Callus induction and plant regeneration From leaf explants of tobacco
Chao yanjie
(Class 2 of Biotechnology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China)
Abstract: Tobacco (Nicotiana tabacum L.) is a mode plant in the research of tissue culture, plant regeneration and other related
fields. Tobacco leaves were used as explants in this experiment. Large amount calli were formed After leaves cultured in basic MS
medium with intermediate ratio of auxin to cytokinin, and the callus induction rate was up to 100%. Some differences were found
between the treatments of lightness and darkness: the callus cultured in lightness represent more tight structure and regular shape
with the color of kelly, while that in darkness show itself less compact and irregular with the color of straw yellow. Subsequently,
shoot regeneration was observed with the low ratio of auxin to cytokinin, which suggests that light wasn’t the necessary element
since shoot formed both in darkness and lightness and the No. of shoots had no significant difference. At the same time, slight
differences were found in surface color of callus and the state of shoots.
Keywords: Callus induction; Tobacco; in vitro; regeneration; shoot; auxin; cytokinin
Abbreviations: MS medium=Murashige and Skoog (1962); BA=6-benzyladenine;
NAA=α-naphthaleneaetic acid
1. Introduction
Cell engineering, as an important component of bioengineering, has been widely applied to the fields of plant
breeding, medicine, animal husbandry and environment protection and has been progressing rapidly in recent years
contributing to social and economic advance. As an applied science and engineering technique, cell engineering involves
genetically modifying cells or with the purpose of providing large-scale commercial production of proteins and other
important biological molecules, by using the theories and techniques of cell biology and molecular biology [1].
Tissue Culture, in vitro culture of cells, tissues or organs, one of most important content of cell engineering is based
on theory of cellular totipotency, whereby Multicellular eukaryotic organisms can be distinguished by the ability of
individual cells to regenerate into an entire organism [2,3], also allows one type of tissue or organ to be initiated from
another type. The regeneration of whole organisms depends upon the concept that all plant cells can, given the correct
stimuli, express the total genetic potential of the parent plant, so this practice involves the culture environment, culture
media, and growth regulators and so on. Both chemical and physical, of the plant cells have to meet by the culture
vessel, the growth medium and the external environment (light, temperature, etc.). Culture media used for the in vitro
cultivation of plant cells are composed of three basic components: (1) essential elements, or mineral ions, supplied as a
complex mixture of salts; (2) an organic supplement supplying vitamins and/or amino acid; (3) a source of fixed carbon;
usually supplied as the sugar sucrose. Date back 1962, MS medium, which was invented by Murashige and Skoog [4], is
an extremely widely used medium and forms the basis for many other media formulations. This classical medium was
also used in this experiment. Plant growth regulators, divided into 5 classes that are auxins, cytokinins, gibberellins,
abscisic acid, and ethylene are the critical media components. And several different culture types most commonly used
in plant transformation studies such as Callus, Cell-suspension cultures, protoplasts, root cultures, shoot tip and
meristem culture, embryo culture and microspore culture [5].
Tobacco was found somewhere in the American continent, but how and when it was first discovered is unknown.
What is certain is that tobacco smoking was practised among the early Mayas. After first cultivated in European country
outside of the Americas in 1500s by Portuguese, tobacco plants were spread quickly. Not waiting for long, Portuguese
introduced smoking into India, Eastern Asia and Japan in 1699 [6]. Since that, tobacco was planted widely. In 1997, the
acreage of tobacco in China was up to 2.16 million hectare [7], 42% of that in the world [8]. Tobacco is one of the most
important economic crop, which has been considerate to fruit fly of plant kingdom because it becomes to a classical
mode plant that could be cultured in vitro and gain regenerated transformation plant easily [9].
2. Materials and Methods
The plant material is aseptic callus of tobacco (Nicotiana tabacum L.) kept in vitro.
Chose fresh leaves as explants. These explants were cut into pieces about 2mm2, and then inoculate into conical
flask filled with the MS basic medium and proper hormone. All the cultures were incubated at 24±2°C in darkness
for a week, and then half of them were placed under cool, white fluorescent tube for callusing or shoot
regeneration with the different treatments.

Corressponding author. Email:[email protected]
2.1 Callus induction
Explants were cultured on callusing medium containing basal MS semi-solid medium with 1.4% agar,
supplemented with 2% sucrose, 0.8% BA and NAA for callus induction. The pH of medium was adjusted to 5.86.0. For each replicate, a total of five leaves were placed separately in culture conical flask containing 30ml of medium,
placed in a dark growth room for callus initiation.
2.2 In vitro shoot regeneration (Organ differentiation and plant regeneration)
The selected calli harvested from callusing media were transferred to differential medium containing basal MS
medium with 2.0mgl-1 BA and 0.5mgl-1 NAA for shoot regeneration. The cultures were maintained at 20-22°C under
cool, white fluorescent lamps with 16h photoperiod.
3. Results and discussion
3.1 Induction of callus
Calli were initiated from tobacco leaves both in darkness and lightness, but different in morphology and physiology.
See table 1 below.
Table 1. Statistics of callus induction from tobacco leaves in darkness and lightness.
