Mohamed and Girgis  Bulletin of the National

Bulletin of the National Research Centre (2023) 47:80

https://doi.org/10.1186/s42269-023-01056-3 Research Centre

RESEARCH Open Access

Factors affecting in vitro tuberization

of potato
Adel El‑Sawy Mohamed1 and Nancy Danial Girgis1*

Abstract
Background Potato (Solanum tuberosum L.) is one of the most economically important annual vegetable crops.
Microtubers can be produced, stored around year and directly transported to market without transferring to fresh
media and without acclimatization. Thus reducing the field cycle to obtain sufficient number of seed potatoes and a
high level of healthy materials. The aim of this study was to investigate effects of different combinations and concen‑
trations of growth regulators i.e., kinetin (2.5 mg/l) and coumarin (20, 40 and 60 mg/l) alone or in addition to varied
concentrations of sucrose (30, 60 and 90 g/l) on in vitro tuberization of potato (Solanum tuberosum) cv. Diamount.
Results The plantlets were propagated using single-node cuttings cultured on Murashige and Skoog medium
containing 0.04 mg/l kinetin and 1.0 mg/l IAA. Shoots (6–7 nodes) from the previous step were cultured into medium
supplemented with a factorial combination of sucrose concentrations (30, 60 and 90 g/l), coumarin concentrations
(0, 20, 40, and 60 mg/l) and two concentrations of kinetin (0 and 2.5 mg/l). Our results showed that kinetin – induced
medium have slightly effect to improve tuberization in vitro. But when kinetin was combined with sucrose, this effect
was better when raised sucrose concentration from 30 or 60 to 90 g/l. The highest percentage of tuberization after
8 weeks, highest number of microtuber were obtained with high concentration of sucrose (90 g/l) together with dark
condition, at 18–20 °C. Also, the highest concentration of sucrose significantly increased the fresh weight of microtu‑
ber. In case of coumarin, results revealed that it has important effect on tuber initiation especially with concentrations
20 mg/l and 40 mg/l; otherwise, the initiation period was minimized after adding kinetin. So, the plantlets must be
continuously incubated in coumarin –induce medium plus kinetin for 8 weeks to affect the tuberization response.
Also, all the other characters of tuberization process on coumarin-inducing medium could be improved by increasing
sucrose concentration to 9 g/l.
Conclusions Generally, results pointed out that the treatment which consists of kinetin (2.5 mg/l) plus 90 g/l sucrose
and 20 mg/l coumarin has the best characteristics of in vitro micro-tuberization.
Keywords Solanum tuberosum L, Sucrose, Kinetin, Coumarin, Microtubers

Background high nutritive value and their low price (Xin et al. 1998).
Potato (Solanum tuberosum L.) is one of the most impor- Potato is an annual herbaceous plant, it is vegetatively
tant vegetable crops all over the world. It is the fourth propagated by tubers. Microtuberization of potato has
largest staple crop next to rice, wheat and barley. In some been one of the successful methods to increase potato
countries, potato is considered the main daily food due to under in vitro conditions (Yu et al. 2000). Microtubers
can be preserved for a long time and thus these could be
*Correspondence: an ideal propagation material (Fufa and Diro 2014; Mahdi
Nancy Danial Girgis et al. 2004). Also, they can be planted directly in the
nancydanial333@yahoo.com soil and produced for around-year. In vitro propagated
1
Department of Plant Biotechnology, Biotechnology Research Institute,
National Research Centre, Giza 12622, Dokki, Egypt potato plantlets are commonly used for the production
of in vitro tubers in potato seed production programs.

