 8% of sucrose.

The effects of hormones and sucrose on microtuberi-

zation were well documented [8] but a few were reported

on the influence of these exogenous factors on microtu-

bers germination.

The residual effect of hormones and sucrose can affect

the dormancy duration of cold stored microtubers and

therefore has an impact on their early germination when

put to normal temperature. Indeed, increasing the sucrose

concentration from 80 to 140 g·L−1 during the tuberiza-

tion process, it is possible to reduce the six weeks dura-

tion of Désirée microtubers dormancy of [9]. Hussey and

Stacey [10] obtained immediate sprouting of micro-tu-

bers produced in long days, while those induced by short

days in the presence of BAP germinate more slowly and

were heterogeneous.

It is important depending on the variety, to know hor-

monal combinations that are most favorable to the dor-

mancy period reduction of microtubers to obtain a ho-

mogeneous and synchronous lifting of germs that can be

cultivated in fields during the windy season. Indeed, this

speculation is a dry-season crop in Senegal.

This present work describes the residual effect of su-

crose and of growth regulators of the tuberization me-

dium on the germination capacity of microtubers after

cold storage at 4˚C.

2. Material and Methods

2.1. Characteristics of Varieties

The basic material is constituted of Elite tubers of 28 - 35

mm caliber and belonging to three varieties of potato:

Aïda, Atlas and Odessa. They were imported from GER-

MICOPA S.A. (France). These varieties were chosen be-

cause of their adaptability to Senegalese agroclimatic

conditions. Tubers-young plants were virus-free accord-

ing to the phytosanitary certificates (Norms of certifica-

tion ISO 9001). Twenty tubers were chosen regarding the

Aïda variety, 30 for the Atlas and 28 for Odessa.

Atlas is a relatively late variety, high yielding and its

productivity is stable even in hard conditions. Its tubers

are of big caliber, less numerous and less sensitive to

desprouting and could support a prolonged storage period.

Odessa is an early variety and has a good yield per hec-

tare. Its tubers are oblong with a yellow skin and a yel-

low flesh and have an ability to support an average con-

servation period. Aïda is a late variety with an early ma-

turity of tubers and a good yielding. Tubers are oblong

and elongated pale-yellow appearance.

2.2. Microtuber Production

Microtubers were obtained by applying the protocol pre-

viously described in Dieme et al., 2011. Explants con-

sisting of mononodal cuttings were cultured on Mura-

shige & Skoog [11] medium with halved macronutrients

(MS/2) and in the presence of 80 g·L−1 or 100 g·L−1 Su-

crose. To test the residual hormonal effect of the culture

medium on the germination of microtubers, culture me-

dia were previously enriched or not (Table 1) with cyto-

kinins (Kin and BAP 1 to 2.5 mg·L−1) and/or Coumarin

(0.025 mg·L−1) for microtuber production. Agar at 8

g·L−1 was used to solidify the media; the pH was ad-

justed to 5.9 before autoclaving at 110˚C for 20 min.

2.3. Storage and Germination of Microtubers

The microtubers of the 3 varieties from different tuberi-

zation medium were harvested aseptically and previously

stored in a cold room at 4˚C ± 1˚C for 4 months. Expe-

riments on microtubers were conducted to determine

their ability to germinate at 28˚C. Three batches of 10

Residual Effects of Sucrose and Hormonal Treatments of the Tuberization Medium on in Vitro

Germination of Potato (Solanum tuberosum L.) Microtubers

1874

Table 1. Media composition for microtubers production

from the microcuttings of the three varieties.

Media Treatments

MT1 MS/2 + Sucrose 80 g·L−1

MT2 MS/2 + Sucrose 100 g·L−1

M1 MS/2 + Kin 1 mg·L−1 + BAP 1 mg·L−1+ Sucrose 80 g·L−1

M2 MS/2 + Kin 1 mg·L−1 + BAP 1 mg·L−1 + Sucrose 100 g·L−1

M3 MS/2 + Kin 2.5 mg·L−1 + BAP 1 mg·L−1 + Sucrose 80 g·L−1

M4 MS/2 + Kin 2.5 mg·L−1 + BAP 1 mg·L−1 + Sucrose 100

g·L−1

M5 MS/2 + Kin 2.5 mg·L−1 + Coum 0.025 mg·L−1

+ Sucrose 80 g·L−1

M6 MS/2 + Kin 2.5 mg·L−1 + Coum 0.025 mg·L−1

+ Sucrose 100 g·L−1

microtubers for each variety, with caliber between 5 and

7 mm, and obtained from different hormonal combina-

tions were used. The microtubers were deposited asepti-

cally on MS (0) medium [11], then incubated in a dark

room at 28˚C ± 1˚C. A completely randomized block was

used for the experiments. The number of germinated mi-

crotubers was counted after 1, 2 and 3 weeks. For each

factor studied, three repetitions were performed.

