American Journal of Plant Sciences, 2012, 3, 1535-1540
http://dx.doi.org/10.4236/ajps.2012.311185 Published Online November 2012 (http://www.SciRP.org/journal/ajps)
1535
The Effect of Plant Growth Re gulator and Active Charcoal
on the Development of Microtubers of Potatoes
Maolin Peng, Xiyao Wang*, Liqin Li
College of Agronomy, Sichuan Agricultural University, Chengdu, China.
Received August 4th, 2012; revised September 15th, 2012; accepted October 7th, 2012
ABSTRACT
With the detoxicated seedling of a potato cultivation breed named “Mire” as the material, the effect of auxins CCC,
6-BA, and active carbon to microtubers of potato (Solanum tubersum L.) was investigated under the in-vitro circum-
stances. The result indicated the exogenous auxins improved the production and quality of microtubers of potatoes. The
effect of induction can be described as CCC > CCC + 6-BA > 6-BA > CK, the number of microtubers in per flask is
8.17 > 7.67 > 7.29 > 5.46, and the number of large potatoes in per flask is 6.33 > 5.17 > 3.17 > 1. In addition, by adding
0.5‰ of active charcoal, the growth period was shortened from 25.0 days to 9.33 days on average, and the amount of
larger potatoes increased 8.54%. These results benefited the growth of microtubers of potato.
Keywords: Plant Growth Regulators; Active Charcoal; Microtubers of Potato
1. Introduction
Potato (Solanum tubersum L.) is crucial crops to econo-
mies. They are cultivated around the world with an av-
erage annual cultivation area beyond 20 million hm2. It
has the fourth greatest global production [1]. China,
which area of cultivation is around 3 million to 3.33 mil-
lion hm2, is the second largest area of potato cultivation
in the world [2]. Potatoes are praised by its short growth
period, high adaptability and production, great potential
of yield increase, and et al. Potatoes mainly reproduce
asexually. Its degeneration causes its quality and quantity
produced to drop [3]. With the development of the plant
meristem culture, it was possible to produce detoxicated
“rejuvenated” potatoes. This could solve the global issue
of seed potato degeneration [2]. Ever since the success of
the induction of microtubers [4], experts around the
world had started thorough studies on this and seen rela-
tively great improvements [5-7]. Microtubers of potatoes
have outstanding advantages as small volume, light
weight, unlimited producing seasons, easy to storage,
faster reproducing rate comparing to average agricultural
production, and high cultivation survival rate[8]. Micro-
tubers can be used to exchange the germplasm resources.
Their production and transportation are more convenient
than other forms of germplasm. Other than this, micro-
tubers play the role of receptors of gene transfer in con-
temporary studies of potato genes engineering. In addi-
tion, it is an ideal method to study the tuber formation
mechanism of potatoes by inducing the formation of po-
tato tubers under in-vitro circumstances [9].
Fundamental principles of the formation of microtu-
bers are extremely complicated, because their formation
is influenced by multiple factors including, temperature
[10], genotype, age, health of the plantlets, as well as
mineral nutrition [11], carbon source [12], exogenous
auxins [13,14], method, the plant growth retardants [15],
other adjunctions, the illumination [16] and other envi-
ronmental factors. However, different breeds of potatoes
have relatively different level adaptability to the planting
conditions. There’s no universal and high effective in-
duction method for microtubers of potatoes, which re-
sulted inconvenience for our agricultural production and
studies. The purpose is to produce high-quality microtu-
bers at a low cost, which is significantly meaningful for
the occurrence of future agricultural production and ex-
perimental work.
2. Materials and Methods
The virus-free seedlings of potato “Mire” was obtained
from the potato Research Centre of Sichuan Agriculture
University. The sterile explants “Mire” were cut into
stem pieces with a single or two axillary buds, and in-
oculated vertically into a flask (100 ml) with 20 ml of
initiation MS medium [17], containing 3% (w/v) sucrose
and 0.7% (w/v) agar according to a standard yam nodal
segment culture protocol [18,19], the pH was adjusted to
*Corresponding author.

