The Effect of Plant Growth Regulator and Active Charcoal on the Development of Microtubers of Potatoes

doi:10.4236/ajps.2012.311185

Paper Menu >>

Journal Menu >>

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.

Email: *[email protected]

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

8163040

Induction time/

Treatment IV

Treatment V

Treatment III

Treatment II

Number

Figure 1. Influence of exogenous auxins on the development

of microtubers “Mire”.

91%

6.17%

1.50%

7.83%

0.2

0.4

0.6

0.8

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.