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- Callus induction and plant regeneration in vitro from leaf explants of mycetia balansae drake
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- KỈ YẾU HỘI NGHỊ SINH VIÊN NGHIÊN CỨU KHOA HỌC NĂM HỌC 2013-2014
CALLUS INDUCTION AND PLANT REGENERATION IN VITRO FROM LEAF
EXPLANTS OF MYCETIA BALANSAE DRAKE
Dang Thi Thao, Class K60A, Faculty of Biology
Supervisor: Assoc. Prof. Dr. Nguyen Xuan Viet
and MSc. Vu Thi Bich Huyen
Abstract: Mycetia balansae Drake, the plant distributes in Vietnam and Laos, contains sweet-tasting
triterpenes and triterpene glycosides which were discovered and published the patent by Jakob and
collaborators (WIPO, 2012). This plant reproduces by seed but rarely find plantlets in nature, so very
difficult to develop planting for commerce. This is first time, the Mycetia balansae Drake was
multiplied by using callus culture technology from leaf explants. The seedlings were served as initial
explant materials. The sterilized seeds in 20% NaOCl solution for 5 min, then in a solution of 10%
H2O2 for 3 min, showed the highest germination rate of seeds (83.33%). The maximum callus
induction was recorded on MS medium supplemented with 2 mg L -1 BAP; 0.2 mg L-1 NAA; 0.5 mg L-1
2,4-D, and the plant regeneration ratio was 53.33% in MS medium supplemented with 2 mg L -1 BAP
and 1.5 mg L-1 Kinetin. The highest percentage of direct regeneration was noticed in the MS medium
supplemented with 2 mg L-1 BAP; 0.2 mg L-1 NAA; 30 g L-1 sucrose; 7g L-1 agar. The regenerated
shoots were rooted in MS medium and successfully transplanted to the land, and watered with 1/10
MS solution in 8 weeks, the highest survival rate of plantlet was 83.63% (plantlets were transplanted
from September to December).
Key words: Callus, direct regeneration, Mycetia balansae Drake, seed, in vitro plantlet.
I. INTRODUCTION
Mycetia balansae Drake (Ra co pua in Vietnamese) was classified in Rubiaceae family
and distributed in some provinces such as Vinh Phuc (Tam Dao), Ha Noi (Ba Vi), Lam Dong
(Lang Hanh), Dong Nai (Bien Hoa, Gia Ray) [3]. This plant has only been used traditionally in
some medicines related to kindney diseases, or replacing for Glycirrhiza uralensis in the
dispensing drugs. It also has been widely used in medium of children‟s cough. In 2012, WIPO
published the patent of Jakob and collaborators, "Orally consumable formulations comprising
certain sweet – tasting triterpenes and triterpene glycosides”. According to the report, the plant
extract contains new triterpenes and triterpene glycosides which make it to have a distinctive
sweet-tasting [4]. Seed propagation of Mycetia balansae Drake is not easy, due to low
germination rate and low viability, so it is very rare in nature. In order to conserve this plant as
well as to use for commercial purposes in cooperation with Tue Linh Co.Ltd, providing a large
number of plants is necessary and in vitro tissue culture technology could be solve this
problem. Therefore, the study on callus induction and plant regeneration in vitro from leaf
explants of Mycetia balansae Drake was carried out.
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Figure 1. Mycetia balansae Drake in nature
II. MATERIAL AND METHODS
1. Material
Seeds of Mycetia balansae Drake were used as initial materials. It was collected
from the Tam Dao mountain of Vinh Phuc provice by Tue Linh Co.Ltd.
2. Methods
2.1. Seed sterilization and seed germination
Seeds of M. balansae were soaked in 70% ethanol for one minute; rinsed three times in
sterile distilled water; followed by treating surface sterilized by immersed in the solutions of
0.1% HgCl2, 10% H2O2, or 20% NaOCl. Finally, they were rinsed three times with sterile
distilled water. Sterillized seeds were placed on germination MS medium supplemented with
0.5 mg L-1 BAP; 30 g L-1 sucrose; 7 g L-1 agar; pH = 5.6 – 5.8; Cultures were maintained at
25 ± 2oC in the dark for 6 weeks.
