Effect of benzyl adenine and sucrose for in vitro microrhizome production in curcuma aromatica salisb

The aim of this research was carried out to rapid multiplication of Curcuma aromatica salisb with large quantities, uniform and good quality, stored and transported easily which are advantageous for in vitro multiplication. Experiment comprised: the effect of tree concentration Benzyl adenin (3, 5, 7 mg/L) and six concentration sucrose (20, 40, 60, 80, 100, 120 mg/L) on the in vitro microrhizome production were investigated.

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EFFECT OF BENZYL ADENINE AND SUCROSE FOR IN VITRO MICRORHIZOME

PRODUCTION IN CURCUMA AROMATICA SALISB

Nguyen Thi Thuy Diem1

1 An Giang University, VNU - HCM

Information:

Received: 04/10/2018

Accepted: 13/08/2019

Published: 11/2019

Keywords:

Sucrose, microrhizomes, in

vitro, Curcuma aromatica,

Benzyl adenin

ABSTRACT

The aim of this research was carried out to rapid multiplication of Curcuma aromatica salisb with large quantities, uniform and good quality, stored and transported easily which are advantageous for in vitro multiplication Experiment comprised: the effect of tree concentration Benzyl adenin (3, 5, 7 mg/L) and six concentration sucrose (20, 40, 60, 80, 100, 120 mg/L) on the in vitro microrhizome production were investigated The experiment was arranged in a completely randomized form, two-factor, 18 treatments and 5 replications The results indicated that shoots of Curcuma aromatica salisb cultured on MS medium supplemented with 80 g/L sucrose and BA 3 - 5 mg/L for to be the most suitable medium for in vitro microrhizomes induction of Curcuma aromatica with time sooner, the rate of in vitro tuber formation, the number of tubers, tuber weight and tuber diameter reached the highest after 16 weeks of culture

1 INTRODUCTION

White turmeric (Curcuma aromatic Salisb) is a

rare medicinal herb plant species, in Zinger family

– Zingiberaceae, this is projecting to develop

herbs plant source in An Giang province from

now onwards to the year 2020 Studies show the

biological active substances extracted from

rhizome in white turmeric contain group acticve

antioxidant such as curcuminoid, flavonoid,

terpenoid In the adopted antioxidant mechanism,

the manufatured product from white turmeric

have biological effects such as anti- inflammatory

stomach medication from wine (Jeon et al., 2010)

The situation artery antherogenesis is reduced

(Lee et al., 2010), prevent to injury

carcinogenesis on skin by ultraviolet ray (Panich

et al., 2010) The power antioxidant mechanism,

white turmeric plant has potential to become

effect medicine treatment assist for many cancer

types On the other hand, white turmeric was demonstrated diffent from essenttial oil into white turmeric plant such as anti- inflammatory, anti platelet aggregation, cough suppression and

kidney protection (Sikha et al., 2015)

In nature, white turmeric occurs from rhizome, after about 210 day to establish the rhizome breed

(Ravindran et al., 2007) The rhizome has a long

domance and germinates the rainy season Beside, the work to maintain source species of white turmeric annually is both costly and effort Often, the disease on the plant such as rhizome is a

negative ordor (by Pythium sp.) and spot leaf by

Taphrina species and Collectrichum are very

damaging to the preservation process breed in field causing the lack of the breed resource in the culture (Nayak & Naik, 2006) Beside, white turmeric is building plan for planting area in An Giang province to make material area This object

is new so the white turmeric breed source is seen

early So, the cultural technology is set up to

produce breed and quality guaranteed to support

seedlings free from disease, uniform to request

breed produce

In making microrhizomes in vitro by method

micropropagation to processing and good

achieved on object such as Solanum tuberosum L.,

Dioscorea composita, Gladiolus spp., Zingiber

officinale Rosc, Curcuma longa,…(Gopal et al.,

1998; Alizade et al., 1998; Dương Tấn Nhựt et

al., 2007; Sharma & Singh, 1995; Sunitibala et

al., 2001) Currently, the studies on microrhizome

in vitro have not been processed in Viet Nam

through the seedling source for production to

support the farmer

2 MATERIALS AND METHODS

2.1 Plant Materials

The white turmeric rhizome (Curcuma aromatica)

to take on Tinh Bien Oriental medicine congress

in An Giang province and take it from the lab of

Agriculture and Natural Resources, An Giang

University to nursery

2.2 Medium and culture conditions

The medium were used MS basal medium

(Musrashige & Sokol, 1962) supplemented with

agar (8 g/l), Myo - Inositol (0,1 g/l) and 1 mg/L

NAA Depending on how the experiment

supplemented with benzyl adenin (BA) and

sucrose in different concentrations The pH of the

media were adjusted to 5.8 using 1M NaOH or

1M HCl The culture bottles were steam sterilized

in an autoclaved at 1210C, 1 atm for 30 min

Cultural conditions: The explants were maintained

in a growth room under white fluorescent light for

a daily photoperiod of 16 hours, with temperature

24 ± 2 0C

2.3 Methods

2.3.1 Culture to make source sample starting

Curcuma aromatica rhizomes were selected from

7 to 8 months of age, uniform and not diseased

Samples were treating with Antracol 70WP

disease medicine for 60 minutes in clean rice husk ash After 15 – 20 days of incubation, the shoots