The Effect of Plant Growth Regulator and Active Charcoal on the Development of Microtubers of Potatoes 1536
pH 5.8. There were 9 nodal explants per flask. And they
were incubated under controlled environmental condi-
tions of 18˚C ± 2˚C. 16/8 light/dark cycles and the illu-
mination intensity at 2000 lx, which were fostered for
about 3 weeks, they were cultured under the same condi-
tions to cultivate sufficient seedlings to induce microtu-
bers. Subsequently, one hundred and eight health ex-
plants were removed from the MS medium and then
plant them into the inducting medium of microtubers (see
Table 1). 6 separate explants were placed into each bot-
tles, and each treatment need 6 bottles; repeat each
treatment thrice in total. Place and foster the cultured
materials into the organized fostering room. Keep the
temperature at 18˚C ± 2˚C, and the illumination intensity
at 2000 lx for 8 hours per day.
Observations were recorded on the number of micro-
tubers and the size of microtubers. Count all microtubers
on the 40th day, and then count the number of unpolluted
potatoes (potato/bottle), individual plant of microtubers
(plant/bottle), and larger potato (diameter > 5 mm), get
average number of each treatments; Analysis of variance
was calculated for the data using the SPSS version 13.0
statistical package for Windows. The Figures 1 and 2
were completed by the Excel 2003.
The formula for the result would be:
Percentage of large potatoes = number of large pota-
toes/total number of potatoes × 100% (Diameter > 5 mm).
Percentage of potato in individual plant = total number
of potatoes fructified per bottle/number of inoculated
individual plant per bottle × 100%.
3. Conclusion and Analysis
3.1. The Influence of Exogenous Auxins on
Microtubers of Potatoes Induction
Judging from the result observation on the 5th day of
induction fostering, the pure CCC treatment fructified
microtubers at the earliest; the lower part of the potato
plantlets’ stem segments started turning purple and ex-
panding. Later on, the same phenomenon was observed
in other treatments. Among all the four different treat-
ments, treatment III had the highest average production
of potatoes, which were 8.17. Treatment V ranked sec-
ond, with an average production of 7.67 potatoes. Treat-
ment IV had the third highest production of potatoes,
which was 7.29, and the plain control group produced
only 5.46 potatoes on average. As seen in Figure 1, the
number of potatoes fructified in the four treatments in-
creased as the inducing time increased. Their relationship
can be classified as: Treatment III > Treatment V >
Treatment IV > Treatment II. Treatment I (CK) con-
tained no exogenous auxins; potatoes planted in this
treatment fruited relatively later and less. Therefore, the
influential level of the four treatments on the induction
Table 1.Composition of inducing subculture me dia.
Treatment Subculture Media PH
I MS+ 8%Sucrose+ 0.8%Agarose 5.8
II MS + 8%Sucrose + 0.5‰Active Carbon
+ 0.8%Agarose 5.8
III MS + 8%Sucrose + CCC 5 mg/L
+ 0.5‰Active Carbon + 0.8%Agarose 5.8
IV MS + 8%Sucrose + 6-BA 5 mg/L
+ 0.5‰Active Carbon + 0.8%Agarose 5.8
V MS + 8%Sucrose + CCC 5 mg/L + 6-BA 5 mg/L
+ 0.5‰Active Carbon + 0.8%Agarose 5.8
0
1
2
3
4
5
6
7
8
9
8163040
Induction time/
D
Treatment IV
Treatment V
Treatment III
Treatment II
Number
Figure 1. Influence of exogenous auxins on the development
of microtubers “Mire”.
91%
3
6.17%
2
1.50%
2
7.83%
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Rate of large potatoRate of potato per plant
Treatment II
Treatment III
Treatment IV
Treatment V
Figure 2. Effect of exogenous auxins on microtubers’ de-
velopment.
effect of the microtubers, can be described as: CCC > 6-
BA + CCC > 6-BA > control group (Figure 1).