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Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 2 of 10

In vitro microtubers production was used to solve some Micropropagation of plantlets

problems such as virus-free potato seed storage, in vivo The plantlets were propagated using single-node cut-
plantlets transplantation, conservation of important cul- tings. Ten explants were cultured in sterilized culturing
tivars and handling healthy germplasm exchange (Kefi jars containing 40 ml of standard medium. The medium
et al. 2000; Rosu et al. 2004; Kanwal et al. 2006). Micro- contained 4.4 g/l Murashige and Skoog medium with
tubers can be produced, stored for around the year and vitamins ( Murashige and Skoog 1962), with vitamins,
directly transported to market without transferring to 30 g/l sucrose, 6 g/l agar, 0.04 mg/l kinetin and 1.0 mg/l
fresh media (Nhut et al. 2004) and without acclimatiza- IAA, the pH was adjusted to 5.8 -6 before adding the agar
tion (Jimerez et al. 1999). Thus reducing the field cycle and autoclaving. The culture jars were closed with polyvi-
to obtain a sufficient number of seed potatoes and a nyl carbonate caps and sealed with household plastic foil
high level of healthy materials (Wróbel 2014). However, and placed in a growth chamber set at 24 °C ± 1 and 16 h
there are limitations in the production of microtuber photoperiod for 4 weeks. The single–node explants were
due to the components of the culture environment and grown to rooted plantlets. Plantlets were cut again into
the low rate of photosynthesis process of the explants or single-node explants and were transferred into a fresh
plantlets. Tuberization is a complex physiological pro- MS medium as described above.
cess controlled by several factors, such as environmen- The multiplication phase was routinely repeated every
tal factors, nitrogen supply, growth regulators (PGRs), 4 weeks by subculturing single-node cuttings until the
genotypes, growth nutrients, photoperiods, temperature, desired numbers of in vitro plantlets for the experiment
source of explant, potato cultivar and sucrose concen- was obtained (El-Sawy et al 2015).
tration (Ramawat and Merillon 2013). In vitro Potato
microtubers derived from single-node cuttings is an
efficient method for handling and storage compared to In vitro tuberization
those obtained from sprouts (Liljana et al. 2012). Most Shoots (6–7 nodes) resulted from the previous step were
studies pointed that tuberization is affected by hormo- cultured into solid MS medium supplemented with a fac-
nal control but the stimulus to tuberization is not unique torial combination of four sucrose concentrations (30,
and can be a single compound some of them having hor- 60 and 90 g/l), four coumarin concentrations (0, 20, 40,
mone activity. Although the physiological mechanism and 60 mg/l) and two concentrations of kinetin (0 and
of potato tuberization between the various hormonal 2.5 mg/l). PH was adjusted to 5.7 before autoclaving for
compounds that regulate the process is still unknown, 20 min at 121° C. Each culture jar (250 ml) received 40 ml
several researchers used different growth regulators to medium and contains five plantlet shoots. Cultures were
stimulate microtuberization (Coleman et al. 2001; Tugrul incubated under completely dark conditions at 18–20° C.
et al. 2001). Potato microtubers induction depends on All the experiments were repeated twice, three repli-
the combination of auxins and cytokinins (Tugrul and cates per treatment with 5 explants for each replicate.
Samanci 2001; Hossain 2005). Some reports suggested The following growth characters were recorded:
that cytokinins have the best effect on potato microtu-
bers formation (Wang and Xiao 2009) such as BAP (Kane 1. Tuberization growth curve during 8 weeks
2011) or Kin (Hoque 2010) or coumarin (El-Sawy et al. 2. Number of days to begin tuberization
2007, 2015). Retardants or growth inhibitors are also 3. Number of days to reach 50% tuberization
needed to inhibit and suppress the activity of gibberel- 4. Percentage of tuberization after 8 weeks
lins so that the energy can be focused on forming potato 5. Number of tubers per plantlet after 8 weeks
tubers (Nuraini et al. 2018). Suppressing gibberellin 6. Fresh weight of microtubers (mg).
activity accelerates the formation of tubers and ultimately
increases their production (Kolachevskaya et al. 2019).
The objectives of the present study are to investigate
the effects of kinetin, sucrose and coumarin on tuberi- Experimental design and statistical analysis
zation process and identify the best microtuberization All experiments were set up in a factorial experiment
media. based on a completely randomized design, and data were
statistically analyzed by one-way analysis of variance
Methods (ANOVA) for testing the differences among treatments
Source of plant materials using least-significant-difference (LSD) test at p < 0.05
Virus-free potato plantlets cv. Diamount were obtained (LSD0.05) according to Gomez and Gomez (1984). The
from plant Biotechnology Dep., Biotechnology Institute, results were expressed using analysis of variance from
National Research Centre, Cairo, Egypt. which mean standard error values (mean ± SE) including
Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 3 of 10