2.2. Callus induction
In vitro leaf pieces (5 mm × 5 mm) were cultured on MS medium supplemented with 2
mg L BAP; 0.2 mg L-1 NAA; 2,4-D (0 – 2 mg L-1); 30 g L-1 sucrose; 7 g L-1 agar, pH = 5.6
-1
– 5.8. In order to callus induction, the cultures were incubated at 26 ±1 ºC in the dark
conditions. Callus formation efficiency was recorded after 6 weeks of incubation.
2.3. Shoot regeneration
Calli were cultured on MS medium contained 2 mg L-1 BAP and Kinetin (0 – 1.5 mg
L ); 30 g L-1 sucrose; 7 g L-1 agar, pH = 5.6 – 5.8. Culltures were maintained at 25 ± 20C
-1
under 16/8 h photoperiod for 8 weeks. The regenerated shoots were subsequently
transferred on MS medium for rooting.
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2.4. Transplanting
In vitro plantlets were transplanted into the pots containing substrates in the garden
and watered with some mineral solutions (1/10 MS solution; 4/10 MS solution; water) for
8 weeks. The substrates GT01; GT02's Quang Minh Co., Ltd; Thuy Cam Co. Ltd; mixture
of garden soil and sand (1:1) and garden soil: sand: coir (2:1:1 ) were signed in PS01,
PS02, PS03, PS04, PS05 in this experiment.
3. Results and Discussion
3.1. Seed sterilization and seed germination
Seed size of M. balansae is very small, so sterilization time and chemical types must
be suitable with seed germination. The result of seed sterilization was recorded after 6
weeks of culture and represented in table 1 and figure 2.
The data of table 1 shows that seed sterilization in HgCl2 solution resulted in no
germination. This result can explain that the seeds of Mycetia balansae Drake are very
sensitive with HgCl2. The loss of germination ability of seeds which was sterilized by
HgCl2 may relate to the toxicity of HgCl2.
In sterilization with only a chemical type one time, neither H2O2 solution nor NaOCl
solution could have high clean explant rates. Sterilization with 20% NaOCl solution (5
min) had a considerable effect to seeds (infect explant percentage dropped to 46.47%) but
the rate of seed germination was very low (21.43%).
Figure 2: Fruits and seeds (A & B), germinated seeds (C) and plantlets
on the MS medium with 0.5mg L-1 BAP (D)
Sterilization with only one chemical two times gave higher germination effect.
Sterilization with 20% NaOCl solution (5 min and 3 min in SS9 solution) obtained high
sterilization effect (infect explant percentage 0%) but the rate of seed germination was just
24.33%. Sterilization in two times of 10% H2O2 solution (5 min; 3 min in SS10 solution)
was more effective than that of 20% NaOCl solution with germination rate 66.67%.
Effect of seed sterilization was the highest in the condition which contains two
chemical (NaOCl in 5 min and H2O2 in 3 min); in this condiction, the highest germination
rate was 83.33%; infect explant percentage was 0%. Therefore, the combination H2O2 with
NaOCl obtained high clean explants rate and high germination percentages. Plantlets were
used as initial material.
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Table 1. Effect of chemicals and strerilization time to seed germination
Sterilization time (min)
Clean explants rate
Solutions Infect
No. of
NaOCl H2O2 HgCl2 No explant
seeds Germinat
(20%) (10%) (0.1%) germina- rate (%)
-ion (%)
tion (%)
SS1 3 300 0.00 0.00 100.00
SS2 5 300 21.43 31.90 46.67
SS3 3 300 0.00 0.00 100.00
SS4 5 300 0.00 0.00 100.00
SS5 3 300 0.00 76.67 23.33
SS6 5 300 0.00 100.00 0.00
SS7 8 300 0.00 100.00 0.00
SS8 12 300 0.00 100.00 0.00
5
SS9 300 24.33 75.67 0.00
3
5
SS10 300 66.67 10.00 23.33
3
5
SS11 300 0.00 100.00 0.00
3
SS12 5 3 300 83.33 16.67 0.00
SS13 5 3 300 0.00 100.00 0.00
SS14 5 3 300 0.00 100.00 0.00
3.2. Callus induction
Leaf pieces were cultured on MS medium supplemented with 2 mg L-1 BAP; 0.2 mg
L NAA; 2,4-D at concentrations (0; 0.5; 1; 2 mg L-1); after 6 weeks of culture, the result
-1
was shown in table 2 and figure 3.