3.2. The Influence of Exogenous Auxins on the
Development
As seen in Table 2, it’s easy to tell, with the help from
the F-test of One-Way Anova, the difference in the
number of fruited potatoes and large potatoes per bottle

The Effect of Plant Growth Regulator and Active Charcoal on the Development of Microtubers of Potatoes 1537
is significant among treatment III, IV, V, and II, which is
the plain control group (Table 2). This proves adding
appropriate amount of exogenous auxins can increase the
induction of microtubers and surpass non-adjunction
experimental groups on both quality and quantity.
As seen in Table 3 and Figure 2, Treatment III’s av-
erage value of fruited potatoes, large potatoes (Table 3),
percentage of potatoes fruited on individual plant and
percentage of large potatoes ranked the highest among
the four. It’s easy to see an extremely significant differ-
ence on the amount of fruited potatoes and large potatoes
per bottle between treatment III and the control group.
There is also an obvious difference on the percentage of
large potatoes, percentage of potatoes fruited on individ-
ual plant and the number of fructified potatoes per bottle
between treatment III and other treatments. After adding
the CCC, the percentage of large potatoes dramatically
increased, proving the CCC greatly promoted the nutrient
accumulation of the microtubers of potatoes. Possibly
because of the lack of CCC, there was no dramatic dif-
ference observed between the number of potatoes fructi-
fied per bottle and the treatment with 6-BA.
3.3. The Influence of Active Carbon on
Microtubers of Potatoes’ Induction
As seen in Table 4, the subculture media with 0.5‰ ac-
tive carbon more rapidly induced bigger potatoes than
the other control group media without it. The large potato
percentage increased by 8.54% and the time needed to
fructify the microtubers dramatically shortened. However,
because of the numerous small potatoes produced in the
control groups, the percentage of potatoes fructified on
each plant in the active carbon media is actually less. In
the early period of the experiment, the colors of the mi-
crotubers in the two different treatments seemed to differ
dramatically. As illustrated in Figures 3 and 4, the mi-
crotubers grown from the subculture medium with active
carbon appeared brown at first. On the other hand, the
ones grown from the control group medium appeared
green. As time went by, the brown lightened. When the
fructified potatoes induced from the subculture media
with active carbon were gathered on the 40th day, they
had turned into green, as seen in Figure 4. The active
toner might have prevented light from reaching the
plantlets, potentially causing this color change. As the
plantlets grew little by little, the preventing effect seemed
to wear off.
4. Discussion
Microtubers’ propagation is controlled by many factors.
When conditions are suitable, it can generate microtubers
without auxins. However, this will lengthen the forming
Table 2. The effect of exogenous auxins on the induc tion on the microtubers.
Factor SS df MS F P-value Fcrit
Between group (laeger potato number) 98.83333 3 32.94444 31.37566** 9.27E−08 3.098391
Within group 21 20 1.05
Between group (potato number) 94.3333 3 31.44444 36.99346** 2.37E−08 3.098391
Within group 17 20 0.85
Note: F-test of one-way anova.
Table 3. Effect of exogenous auxins on microtubers’ induction and development.
Treatment Microtuber of per flask Large potato Rate of lager potato (%) Rate of potato per plant (%)
II (CK) 5.46B 1B 18.32% 91%
III 8.17A 6.33A 77.48% 136.17%
IV 7.29A 3.17B 43.48% 121.5%
V 7.67A 5.17A 67.41% 127.83%
Note: Duncan’s single factor test was performed at the 0.01 level.
Table 4.The effect of ac tive carbon on microtubers of potatoes’ induction.
Treatment Time/d Rate of large potato (%) Rate of potato per plant (%) Color
I (CK) 25.07 18.96% 108.33% green
II 9.33 27.5% 91% brown

The Effect of Plant Growth Regulator and Active Charcoal on the Development of Microtubers of Potatoes 1538
Figure 3. The effect of no active carbon on microtubers of
potatoes’ induction.