2.4. Statistical Analyses

Multiple comparisons of averages and rates have been

done, after analysis of variance, using the test of Student,

Newman, and Keuls at the probability threshold of 5%

(SPSS software).

3. Results

3.1. Influence of the Sucrose in the Tuberization

Medium

The different varieties of microtubers obtained with su-

crose concentrations of 80 g·L−1 and 100 g·L−1 are kept

in a cold room and then put in 28˚C ± 1˚C for germina-

tion and the germination rate of microtubers determined

during 3 weeks (Figures 1 and 2).

The effect of high concentrations of sucrose is ex-

pressed mainly by reducing the duration of dormancy.

Indeed, to obtain 70% germination after 3 weeks at 28˚C,

80 g·L−1 of Sucrose in the medium of tuberization is

proved sufficient for Aïda variety. Concentration of 100

g·L−1 sucrose lowers the germination rate to 53.33%. In

addition, the germination process begins earlier, in the

presence of Sucrose at 80 g·L−1. However, for the varie-

ties Atlas and Odessa, the best germination rate is obtained

in the presence of 100 g·L−1 of Sucrose in the initial in

vitro tuberization medium with 100% and 66.66% of

microtubers germinated.

Figure 1. In vitro germination of potato microtubers after

21 days of incubation in MT1 medium at 28˚C in darkness.

MT1

Germination

(

)

MT2

100

ïda Atlas Odessa Varieties

Figure 2. Effect of sucrose of the tuberization medium on

the microtuber germination of 3 potato varieties (Aida,

Atlas and Odesa), after 3 weeks of incubation at 28˚C. MT1

= MS/2 + Sucrose 80 g·L−1; MT2 = MS/2 + Sucrose 100

g·L−1. Treatments followed by the same letter are not sig-

nificantly different at probability level of P <0.05 by Stu-

dent-Newman-Keuls (SNK).

3.2. Combined Effect of Kinetin and BAP and

Sucrose in the Tuberization Medium

The combined effect of kinetin and BAP on Aida variety

was studied in the presence of two concentrations of Su-

crose (80 g·L−1 and 100 g·L−1). The highest rate of mi-

crotubers germination (86.66%) was obtained with the

medium M3 [Kin 2.5 mg·L−1 + BAP 1 mg·L−1 + Sucrose

80 g·L−1] while the combination M1 [Kin 1 mg·L−1 +

BAP 1 mg·L−1 + 80 g·L−1 Sucrose] allows a significantly

lower germination rate, of 83.33%. Increasing the con-

centration of Kinetin from 1 to 2.5 mg·L−1 helped to raise

slightly the germination rate (Figure 3).

However, increasing the sucrose concentration of 80

g·L−1 up to 100 g·L−1 in the best medium combination

has reduced the germination rate to 66.66%. The de-

crease in hormonal concentration of Kinetin 2.5 mg·L−1

Residual Effects of Sucrose and Hormonal Treatments of the Tuberization Medium on in Vitro

Germination of Potato (Solanum tuberosum L.) Microtubers

1875

MT1

Germination

(

)

MT2

Aïda

Atlas

Odessa

M1 M3 M5 M2 M4 M6

Tuberization media

100

bcde

abcd abcd

abcde abcde

abc

def

def def

g g

efg

cdef cdef

cdef

Figure 3. Hormonal effects of the tuberization medium on

microtubers germination of potato varieties (Aida, Atlas

and Odessa) at 28˚C. MT1 = MS/2 + Sucrose 80 g·L−1; M1 =

MS/2 + Kin 1 mg·L−1 + BAP 1 mg·Lw−1 + Sucrose 80 g·L−1;

M3 = MS/2 + Kin 2.5 mg·L−1 + BAP 1 mg·L−1 + Sucrose 80

g·L−1; M5 = MS/2 + Kin 2.5 mg·L−1 + Coum 0.025 mg·L−1 +

Sucrose 80 g·L−1; MT2 = MS/2 + Sucrose 100 g·L−1; M2 =

MS/2 + Kin 1 mg·L−1 + BAP 1 mg·L−1 + Sucrose 100 g·L−1;

M4 = MS/2 + Kin 2.5 mg·L−1 + BAP 1 mg·L−1 + Sucrose 100

g·L−1; M6 = MS/2 + Kin 2.5 mg·L−1 + Coum 0.025 m g·L−1 +

Sucrose 100 g·L−1; Treatments followed by the same letter

are not significantly different at probability level of P < 0.05

by Student-Newman-Keuls (SNK).

to 1 mg·L−1 in the presence of BAP 1 mg·L−1 gave a

higher rate (83.33%) than the M1 medium [Kin 1 mg·L−1

BAP + 1 mg·L−1 + 100 g·L−1 sucrose] (80%).