of white turmeric began to appear

The shoots were divided from rhizome washed, roots cut and leaves cleaned, 1/2 leaf cut and clean washed Next, the shoots were soaked in light soap after immature buds were cleaned with running tap water Explants were then immersed

in 70% ethanol for 1 min and the explants were rinsed three times with distilled water Next, the explants were soak with 20% Ca(OCl)2 for 15 minutes, after the explants were rinsed three times with distilled water The explants were cut, divided to take the shoot top and culture in bottle contain medium MS not supplemented plant growth regulator to create a material source to use for experimentation in making microrhizome

2.3.2 Experimental arrangement

Excised buds of Curcuma aromatica were high

about 4-5 cm, initially cultured on MS basal medium After 14 days of culture, the shoots became root formations and were transfered to a culture medium MS supplement with BA and sucrose in different concentrations for microrhizome induction

The experiment was arranged in Completely Block Design (CBD), two factor, including 3 concentrations of BA (3, 5 and 7 mg/L) and 6 concentrations of sucrose (20, 40, 60, 80, 100 and

120 g/L) The experiment consists of 18 treatments, each treatment was repeated 5 times, each time was 5 the culture bottle (1 explant/ the culture bottle)

2.3.3 Observations recorded for experiments The time microrhizome induction (weeks after

cultured - WAC), rate tuber formation (%), the number average tuber/explants, the weight microrhizome (g), diameter microrhizome (cm) Observations were done every week starting from the first week after culture initiation to 16 weeks

after culture initiation

2.3.4 Data analysis

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Data were analyzed using the Statistical SPSS

software version 20.0 and analysis of variance

(ANOVA) was used to investigate if there is any

significant difference A mean separation test was

conducted using Duncan multiple range

3 RESULT AND DISCUSSION

3.1 The time microrhizome induction in vitro of

white turmeric shoots

The time set up microrhizome in vitro had interact

between BA concentration and sucrose The time

set up microrhizome induction in vitro earliest on

two experiment to supplemented 3 mg/L BA combine 80 mg/L sucrose and 5 mg/L BA combine 60 mg/L sucrose in 5 weeks after being culturation (Fig 1.), it was statistically different in significance at 1% compared to the remaining treatments

Table 1 Effect of concentration of BA and sucrose on the time microrhizome white turmeric induction in vitro

Sucrose

concentrations

(g/L)

(Sucrose)

3 5 7

20 11.00 d 10.00 e 12.00 c 11.00 b 40 9.00 f 8.00 g 9.00 f 8.67 c 60 6.00 i 5.00 k 7.00 h 6.00 e 80 5.00 k 5.67 j 6.00 i 5.56 f 100 7.00 h 7.00 h 9.00 f 7.67 d 120 13.00 b 13.00 b 15.00 a 13.67 a Average (BA) 8.50 b 8.11 c 9.67 a F (Experiment) **

F (BA) **

F (Sucrose) **

F (BA x Sucrose) **

CV (%) 1.54

Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1%

Fig 1 In vitro microrhizomes induction of white turmeric after 5 weeks of incubation on culture medium

The effect of BA concentration to time induction

to microrhizome production in vitro In

concentrate 5 mg/L BA has time to show

microrhizome in earliest (8.11 weeks after

cultured), the difference was statistically

significant at 1% compared with 3 mg/L BA and 7

mg/L BA concentration in a culture medium It

suggest that the time induction microrhizome of

BA early or later then depended on BA

concentration in supplemented in a culture

medium In the BA concentration suitable for

induction microrhizome formation in vitro were 5

mg/L, the microrhizome were formation earlier,

average about 8.11 weeks after cultured, the

shoots start set up microrhizome in 3 mg/ L BA

concentration and 7 mg/L BA concentration after

8.50- 9.67 weeks after cultured the new

microrhizome was establish

The sucrose concentration to effect come on the

time induction microrhizome production in vitro

The sucrose on 80 g/L concentration were time

induction production earliest was 5.56 weeks after

cultured, the difference was statistically

significant at 1% compared the sucrose

concentration in the other in a culture medium

When the sucrose concentration were increase from 20 – 80 g/L then the time induction microrhizome production to shorten from 11 weeks after cultured to decrease 5.56 weeks after cultured But, when the sucrose concentration were increased continuous up 100 – 120 g/L then the time induction microrhizome production from 7.67 weeks after cultured to increase 13.67 weeks after cultured show new microrhizome

In general, the time induction microrhizome production were MS medium supplement 1 mg/L NAA combine 3 – 5 mg/L BA and 60 -80 g/L sucrose in earliest, the microrhizome was show 5 – 6 weeks after culturation

microrhizome of shoot white turmeric

The results on table 2, the rate of explant to microrhizome established in 16 weeks after cultured to have interact between BA concentration and sucrose The explant culture rate were highest in experiment MS supplemented