Figure 4. The effect of active carbon on microtubers of po-
tatoes’ induction.
time and lower the quantity of large potatoes produced,
which hinders the producing industry. Adding exogenous
auxins is a good way to reduce fructifying time and en-
hance the quality and yield of microtubers. There are
many different exogenous auxins to choose from, but the
CCC and the 6-BA are the most frequently found ones in
China [20]. Cytokinins can promote potatoes’ tuberiza-
tion and are considered to be tuber-inducing factors [21-
25]. Among these; 6-BA is the most significant one,
which is considered to promote the cell division and
growth, to stimulate the activity of some enzymes, and to
make the nutrients be transported to the parts of cyto-
kinins more easily. So it performs to increase the potato
number and weight at the same time [26]. As a plant
growth inhibitor, the CCC is most frequently used for
microtuber induction. It is also considered a dominant
regulator. The CCC remarkably helps to fructify the tu-
bers, to increase the production, and to enhance the effect
of cytokinins. The contents of sucrose and starch in-
creased in tubers when treated with CCC [27]. In the
experiment, microtubers were formed in the treatments
of 6-BA, 6-BA and CCC. 6-BA increased the number of
microtubers, however, the number of large potato was
lower than the treatment of CCC and 6-BA. This indi-
cated that CCC played a main role. Although microtubers
were formed both in the treatments of CCC and 6-BA +
CCC, the former was proved more efficient than other
treatments, for it had the highest number of potatoes
produced, highest number of large potatoes produced,
highest rate of large potatoes and highest number of sin-
gle-plant potatoes produced. Especially, its number of
large potato produced was nearly twice of the 6-BA
treatment. According to Bai [26], adding a certain con-
centration of exogenous auxins does well in improving
the yield and quality of microtubers .The effect is 6-BA >
6-BA + CCC > CCC > CK. But the experiment of Pang
gets completely opposite conclusion, which is CCC >
CCC + 6-BA > 6-BA, and has an extreme influence of no
root, no elongation, no microtubers with the treatment of
6-BA [26]. The result of our experiment is CCC > CCC
+ 6-BA > 6-BA > CK, which is similar to the experie-
ment conducted by Pang [28], the different concentration
of CCC plays a positive role in microtubers, but if the
concentration of CCC is too high, it can be inhibited by
6-BA in some degree. Compared with the experiment of
Bai, the materials are different, which may lead to the
different result. Activated charcoal is an adsorbent,
which has a significant effect on the microtuber induc-
tion. It can increase tuber yield and greatly shorten the
time of microtuber induction [29,30]. The color of early
potato in the treatment with active charcoal was brown at
first, and the color of microtubers in the treatment with-
out it appeared green. The reason for this might have
been that the activated charcoal blocked much of the
lighting, and the activated charcoal kept a balance be-
tween the major elements and the minor elements in the
culture medium.
In the experiment, we have found the growth of mi-
crotubers is related to the age and health of its explant.
Older vines’ nodes are more easily fructified than youn-
ger ones. The rate of lower vines appears higher than it
of upper vines. They have found similar phenomena ear-
lier [31]. However, the terminal bud is relatively easy to
germinate under illumination circumstances. Therefore,
the dark treatment is necessary to the explants.
REFERENCES
[1] FAO, “Potatoes in the 1990s: Situations and Prospects of
World Potato Economy,” Publications, Food and Agri-
culture Organization, 1998.
[2] J. He, Y. Chen and Y. S. Zhang, “Induction of Potato
Microtuber in Vitro,” Journal of Yunnan University, Vol.
1, No. 23, 2001, pp. 62-64.
[3] K. Kawakami, “The Physiological Degeneration of Potato
Seed Tubers and Its Control,” Potato Research, Vol. 5,
No. 1, 1962, pp. 40-49.
http://link.springer.com/article/10.1007%2FBF02367660
[4] Y. Kim, “In Vitro Tuber Formation from Potato,” Seoul
University Publishing, Seoul, 1982.

The Effect of Plant Growth Regulator and Active Charcoal on the Development of Microtubers of Potatoes 1539
[5] M. J. Hossain and N. Sultana, “Effect of Benzyl Amino
Purine (BAP) and Chloro Choline Chloride (CCC) on in
Vitro Tuberization of Potato,” Bangladesh Journal of Ag-
riculture Research, Vol. 4, No. 23, 1998, pp. 685-690.