Table 1 Effect of Kinetin on the development of in vitro potato after 8 weeks from culture. However, 74.4% of plantlets
microtubers cultured on free-kinetin medium produced microtubers.
Characters of microtuberization Without kinetin With kinetin These microtubers produced on kinetin-induced medium
were heavier than those produced on the free medium by
No. of days to begin tuberization 12 ± 0.577a 8 ± 0.577b rate of 19.2%. The plantlets cultured on kinetin-induced
a
No. of days to reach 50%tuberization 30.3 ± 0.723 24.5 ± 0.348b medium began tuberization 8 days after cultivation,
b
% of tuberization after 8 weeks 74.4 ± 1.131 81.8 ± o.608a needed about 24.5 days to achieve 50% tuberization, but
a
No of tubers per plantlet after 8 w 1.4 ± 0.066 1.5 ± 0.066a they needed 30.3 days on kinetin-free medium. Other-
b
Weight of microtuber (mg) 50.6 ± 0.457 62.6 ± 0.288a wise, the tuberized plantlets produced slightly number
V values are means ± SE, LSD test at level (p < 0.05) of microtubers (1.4 and 1.5/planlet). Our results showed
Values with the same superscript letters (a,b,c or d) are not significantly different that kinetin–induced medium had a slightly effect to
from each other whilst, values with different superscript letters are significantly
different
improve tuberization in vitro of potato. Data in Fig. 1
showed that in both treatments the rate of tuberization
was nearly closed for each. Tuberization is a complex
superscript letters were computed for comparison developmental phenomenon including many physiologi-
between treatments indicating the presence of significant cally regulated processes. Tuberization of potato plants
difference at the 0.05 probability level. would be better understood under in vitro conditions as
several morphogenic regulators and environmental fac-
Results tors can be controlled.
Effect of kinetin
Microtuberization process on kinetin–induced medium Effect of sucrose
and non-kinetin –induced medium was evaluated after The carbon source in culture media is primary factor
8 weeks incubation period under complete dark condi- for microtuberization. Many investigators have used
tion at 18- 20 C°. The results are shown in Table 1 and different sugars with different concentrations in the
Fig. 1, 81.8% of cultured plantlets produced microtubers medium. Sucrose is the most sugar have been used in
when cultured on kinetin–induced medium (2.5 mg/l) in vitro tuberization. In this study, potato plantlets were

with Kinetin without Kinetin

100 81 81.8 80 70 73.5 74.5
74 78 63
55
% of tuberizaon

% of tuberizaon

80 65 60
60 47.5 40
27.5 40
40 20
20 20
1.8 0
0 0
first 2nd 3rd 4th 5th 6th 7th 8th first 2nd 3rd 4th 5th 6th 7th 8th
week week
A B
Fig. 1 Tuberization frequency (%) on culture media with 2.5 mg/l Kinetin (A) or without Kinetin (B) every week

Table 2 Effect of sucrose on development of in vitro potato microtubers

Characters of microtuberization Sucrose concentration (g/l)
30 60 90

No. of days to begin tuberization 16 ± 0.577a 8 ± 0.577b 6.5 ± 0.577b

No. of days to reach 50% tuberization 39.6 ± 0.781 a
23.3 ± 0.260 b
19.4 ± 0.451c
% of tuberization after 8 weeks 58 ± 3.403 c
88 ± 1.233 b
89 ± 2.791a
No of tubers per plantlet after 8 w 1.3 ± 0.040 b
1.6 ± 0.036 a
1.6 ± 0.036a
Weight of microtubers 26.8 ± 0.463 c
49 ± 0.577 b
66.3 ± 0.276a
Values are means ± SE, LSD test at level (p < 0.05)
Values with the same superscript letters (a,b,c or d) are not significantly different from each other whilst, values with different superscript letters are significantly
different
Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 4 of 10

213
214 30 g/l sucrose 60 g/l sucrose 90 g/l sucrose
215 80 100 80 83 85 88 100 82.586 88 89
55 5858.3 72 72