Flask Number
No. Of explants
Light Condition
1
2
3
4
5
6
5
5
5
5
5
5
Lightness
Lightness
Lightness
Darkness
Darkness
Darkness
Induction rate of
callus (%)
100%
80%
100%
100%
100%
100%
Status of Callus
a
b
c
d
e
f
Contamination
(%)
0
0
0
0
0
0
a. Big¶ and medium for one respective, small for three. The small calli are compact, and have the regular shape, while big one is irregular.
b. An explant is died because it’s embedding in the media. Two are small and two are big. Calli are verdant and part covered white shoot, while callus
near the media has the color of light kelly.
c. Two are medium; one is small; two are very tiny. The tiny ones are round cellular agglomerate with the color of light kelly.
d. Five for big. All represent semi-transparent, most covered with white villiform. One of them has a brown spot.
e. One is small, and the others are medium. All are buff, decorated with a little white villiform surface, and represent a tight structure and regular
shape.
f. Three for big, one is medium, and one is small. All are buff, spotted white color. There is also a brown speckle in the big one. The shape of them is
irregular.
¶.small<ø1cm, medium≒ø1cm, big>ø1cm
The results suggest that the intermediate auxin (NAA) to cytokinin (BA) ratio (1:1) generally favors callus formation
indeed. Calli were initiated both in lightness and darkness. It seems that the light condition has not obviously and
remarkable influence in callus induction. But light had effect on the state of callus. The callus cultured in lightness
represent more tight structure and regular shape with the color of kelly, while that in darkness show itself less compact
and irregular with the color of straw yellow.
There are many reports about the studies on the regulation of plant hormones using tobacco callus system [10]. Some
research made the conclusion, which suggests Cytokinins is necessary for callus differentiation, and the ratio of the
auxin to the cytokinin determining the type of culture established or regenerated [11]. Low auxin to cytokinin ratio begets
shoot formation; intermediate ratio results in callus formation and high ratio induces root formation.
3.2 in vitro shoot regeneration and differentiation
Buds outgrow in every callus, 100% rate of differentiation, proves that low auxin to cytokinin ratio (1:4) results in the
shoot formation. But the statistics of shoot regeneration suggests that no significance difference was found between the
treatments of darkness and lightness (See table 2). Light maybe play a role in effect on the plant endogenesis hormone,
which regulate the growth, metabolism and differentiation. The only difference is the state of shoots: the callus which
came out some vigorous shoots covered by green in Lightness, while that in darkness covered with fewer bud spots in
white and no big shoot. Other researchers also found that shoots formed in darkness [12], and one study on effect of light
and hormone discovered that light has not certain relation with shoot regeneration since the shoot formed when proper
amount of hormone is add to the culture medium [13].
Table 2. Statistics of tobacco shoot regeneration from callus induction in darkness and lightness.
Flask Number
Callusing in
darkness/lightnes
s
Rate of
differentiation¡
(%)
100
No. of shoots/per
callus
Status of shoot
Many bud spot, 3 big
shoots (1.5cm)
5 big shoots with 2-3
2
Lightness
100
19
leaves
5 big shoots, with the
surface covered
3
Lightness
100
40
green.
Few green bud spot.
Surfaces almost are
4
Darkness
100
50
white.
No big shoot.
5
Darkness
100
17
represent in white.
No big shoot, with
6
Darkness
100
40
few green bud spots.
¡.Rate of differentiation(%)= No. of callus with shoot differentiated/all callus×100
0.07<<F0.05=7.71. F analyse shows that there is no difference between lightness and darkness condition.
1
Lightness
65
Contamination
(%)
0
0
0
0
0
0
ACKNOWLEDGMENTS
I'd like to thank Ms. Liu Jun for teaching us fundamental knowledge of Cell Engineering and basic experiment skills.
Also thank Mr. Hu Jianbin and Zhu Qing for careful guidance in laboratory.
Reference
1. Chen, Z. N.; Xing, J. L.; Bian, H. J. et al. Application of cell engineering technology to the tumour immunotherapeutic drug. Cell biology
International 2001.Vol 25, Issue10:1013-1015
2. Buss LW. The evolution of individuality. Princeton University Press (Princeton), 1987
3. Kaplan DR, Hagemann W. The relationship between cell and organism in vascular plants: Are cells the building blocks of plant form? BioScience
1991,41: 693-703
4. Murashige, T. and F. Skoog. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 1962,15: 473-479.
5. http://www.forces.org/writers/james/files/history.htm
6. http://www.cn-yaas.net/
7. Zhao Jianying(赵剑英). Economics Daily(经济日报),2003.02.25:P6 available on http://www.chinaed.com.cn
8. Anonymous. Plant tissue culture, chaper2
9. Jiao Zhen, Qin Guangyong, Huo Yuping. Callus inducement from Tobacco leaf and it’s tolerance to Vacuum. Journal of Zhengzhou University
2003, Vol:35(1):46-48
10. Dodds.J.H. Experiments in plant tissue culture. Cambridge University Press 1984:36-50
11. 张同庆,姚根怀,吴中心. 激素对烟草愈伤组织分化影响的研究. 烟草科技 1992(6):33-36
12. 许智宏, 王雄, 刘桂云. 烟草叶组织培养中器官形成的研究. 植物生理学报 1978:177-182
13. Pen Zhan-sheng, Chen ji-yao, Cai huai-xin. The regulation effect of the light and hormones on morphogenesis in tobacco leaf tissue cultures. Acta
biologiae experimentalis sinica 1989(22), No.3:279-285