[6] J. Gopal, J. L. Minocha and H. S. Dhaliwal, “Microtuber-
ization in Potato (Solanum tubersum L.),” Plant Cell Re-
ports, Vol. 17, No. 10, 1998, pp. 794-798.
doi:10.1007/s002990050485
[7] R. Estrada, P. Tovar and J. H. Dodds, “Introduction of in
Vitro Tubers in a Broad Range of Potato Genotypes,”
Plant Cell, Tissue and Organ Culture, Vol. 7, No. 1, 1986,
pp. 3-10. doi:10.1007/BF00043915
[8] J. L. John, W. H. Courtney and D. R. Decoteau, “The
Influence of Plant Growth Regulators and Light on Mi-
crotuber Induction and Formation in Dioscorea alata L.
Cultures,” Plant Cell, Tissue and Organ Culture, Vol. 34,
No. 3, 1993, pp. 245-252. doi:10.1007/BF00029713
[9] W. S. Hao, Y. X. Zhao and Q. H. Gao, “The Progress of
Potato Microtubers Induction in China,” Inner Monglia
Agricultural Science and Technology, Vol. 6, 2002, pp.
4-7.
[10] M. E. Hoque, “In Vitro Tuberization in Potato (Solanum
tuberosum L.),” Plant Omics Journal, Vol. 1, No. 3, 2010,
pp. 7-11.
[11] D. Sarkar and P. S. Naik, “Effect of Inorganic Nitrogen
Nutrition on Cytokinin-Induced Potato Microtuber Pro-
duction in Vitro,” Potato Research, Vol. 41, No. 3, 1998,
pp. 211-217. doi:10.1007/BF02358191
[12] O. O. Paul, K. Claire and D. Jacques, “Effects of Reduc-
ing Sugar Concentration on in Vitro Tuber Formation and
Sprouting in Yam (Dioscorea cayenensis-D. rotundata
Complex),” Plant Cell, Tissue and Organ Culture, Vol.
99, No. 1, 2009, pp. 55-59.
doi:10.1007/s11240-009-9575-1
[13] O. Mahmoud, C. S. Farhad and N. Paul, “Effects of Tem-
perature Fluctuation during in Vitro Phase on in Vitro
Microtuber Production in Different Cultivars of Potato
(Solanum tubersum L.),” Plant Cell, Tissue and Organ
Culture, Vol. 98, No. 2, 2009, pp. 213-218.
doi:10.1007/s11240-009-9554-6
[14] K. Pruski, T. Astatkie, P. Duplessis, T. Lewis, J. Nowak
and P. C. Struik, “Use of Jasmonate for Conditioning of
Potato Plantlets and Microtubers in Greenhouse Produc-
tion of Minitubers,” American Journal of Potato Re-
search, Vol. 80, No. 3, 2003, pp. 183-193.
doi:10.1007/BF02855690
[15] P. Kris, A. Tess and N. Jerzy, “Jasmonate Effects on in
Vitro Tuberization and Tuber Bulking in Two Potato Cul-
tivars (Solanum tubersum L.) under Different Media and
Photoperiod Conditions,” In Vitro Cellular & Develop-
mental Biology—Animal, Vol. 38, No. 2, 2002, pp. 203-
209.
http://rd.springer.com/article/10.1079/IVPIVP2001265
[16] Janet E. A. Seabrook, “Light Effects on the Growth and
Morphogenesis of Potato (Solanum tubersum) in Vitro: A
Review,” American Journal of Potato Research, Vol. 82,
No. 5, 2005, pp. 353-367.
http://rd.springer.com/article/10.1007/BF02871966
[17] T. Murashige and F. Skoog, “A Revised Medium for
Rapid Growth and Bioassays with Tobacco Tissue Cul-
tures,” Physiologia Plantarum, Vol. 15, No. 3, 1962, pp.
473-497. doi:10.1111/j.1399-3054.1962.tb08052.x
[18] S. H. Mantell, S. Q. Haque and A. P. Whitehall, “Clonal
Multiplication of Dioscorea alata L. and Dioscorea ro-
tundata Poir. Yams by Tissue Culture,” The Journal of
Horticultural Science, Vol. 53, No. 2, 1978, pp. 95-98.