% of tuberizaon

% of tuberizaon
% of tuberizaon

216 60 45 80 59 80
56
217 30 60 60
40 35 34
218 15 20 40 40
20 20 20 1.3
219 0 0
0 0 0
220 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
week week week
221
Fig. 2 Tuberization frequency (%) under the effect of different sucrose concentrations every week

cultured on media containing 30, 60 and 90 g/l sucrose investigated the effect of applying Kinetin at 2.5 mg/l
under dark condition at 18–20 °C. The results shown in in combination with different sucrose concentrations
Table 2 and Fig. 2, indicated that the cultured plantlets (30, 60 and 90 g/l). Results shown in Table 3 showed
began to tuberize after 16, 8 and 6.5 days from cultur- that effect of kinetin depends on sucrose content in
ing date depending on sucrose concentration 30, 60 and the medium. Data in Table 3 and Fig. 3 indicated that
90 g/l, respectively. These plantlets took about 39.6, 23.3 increasing sucrose concentration in the medium either
and 19.4 days to obtain 50% tuberized plantlets. This with or without adding kinetin promotes microtuberi-
rate reached to 58%, 88% and 89% of tuberization after zation in dark conditions at 18–20 °C. After 8 weeks,
8 weeks of culturing in the case of using 30, 60 and 90 g/l all cultured plantlets tuberized (100% tuberization) but
sucrose as shown in Fig. 2. Using 90 g/l sucrose gave the the number of tuber /plantlet was 1.5 in case of adding
highest number of microtubers. Thus, it can be noticed kinetin with 90 g/l sucrose more than without kinetin
that high sucrose concentrations with dark conditions (1.3 tubers/plantlet). Also, the fresh weight of micro-
and relatively low temperature (18–20 °C) can be used tuber in the case of using 90 g/l sucrose increased by
to produce microtubers. Also, the highest concentra- 46.5%. While with 60 g/l sucrose was 51%. Our results
tion of sucrose significantly increased the fresh weight of pointed that kinetin has a higher inductive and stimu-
microtuber as high fresh weight (66.3 mg/l) was obtained latory effect at intermediate with high sucrose means
by using 90 g/l sucrose as shown in Table 2. But it was kinetin was shown to act mainly on tuber initiation
lower in the case of using 60 g/l and 30 g/l sucrose (49 thereby increasing tuber number. External sucrose is a
and 26.8 mg/l, respectively). very important factor since it provides the main source
of energy under these experimental conditions. Fur-
Interaction between kinetin and sucrose thermore, microtuberization has been affected by both
Our results pointed out that kinetin has slightly effect high concentrations of sucrose and growth regulators.
to improve microtuberization in vitro as mentioned Although the important role of high sucrose in tuberi-
before. This effect was better when sucrose concen- zation induction, it was not an acceptable factor for the
tration rises from 30 or 60 to 90 g/l in the medium as formation process of tuber because of its high level in
shown in Table 3 and Fig. 3. In our experiment, we the culture medium.

Table 3 The effect of interaction between sucrose and kinetin on development of in vitro potato microtubers
Characters of microtuberization Sucrose concentration (g/l)

30 60 90
With kin Without kin With kin Without kin With kin Without kin

No. of days to begin tuberization 18 ± 0.57735b 25 ± 0.57735a 8 ± 0.57735c 8 ± 0.57735c 8 ± 0.57735c 8 ± 0.57735c
No. of days to reach 50% tuberization 25 ± 0.57735b 39 ± 0.5773a 16 ± 0.5773e 20 ± 0.5773c 13 ± 0.5773f 18 ± 0.5773d
% of tuberization after 8 weeks 75 ± 1.258306 b 67 ± 0.737111c 100 ± 0.5773a 75 ± 0.57735b 100 ± 0.5 a 100 ± 0.57735a
No of tubers per plantlet after 8 w 1.2 ± 0.5773a 1 ± 0.5773a 1.3 ± 0.5773 a 1.2 ± 0.5773 a 1.5 ± 0.5773 a 1.3 ± 0.5773 a
Weight of microtubers 16.6 ± 0.288675e 11.8 ± 0.1154f 57.7 ± 0.3464c 28.3 ± 0.3464b 63 ± 1.73205a 33.7 ± 0.17320d
Values are means ± SE, LSD test at level (p < 0.05)
Values with the same superscript letters (a,b,c or d) are not significantly different from each other whilst, values with different superscript letters are significantly
different
Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 5 of 10