With Atlas variety, the sucrose concentration in the

culture medium most likely to give better germination

rate of microtubers was 100 g·L−1 with 100% of micro-

tubers germinated after three weeks of incubation. The

effect of hormones on the germination of microtubers

was also appreciated in the presence of 80 g·L−1 and 100

g·L−1 Sucrose (Figure 3). The experiment revealed a ger-

mination rate of 90% of microtubers obtained in the M2

tuberization medium [Kin 1 mg·L−1 + BAP 1 mg·L−1 +

Sucrose 100 g·L−1]. By increasing the concentration of

Kinetin from 1 to 2.5 mg·L−1 in the hormonal combina-

tion, we noticed a decrease in microtubers germination

with a maximum rate of 60%. The reduction in sucrose

concentration also causes a decrease in the germination

rate (73.33%) but it does not seem significantly different

from 90% (Table 1).

The experiment, in Odessa variety, was performed

under the same experimental conditions as those used for

Aida variety. For Odessa variety, the M4 combination

[Kin 2.5 mg·L−1 + BAP 1 mg·L−1 + Sucrose 100 g·L−1]

gave a germination rate of 53.33% after three weeks of

incubation at 28˚C ± 1˚C in darkness (Figure 3).

The highest germination rate (23.33%) in the presence

of sucrose 80 g·L−1 is obtained with the combination Kin

2.5 mg·L−1 + BAP 1 mg·L−1. So, this concentration of

sucrose is not favorable for the germination of microtu-

bers of this variety in comparison to Aida variety.

3.3. Combined Effects of Kinetin and Coumarin

and Sucrose in the Tuberization Medium

For Aida variety, microtubers obtained from the M5

combination [Kin 2.5 mg·L−1 + Coum 0.025 mg·L−1 + 80

mg· L −1 Sucrose], offer a germination rate of microtubers

of 93.33% after a three weeks incubation time. The in-

crease in sucrose concentration at this hormonal combi-

nation (M6 medium) gave a low rate of 60% (Figure 3

and Table 2). Compared to other hormonal combinations,

it can be seen that the M5 combination, [Kin 2.5 mg·L−1+

Coum 0.025 mg·L−1 + 80 g·L−1 Sucrose], gave more

germinated microtubers than the combinations M1 [Kin 1

mg· L −1 + BAP 1 mg·L−1] and M3 [Kin 2.5 mg·L−1 +

BAP 1 mg·L−1].

For Atlas variety, the substitution of the BAP by Cou-

marin, in the combination Kin 2.5 mg·L−1 + BAP 1

mg· L −1 + 80 or 100 g·L−1 Sucrose, can increase the ger-

mination rate three weeks after incubation, with a maxi-

mum of 80% when microtubers originated from the M5

hormonal combination [Kin 2.5 mg·L−1 + Coum 0.025

mg· L −1 + sucrose 80 g·L−1]. However, for the Atlas va-

riety, the combination M2 [Kin 1 mg·L−1 + BAP 1

mg· L −1 + Sucrose 100 g·L−1] gave the best germination

rate (90%).

Regarding the Odessa variety, the replacement of the

BAP by Coumarin in the combination [Kin 2.5 mg·L−1 +

BAP 1 mg·L−1 + Sucrose 100 g·L−1] was not beneficial

for this variety because there is a fall of the germination

rate from 53.33% to 46.66%. For a best germination rate

of Odessa microtubers after cold storage (Figure 3 and

Table 2), microtuberization must be realized in a me-

dium containing 100 g·L−1 Sucrose and supplemented

with Kin 2.5 mg·L−1 + BAP 1 mg·L−1. The Odessa vari-

ety gave a maximum rate of germinated microtubers

equal to 53.33% with the medium M4 [Kin 2.5 mg·L−1 +

BAP 1 mg·L−1 + 100 g·L−1 Sucrose] against 66.66% for

the microtubers formed in the medium MT2 without

hormones but supplemented with 100 g·L−1 Sucrose.