Table 2. Effect of 2,4 –D concentrations on callus induction
MS supplemented with 2 mg L-1
Induction
BAP; 0.2 mg L-1 NAA and 2,4-D No. of
rate of Status of callus
-1 explants
2,4-D (mg L ) callus (%)
Direct shoot
0.0 60 100
regeneration
White, tough, porous
0.5 60 100
calli
1.0 60 0 Small pieces of leaf
2.0 60 100 Friable calli
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Figure 3. Callus and shoots of direct regeneration in MS medium supplemented
with 2 mg L-1 BAP; 0.2 mg L-1 NAA and 2,4-D
A. Without 2,4- D (direct regenerated shoots) C. 1 mg L-1 2,4-D (no callus)
B. 0.5 mg L-1 2,4-D (white, tough, porous calli) D. 2.0 mg L-1 2,4-D (friable calli)
Data on table 2 shows that the induction rate of callus in MS medium supplemented with
0.5 or 2 mg L-1 2,4 - D were 100%; however, quality of calli was very different. In MS medium
supplenmented with 0.5 mg L-1 2,4 - D , there was rapid callus induction and calli were white,
tough, porous and high regenerative ability. In constrast, calli in medium with 2 mg L-1 2,4 - D
were more friable than those in medium with 0.5 mg L-1 2,4 D. Calli were not inducted in MS
medium supplemented with 1 mg L-1 2,4 - D (in which calli of Mycetia sinensis were fromed
following Lu.Y‟s report). Thus, MS medium added 0.5 md L-1 2,4 - D was the best suitable
medium to produce callus from M. balansae Drake in vitro leaf.
Leaf pieces in medium without 2,4 - D overed a short period of callus induction, then
it directly regenerated shoots with 7 – 9 shoots per callus. This result can save time as well
as money in in vitro culture of M. balansae.
3.3. Shoot regeneration from callus
Calli were transfered on MS medium supplemented with 2 mg L-1 BAP; Kinetin (0 –
1.5 mg L-1) to evaluate the regenerative ability of callus. The data was recorded after 8
weeks of culture in table 3.
Table 3. Influce of Kinetin to regenerative ability of shoots from callus
MS supplemented with 2 No. of No. of No. of average
mg L-1 BAP and 2,4-D Regeneration shoots/
initiation regenerative
rate (%)
Kinetin (mg L-1) callus callus callus
0 30 11 36.67 3.60
0.5 30 2 6.67 1.31
1 30 8 26.67 3.49
1.5 30 16 53.33 6.05
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.
Figure 4. Shoots regenerated from callus in MS medium supplemented with 2 mg L-1
BAP and without Kinetin (A); 1 mg L-1 Kinetin (B); 1.5 mg L-1 Kinetin (C)
Regenerated shoots in MS medium without Kinetin and 2 mg L-1 BAP were formed
early and regeneration rate was 36.67%. It indicated that the levels of endogenous
hormones were quyte high in this leaf tissue. Using Kinetin with low concentration made
regeneration rate to decline. In MS medium supplemented with 1 mg L-1 Kinetin,
regeneration rate was 26.67%, number of average shoots/callus was 3.49; however,
regenerated shoots was formed slowly, was greenish and puny. In MS medium using 1.5
mg L-1 Kinetin, regeneration rate reached 53.33%. This result explained that stablishment
of balance between the level of BAP and that of Kinetin in cultured leaf tissue made the
efficiency of shoot regeneration to increase in in vitro multiplication. Further studies are
being conducted to result in the final conclusion.
This regenerated shoots from callus and the shoots from direct regeneration
(following the experiment of callus induction 3.2) were cultured on MS medium to root
and to compare growth of the shoot types. After 4 weeks of culture, vitality of shoots from
direct regeneration was better than that of regenerated shoots from callus. The leaves of
plantlets from direct regeneration were greener and had bigger size (Figure 5).