Fig. 3 The effect of interaction between sucrose and kinetin on the tuberization frequency (%) every week

Table 4 Effect of coumarin on development of in vitro potato microtubers

Characters of microtuberization Coumarin concentration ( mg/l)
0 20 40 60

No. of days to begin tuberization 13.7 ± 5.666a 11.3 ± 3.33a 11.3 ± 3.333a 13.7 ± 5.66a
No. of days to reach 50% tuberization 30.7 ± 0.351b 22.3 ± 3.21b 25.3 ± 2.426b 45 ± 5.507a
% of tuberization after 8 weeks 65.2 ± 10.63a 83.3 ± 8.81a 80.6 ± 10.007a 60 ± 5.773a
No of tubers per plantlet after 8 w 1.2 ± 0.057a 1.5 ± 0.152a 1.5 ± 0.173a 1.2 ± 0.1a
Weight of microtubers 24.6 ± 6.333c 69 ± 0.577a 50.2 ± 0.986b 27 ± 0.577c
Values are means ± SE, LSD test at level (p < 0.05)
Values with the same superscript letters (a,b,c or d) are not significantly different from each other whilst, values with different superscript letters are significantly
different
Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 6 of 10

Also, the addition of the growth regulators in media Interaction between Coumarin and kinetin
for induction of microtuber makes it difficult to ensure The effects of kinetin and coumarin-induced tuberiza-
the certain role of sucrose in tuberization. tion are shown in Table 5 and Fig. 5 showed that kine-
tin slightly stimulated tubrization. During the course
Effect of coumarin of our studies, we investigated four concentrations of
The coumarin effect on tuberization is shown in Table 4. coumarin (0, 20, 40 and 60 mg/l) added to the media
Tuberization was observed with 20, 40, 60 mg/l of cou- plus kinetin at 2.5 mg/l for evaluation of tuberization
marin concentrations but it is more effective at low con- process in potatoes under dark conditions at 18–20 °C.
centration (20 mg/l). At 40 mg/l and 60 mg/l the effective Tuberization was stimulated on coumarin-induced
rate decreased gradually compared with 20 mg/l. The with this concentration with different stimulated rates
tuberization percent was 65.2, 83.3, 80.6 and 60 at 0, 20, depending on coumarin concentrations as mentioned
40 and 60 mg/l of coumarin, respectively. It does mean before. The effect was more effective when added kine-
that characteristics of the tuberization process on cou- tin (2.5 mg/l) to the medium.
marin-induced media occurred with 20 mg/l as shown in As shown in Table 4, one of our objectives in this
Table 4 and Fig. 4. without any other growth regulators, study was comparison of coumarin effect on tuberiza-
especially cytokinin. tion with or without addition of kinetin (2.5 mg/l) to

0mg/l coumarin 20mg/l coumarin

80 65.5 65.5 100 80 83
53 54 60 65 71 73
%of tuberizaon

%of tuberizaon

60 40 54
40 22 50 28
20 0 1
0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
week week

40 mg/l coumarin 60 mg/l coumarin

100 76 80.6 80
55 60 60
60 65 45 51
%of tuberizaon

%of tuberizaon

60
44 31
50 22 30 40 17
0 20 1
0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
week week

Fig. 4 Tuberization frequency |(%) under the effect of different coumarin concentrations (0, 20, 40 and 60 mg/l) every week

Table 5 Effect of interaction between Coumarin and kinetin: (2.5 mg/l) on development of in vitro potato microtuberization
Characters of microtuberization Coumarin concentration (mg/l)
0 20 40 60