4. Discussion

4.1. Effects of Sucrose Concentration of the

Tuberization Medium on the Microtubers

Germination

The sucrose concentration giving the best germination

rate varies depending on the variety. Indeed, 80 g·L−1 Su-

crose gave the best germination rate for the variety Aida

while those of the varieties Odessa and Atlas are ob-

tained in the presence of 100 g·L−1 Sucrose in the initial

in vitro tuberization mediumhe Atlas variety provides . T

Residual Effects of Sucrose and Hormonal Treatments of the Tuberization Medium on in Vitro

Germination of Potato (Solanum tuberosum L.) Microtubers

1876

Table 2. Residual effects of Sucrose and hormonal combinations on the microtubers germination of different varieties, after 3

weeks of incubation at 28˚C.

Germination (%)

Media Treatments

Aïda Atlas Odessa

MT1 MS/2 + Sucrose 80 g·L−1 70.00 cdef 76.66 bcde 20.00 h

MT2 MS/2 + Sucrose 100 g·L−1 53.33 fg 100.00 a 66.66 def

M1 MS/2 + Kin 1 mg·L−1+ BAP 1 mg·L−1 + Sucrose 80 g·L−1 83.33 abcd 73.33 bcdef 10.00 h

M2 MS/2 + Kin 1 mg·L−1 + BAP 1 mg·L−1 + Sucrose 100 g·L−1 80.00 abcde 90.00 abc 26.66 h

M3 MS/2 + Kin 2.5 mg·L−1 +BAP 1 mg·L−1 + Sucrose 80 g·L−1 86.66 abcd 70.00 cdef 23.33 h

M4 MS/2 + Kin 2.5 mg·L−1+ BAP 1 mg·L−1 + Sucrose 100 g·L−1 66.66 def 60.00 efg 53.33 fg

M5 MS/2 + Kin 2.5 mg·L−1 + Coum 0.025 mg·L−1 + Sucrose 80 g·L−1 93.33 ab 80.00 abcde 43.33 g

M6 MS/2 + Kin 2.5 mg·L−1 + Coum 0.025 mg·L−1 + Sucrose100 g·L−1 60.00 efg 66.66 def 46.66 g

Student-Newman-Keuls (SNK) analysis of difference in rates: in columns, treatments followed by the same letter are not significantly different at a probability

level of 0.05.

a better ability to germinate than the other varieties.

The effect of high concentrations of sucrose is mainly

expressed on reducing the duration of microtuber dor-

mancy. Indeed, the more important is the concentration,

the earlier the microtubers germinate. Thus, with Odessa

and Atlas varieties, microtubers obtained in the culture

medium containing 100 g·L−1 Sucrose gave a higher ger-

mination percentage than those obtained with 80 g·L−1.

These results are consistent with those of Désiré et al.

[9,12] who found that, by increasing the sucrose con-

centration from 80 to 140 g·L−1 in the phase of tuberiza-

tion, it is possible to reduce the duration of dormancy of

Désiré variety microtubers to 6 weeks. High concentra-

tions of sucrose (140 g·L−1) and cold conditions can

shorten the dormancy of microtubers. Storage at 4˚C be-

fore transfer to 19˚C is also favorable for the germina-

tion of microtubers whatever the time of tuberization. In

all cases, the longer is the storage duration at 4˚C, the

more rapid and homogeneous is the germination [12].

The influence of carbohydrate nutrition on the duration

of dormancy has not been highly documented. Van Itter-

sum [13] showed a decrease in the duration of the dor-

mancy of 5 to 8 days for tubers obtained from plants

grown in the field, when the nitrogen is added during the

culture.

For Aida variety, increasing the sucrose concentration

in the culture medium from 80 to 100 g·L−1 did not affect

the germination of microtubers because we had a germi-

nation percentage at 100 g·L−1 less than that of microtu-

bers obtained at 80 g·L−1. These results contrast to those

found in varieties of Odessa and Atlas, and those of Dé-

siré et al. [11]. We can therefore argue that the effect of

high concentrations of sucrose on reducing the duration

of dormancy depends on varieties.

4.2. Effects of Hormone Concentrations and

Sucrose in the Tuberization Medium on the

Germination of Microtubers

In vitro tuberization of potato is cumulatively controlled

by the carbon source (sucrose) and growth regulators.

The influence of hormones on the germination of micro-

tubers has not been much reported in the literature.