Therefore, there were two ways to form in vitro plantlets: direct regeneration and
plant regeneration from callus. With direct regeneration, culture time was shorter and
shoots grew well, so in vitro multiplication of M. balansae should use this way.
Figure 5. Plantlets in MS medium (direct regenerated shoot ( A)
and regenerated shoots from callus (B))
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3.4. Effect of the plant substrates and minerals to survival and growth of plantlets
Plantlets with 3 – 4 leaves were washed agar and transplanted to plant substrates.
After 4 weeks, the length of plantlet reached 3 cm after plants were watered with different
mineral concentrations (water, 1/10 MS, 4/10 MS) to evaluate their growth. The result was
reported after 10 weeks in tables 5 and 6.
As can be seen from the data in table 5, the best survival and growth of plantlets was in
Thuy Cam substrate (the highest survival rate 83.63%) and the seconds was 60% in mixture of
garden soil and sand (1:1) substrate. The other substrates were unsuitable for this plant because
of low survival rate (33.33 - 48.57%).
Table 5. Effect of the plant substrates to survival rate of plantlets
No. of No. of
Plant No. of dead
plant survivable Survival rate (%)
substrate plants
transferred plants
PS01 70 34 36 48.57
PS02 70 25 45 35.71
PS03 110 92 18 83.63
PS04 40 24 16 60.00
PS05 30 10 20 33.33
Table 6. Effect of the concentration of minerals to growth of plantlets
Growth evaluation
No. of No. of The
Survival The number
Solutions experiment dead number of
rate (%) of average
plants plants average of
leaves
height(cm)
Water 30 1 96.67 2.79 2.95
1/10 MS 30 1 96.67 4.69 4.24
4/10 MS 30 3 90.00 3.39 2.78
Figure 6. Plantlets in garden were watered different solutions (A)
and placed in Thuy Cam substrate (B)
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The results presented in Table 6 shows that plantlets were better growth when they
were watered 1/10 MS solution. The average plant length increased about 4.69 cm, the
average number of leaves increased 4.24. Using higher concentration 4/10 MS, the survival
rate reduced. Consequently, the substrate of Thuy Cam Co. Ltd and 4/10 MS solution were
found to be the best suitable substrate supporting for growth in outside condition.
III. CONCLUSIONS
Seeds sterilization in 20 % NaOCl for 5 min, then in 10 % H2O2 for 3 min was the best
sterilization condition, non infect explants, and the germination rate of seeds was 83.33%.
The maximum callus induction was obtained in MS medium supplemented 2 mg L-1
BAP; 0.2 mg L-1 NAA; 0.5 mg L-1 2,4-D and the plant regeneration rate was 53.33% in
MS medium supplemented 2 mg L-1 BAP; 1.5 mg L-1 Kinetin.
The proportion of shoot direct regeneration was 100% and from 7 to 9 shoots per
explant on MS medium supplemented with 2 mg L-1 BAP and 0.2 mg L-1 NAA. The plant
from direct regeneration showed a growing better than that the regenerated plant from callus.
The highest survival rate of plantlets (83.33%) obtained on the substrate of Thuy
Cam Co.Ltd with 1/10 MS solution for 4 weeks.
REFERENCES
[1] M.R. Ahuja, Somatic cell genetics of woody plants, Kluwer Academic Publishers,
The Netherlands, 28 pages, 1988.
[2] N. Mark, K. Sounthone, S. Bouakhaykhone, T. Philip, S. Khamphone, L. Vichith, A.
Kate, A checklist of the Vascular plants of Lao, Royal Botanic Garden Edinburgh
310, 2007.
[3] Pham Hoang Ho, An illustrated flora of Vietnam, Vol .3. Tre Publishing house, Ho
Chi Minh, Vietnam, 185 pages, 2000.
[4] L. Jakob, R. Katharina, O. Katja, W. Ludger, W. Sabine, T.V.Sung, N.T. Anh, N.V Trai
for “Orally consumable formulation comprising certain sweet-tasting triterpenes and
triterpene glycosides”, World Intellectual Property Organization, 2012.
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