No. of days to begin tuberization 11.3 ± 3.333a 6 ± 2a 6 ± 2a 8 ± 0.577a

No. of days to reach 50% tuberization 18.6 ± 2.905b 18 ± 1.527b 22 ± 3.605a 32.6 ± 8.685a
% of tuberization after 8 weeks 87.2 ± 8.935a 91.7 ± 8.33a 83.6 ± 12.09a 66.7 ± 13.059a
No of tubers per plantlet after 8 w 1.3 ± 0.057b 1.7 ± 0.145a 1.6 ± 0.20ab 1.3 ± 0.152b
Weight of microtubers 45.8 ± 3.372b 75.3 ± 2.91a 46.6 ± 12.04b 45.3 ± 5.126b
Values are means ± SE, LSD test at level (p < 0.05)
Values with the same superscript letters (a,b,c or d) are not significantly different from each other whilst, values with different superscript letters are significantly
different
Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 7 of 10

100 0 mg/l coumarin 100 20 mg/l coumarin 91.7

84.3 87 87.2 80.3 83.3
86
75.7 73.3
80 80
60.7 58.7
60 52.7 60

%of tuberizaon
%of tuberizaon

36
40 40
25.3
20 20
0 2
0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
week week

100 40mg/l coumarin 70 60mg/l coumarin 63.3 63.3 66.7

80.7 81.7 82.6 83.6 58.3
74.7 60 48.7
80
50 38.7
60 53.3

%of tuberizaon
40
%of tuberizaon

40 30 30 22.7
20
20 5 10 0
0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
week week

Fig. 5 Tuberization frequency (%) under effect of interaction between Coumarin (0,20,40 and 60 mg/l) and kinetin: (2.5 mg/l) on the development
of in vitro potato micro-tuberization

medium to induce tuberization. We found that plant- Shoots cultured on high nitrogen media tuberized only
lets tuberized in case of kinetin addition earlier than in case of increasing carbohydrate level (sucrose con-
that without kinetin. This means that the initiation centration). The inhibition of high nitrogen of coumarin-
period was minimized after adding kinetin. The data of induced tuberization could be changed by increasing the
this study show that the plantlets must be continuously level of carbohydrates. Tuberization percent was com-
incubated in coumarin –induce medium plus kinetin pared in medium supplemented with 30, 60 and 90 g/l
for 8 weeks to affect the tuberization response (Table 5 sucrose as shown in Table 6
and Fig. 5).
Our data pointed that axillary shoots formed tubers The optimal medium for tuberization
easily on media containing coumarin or kinetin, whereas As mentioned before, our results showed that kinetin-
other tested growth regulators were very inconsistent in induced medium has a slightly effect to improve tuberi-
stimulating tuberization. zation in vitro. But when kinetin was combined with
sucrose, this effect will be better when raised sucrose
concentration to 90 g/l. Added to that, all the character-
Effect of sucrose on coumarin‑inducing media istics of tuberization process could be improved by add-
Results shown in Table 6 indicate that the concentrations ing coumarin at 20 mg/l. The plantlets were cultured on
of sucrose of the culture media could affect coumarin in tuberized medium consisting of kinetin (2.5 mg/l) plus
the tuberization process. So, the plantlets were cultured sucrose (90 g/l) and coumarin (20 mg/l) and incubated
on the coumarin-induced media tuberization. These under the complete dark conditions at 18–20 C° gave the
plantlets produced more tubers in the case of using 90 g/l best characteristics of tuberization. The results are shown
sucrose than 60 g/l or 30 g/l (100, 100 and 66.7, respec- in Table 7.
tively). Also, all the other characteristics of tuberization
process on coumarin-inducing medium could be affected Discussion
by sucrose concentration. It does mean that carbohydrate Growth regulators are widely used for in vitro multi-
level in culture media for the plantlets could significantly plication during plantlet production and for micro-
modify the effect of nitrogen in the potato shoot media. tuberization. Some authors consider cytokinins to be
Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 8 of 10

Table 6 Effect of sucrose on coumarin-inducing media on development of in vitro potato micro-tuberization

Characters of microtuberization Sucrose conc Coumarin conc. (mg/l)
0 20 40 60

No. of days to begin tuberization 3 18 ± 0.577b 18 ± 0.577b 18 ± 0.577b 25 ± 0.577a