Chinchilla [14] showed that GA3 reduced the period of

dormancy and promoted the formation of very elongated

sprouts on classic tubers. Dieng [15] and Desire et al. [9]

confirmed in their work that GA3 does not allow the lift-

ing of the dormancy in the classic tubers [16-18] but only

stimulates the germination at the end of dormancy cycle

[19,20].

For Aida variety, the combination of BAP + kinetin +

80 g·L−1 Sucrose in the in vitro tuberization medium has

allowed an earlier germination of microtubers with a

maximum germination rate of 86.66% compared to that

of microtubers obtained in MS (0) medium supplemented

with 8% sucrose. The increase in sucrose concentration

at these combinations reduces the germination rate to

80%.

For Odessa and Atlas varieties, the addition of BAP +

kinetin was not beneficial for the germination of micro-

tubers because we noticed a decrease in the rate of ger-

mination. The addition of growth regulators was not ne-

cessary to obtain a better germination rate. The contribu-

tion of hormones on the germination of microtubers va-

ries according to varieties. As for Aida, the hormonal

combination, Kin 2.5 m·L−1 + Coum 0.025 mg·L−1 gave

the best germination rate of microtubers compared to that

Residual Effects of Sucrose and Hormonal Treatments of the Tuberization Medium on in Vitro

Germination of Potato (Solanum tuberosum L.) Microtubers

1877

one obtained in tuberization media without hormones.

Regarding Atlas variety, the contribution of growth re-

gulators is not required; no hormonal combination has

given a germination rate higher than that of tuberization

control media (MT1 and MT2). According to Sidikou et

al. [18], in vitro tuberization of the Atlas variety is rather

optimal in the presence of BAP alone at a high concen-

tration of 5 mg/L. Cytokinins are known to have a signif-

icant impact on the size and weight of microtubers formed

[21,22]. Hussey and Stacey [10] previously obtained an

immediate germination of microtubers produced in long

days period, whereas those induced by short days, in the

presence of BAP, germinated more slowly and heteroge-

neously. However, different results were obtained by

studying the same factors [23].

With Odessa variety, if the concentration of kinetin is

increased from 1 to 2.5 mg·L−1 with 8% Sucrose, a slight

increase in germination was noticed whereas with 10%

Sucrose, a reduction of this rate was obtained. However,

for Atlas the increasing of the concentration of kinetin to

2.5 mg·L−1 causes a significant reduction in the speed

and rate of germination (Figure 3). In a previous study

[5], the in vitro method of vegetative multiplication used,

allows respecting easily, the imperatives of conformity

such as permanency and genetic stability for different

potato varieties. Microtuberization of explants requires

an important provision of sucrose and of growth regula-

tors. It was more precocious when the culture medium

was enriched with cytokinins. These results were in ac-

cordance with those of [24,25]. The weak mass of mi-

crotubers harvested with Atlas variety was to be corre-

lated to its status of late variety, which did not com-

pletely achieve its period of dormancy and non optimal

hormonal combinations into the tuberization media. In

previous works, it was demonstrated that the more the

potatoes tuber are physiologically old, the more rapid is

their germination [9,26]. When microtubers are physio-

logically young, they are either dormant or in a slight

phase of germination. Then, with aging, their germina-

tion vigor increases and therefore germination is acceler-

ated. The effect of the size of microtubers on germination

was studied by Désiré et al. [11]. Indeed when the mi-

crotuber diameter increases, the germination capacity is

important.

The combined effect of Kinetin and Coumarin was

very successful for Aida variety because the speed of ger-

mination was important and the rate is optimal (93.33%).

This combination was not very effective for Odessa and

Atlas varieties because the maximum germination rates

are lower than those obtained in the presence of cyto-

kinins. Couillerot [19] showed that gibberellic acid is

incorporated in plant metabolism and therefore, it is not

excluded that it modifies some physiological phenomena,

namely the germination of microtubers. Similarly, it can

be suggested that Cytokinins and Coumarin can modify

some physiological phenomena such as germination of

microtubers.

With regard to the emergence and homogeneous growth

of shoots stemming from Aida and Atlas microtubers, we

can correlate them to a consequence of a similar physio-

logical age inherent to the method of in vitro culture that

synchronizes and harmonizes their development. The re-

sults allow therefore considering the possibility of a large

multiplication of microtubers in a large scale, in which

germinative capacity is not only important, but in which

plants would have a synchronous development and growth.

5. Acknowledgements

We address our gratitude to “Fond National de Recherches

Agricoles & Agro-Alimentaires” (FNRAA) which al-

lowed the financial support to complete successfully this

study, as part of the project No 09/AP 03M0010202.

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