[19] S. H. Mantell and S. A. Hugo, “Effects of Photoperiod,
Mineral Medium Strength, Inorganic Ammonium, Su-
crose and Cytokinin on Root, Shoot and Microtuber De-
velopment in Shoot Cultures of Dioscorea alata L. and D.
bulbifera L. Yams,” Plant Cell, Tissue and Organ Cul-
ture, Vol. 16, No. 1, 1989, pp. 23-37.
doi:10.1007/BF00044069
[20] H. Q. Wang and L. T. Xiao, “Effects of Chlorocholine
Chloride on Phytohormones and Photosynthetic Charac-
teristics in Potato (Solanum tubersum L.),” Journal of
Plant Growth Regulation, Vol. 28, No. 1, 2009, pp. 21-27.
doi:10.1007/s00344-008-9069-0
[21] J. Martine and C. Mario, “Effects of Some Growth Gegu-
lator on in Vitro Tuberization in Dioscorea Alata L.
‘Brazo Fuerte’ and D. abyssinica Hoch,” Plant Cell Re-
ports, Vol. 11, No. 1, 1992, pp. 34-38.
http://www.springerlink.com/content/b52e1efd7fb94e9e/
[22] C. E. Palmer and O. E. Smith, “Cytokinins and Tuber
Initiation in the Potato Solanum tubersum L.,” Nature,
Vol. 221, 1969, pp. 279-280. doi:10.1038/221279a0
[23] C. S. Mauk and A. R. Langille, “Physiology of Tuberiza-
tion in Solanum tubersum L.,” Plant Physiology, Vol. 62,
No. 3, 1978, pp. 438-442. doi:10.1104/pp.62.3.438
[24] H. Obata-Sasamoto and H. Suzuki, “Activities of En-
zymes Relating to Starch Synthesis and Endogenous Lev-
els of Growth Regulators during Tuberization of Isolated
Potato Stolons Cultured in Vitro,” Zeitschrift für Pflan-
zenphysiologie, Vol. 95, No. 1, 1979, pp. 69-75.
http://www.sciencedirect.com/science/article/pii/S004432
8X7980029X
[25] G. Hussey and N. J. Stacey, “Factors Affecting the For-
mation of in Vitro Tubers of Potato (Solanum tuberosum
L.),” Annals of Botany, Vol. 53, No. 4, 1984. pp. 565-578.
http://aob.oxfordjournals.org/content/53/4/565.short
[26] S. X. Bai, Z. M. An, X. Z. Feng and J. Wang, “Studies on
the Factors Affecting the Induction of in Vitro Potato Mi-
crober,” China Potato, Vol. 5, No. 15, 2001, pp. 271-273.
[27] H. Y. Yang, D. Wang and X. Pan, “Effect of Hormone
and Culture Methods on Potato Microtuber Induction,”
Journal of Gansu Agricultural University, Vol. 2, 2008,
pp. 74-77.
[28] S. Pang, G. N. Fang, J. X. Li and J. X. Zhao, “Effect of
Kinds Growth Regulator on Potato Micro-Tubers Induc-
tion,” Journal of Changjiang Vegetables, Vol. 20, 2009,
pp. 25-27.
[29] C. Z. Xu, X. J. Zhang and A. J. Liu, “Effect of Activated
Chroal on Explant Growth and Tuberization in Potato,”
Review of China Agricultural Science and Technology,
Vol. 5, 2003, pp. 106-107.
[30] R. X. Liu, “Effect of Activated Charcoal and Inorganic

The Effect of Plant Growth Regulator and Active Charcoal on the Development of Microtubers of Potatoes 1540
Nutrition on the Induction of the Test-Tube Potato,” Plant
Physiology Journal, Vol. 37, 2001, pp. 295-298.
[
31] Y. Luo, H. Tian and T. Zhang, “Effect of Coumarin on
Microtuber Induction in Potato,” China Potato, Vol. 1,
No. 14, 2000, pp. 4-8.