6 8 ± 0.577c 8 ± 0.577c 8 ± 0.577c 8 ± 0.577c
9 8 ± 0.577c 8 ± 0.577c 8 ± 0.577c 8 ± 0.577c
No. of days to reach 50% tuberization 3 25 ± 3.282e 46 ± 1.527c 39 ± 1d 57 ± 0.577a
6 13 ± 1h 21 ± 0.577f 51 ± 1.527b 27 ± 1.527e
9 18 ± 0.577fg 15 ± 0.577gh 27 ± 1.154e 25 ± 0.577e
% of tuberization after 8 weeks 3 75 ± 0.577b 50 ± 2.886de 66.7 ± 0.866c 40 ± 2.886e
6 100 ± 0.577a 60 ± 2.886cd 60 ± 2.886cd 100 ± 0.577a
9 75 ± 1.527b 85.7 ± 8.256b 100 ± 0.577a 80 ± 11.547b
No of tubers per plantlet after 8 w 3 2.3 ± 0.440a 1 ± 0.577c 1 ± 0.577c 1 ± 0.577c
6 1.6 ± 0.115b 1.7 ± 0.057abc 1 ± 0.577c 1.4 ± 0.115bc
9 1.3 ± 0.057bc 1.7 ± 0.1abc 2.1 ± 0.057abc 1.8 ± 0.264abc
Weight of microtubers 3 16.6 ± 0.288i 55 ± 1.154d 52 ± 1.154e 6 ± 0.577j
6 33.7 ± 0.317h 33.7 ± 0.317h 33.7 ± 0.317h 39.7 ± 0.115f
9 63 ± 1.732c 98.8 ± 0.461b 35.2 ± 0.115g 106 ± 1.154a
Values are means ± SE, LSD test at level (p < 0.05)
Values with the same superscript letters (a,b,c or d) are not significantly different from each other whilst, values with different superscript letters are significantly
different

involved in tuberization. In general, kinetin advance- on microtuberization induction (Sota et al. 2020). Many
ment and increase in microtuberization are in agree- researchers reported that sucrose is used for in vitro
ment with these reports of cytokinin effects. Although, tuberization of potatoes, especially with high con-
several authors attributed an important role to cyto- centrations for enhancing this process. In the present
kinins such as kinetin (Coleman et al. 2001; Aksenova investigation, the highest number of microtubers was
et al. 2009). Otherwise, Kinetin increases the number produced using 9% sucrose. Bhojwani (2001) found that
of microtubers due to its effect on cell elongation and microtuberization of potatoes can be induced by trans-
tuberization (Romanov et al. 2000). However, addition ferring the shoots from MS medium containing 3%
of kinetin (2.5 mg/l) to the medium was less effective sucrose to the medium supplemented with 10% sucrose
under dark conditions instead of light. Also, Sucrose at
high concentrations between 6–12% was found to be
Table 7 Characters of tuberization process on optimal medium the most critical stimulus for tuber induction, growth
of potato tubers, increasing number of microtubers
Character Value with incubation period length (El Fatih et al 2004,
No. of days to begin 2 Wazir et al. 2015, Hossain et al. 2015 and Khan et al.
tuberization 2018). Our results indicated that microtuberization
No. of days to reach 50% 14 in vitro can be formed in media containing 6% or 9%
tuberization sucrose and 2.5 mg/l kinetin under dark conditions and
% of tuberization after 96% occurred broadly with findings obtained by Hussey and
8 weeks
Stacey (1984). Coumarin has long been known to be
No of tubers per plantlet 1.5
after 8 w an inhibitor of growth processes in plants such as ger-
Weight of microtuber 87.2 mination and root growth. However, it was clear that
(mg) coumarin has stimulation effect on growth, compared
Tuberization curve with Indole acetic acid (IAA) but it has a different
mode of action (Neuman 1959). Coumarin has physi-
ological effect as it delayed the senescence process,
maybe by blocking the loss of chlorophyll (Knypl 1967).
Coumarin and cis-o-coumarinic acid lactone readily
promote in vitro tuber formation of axillary shoots of
Mohamed and Girgis Bulletin of the National Research Centre (2023) 47:80 Page 9 of 10

potato sprouts. Histological data by Reeve et al. 1969 Received: 16 March 2023 Accepted: 2 June 2023
proved that initiation and growth of tuber are due to
cell enlargement and changes in the planes of cell divi-
sion. In 1979, Stallknecht and Fransworth reported that
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