1 mg/L NAA with 3 mg/L BA and 80 g/L sucroser to achieve 91.67%, the difference was statistically significant at 1%

Table 2 Effect of BA concentration and sucrose on the explant rate microrhizome formation (%) in white turmeric

in vitro in the 16 weeks after cultured

Sucrose oncentration

(g/L)

(Sucrose)

F (BA x Sucrose) **

Note: The following letters were the same, the difference was not statistically significant: ** = the difference was statistically significant at 1%

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The sucrose concentration to effect come the time

induction microrhizome production in vitro In the

sucrose 80 g/L concentration for the explant rate

microrhizome formation achieved highest 75%,

the difference was statistically significant at 1%

compared the sucrose concentration different in

the culture medium

The BA concentration to effect come the explants

rate microrhizome formation in vitro On the 5

mg/L BA concentration for the explants rate

microrhizome formation were highest (48.61%),

the difference was statistically significant at 1%

compared with MS medium supplemented 3 mg/L

BA and 7 mg/L BA in the culture medium

In the result to table 2, when BA concentration to

increase from 3 mg/L to 5 mg/L, the explants rate

microrhizome formation to increase from 41.67%

to 48.61% When BA concentration increase on 7

mg/L, the explants rate microrhizome formation

to go down 34.72% In the sucrose concentration,

when the sucroser concentration to increase from

20 to 80 g/L then the explants rate microrhizome

formation to increase from 25% to 75% But when

the sucrose concentrate increased on 100 -120 g/L

then the explant rate microrhizome formation go

to down form 50% to 25% It suggest that, the BA

concentration and high sucrose were inhibition to

microrhizome formation in vitro

Finally, white turmeric shoots to culture on the

MS medium to have 1 mg/L NAA supplemented

3 mg/L – 5 mg/L BA combine with 80 g/L

sucrose for the explants rate microrhizome

formation was highest to achieve from 75% to

91.67%

3.3 The number of shoot white turmeric in vitro

to increase

In the 16 weeks after cultured, the results on table

3, the number of microrhizome to effected of BA

concentration and sucroser content

BA concentration to effect to the number of microrhizome In 5 mg/L BA concentration for the number of microrhizome to achieve highest was 1.94 microrhizome/explants, the difference

was statistically significant at 1% as compared to

BA concentration 3 mg/L and 7 mg/L in the culture medium

The sucrose concentration to effect come number

microrhizome in vitro The sucrose concentration

was 80g/L achive then the number highest baby microrhizome was 3 microrhizome/explants, the

difference was statistically significant at 1% as

compared the sucrose concentration other in the culture medium

The compared between experiment, the results on table 3, the number of microrhizome to highest increase in 3 mg/L BA concentration with 80 g/L sucrose then achieved 3.67 microrhizome/ explants, the difference was statistically

significant at 1% as compared the other

experiment

In general, the results in table 3, when increased

BA concentration from 3 mg/L to 5 mg/ L, the number of microrhizome to increase from 1.67 microrhizome/ explants to 1.94 microrhizome/ explants When increase BA concentration up to 7

mg/L, the number of microrhizome in vitro

become go to down only achieve 1.39 microrhizome/ explants For sucrose, when the sucrose concentration to increase from 20 to 80 g/

L then the number of microrhizome to increase from 1 microrhizome/explants to 3 microrhizome/ explants But when the sucrose content up to 100

-120 g/L then the number microrhizome formation

go to down from 2 microrhizome/ explants to 1 microrhizome/explants So, the white turmeric shoots to culture on MS medium contain 1 mg/L NAA to supplemented 3 – 5 mg/L BA concentration combine with 80 g/L sucrose are microrhizome to achieve highest (Fig.2)

Fig 2 The development of microrhizome in vitro on the white turmeric shoot on medium

supplemented 3 -5 mg/ L BA and 80 g/ L sucrose at 16 weeks after cultured Table 3 Effect of BA concentration and sucrose on the number of to increase on microrhizome in

vitro after 16 weeks after cultured

Sucrose

concentration (g/L)

(Sucrose)

F (BA x Sucrose) **

Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1%

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3.4 The weight of microrhizome white turmeric

in vitro

The results on table 4 represent the 16 weeks

after cultured, the sucrose content to have effect

on weight increase of microrhizome in vitro The

sucrose content on 80 g/L to achieve highest

weight is 0.98 gram/microrhizome, the difference

was statistically significant at 1% as compared the

sucrose other in the cultural medium The weight

microrhizome in vitro on sucrose content to 40

mg/L and 100 mg/L not the different was

statistically significant

The results were statistically significant about

rhizome in vitro weight to increase all

experiments The rhizome in vitro weight to

highest achieve on MS medium supplemented 3

mg/L BA and 80 g/L sucrose (1,08

gram/microrhizome), the different was

statistically significant at 1% as compared the

other experiment Between on the experiment to contain 80g/ L sucrose combine with BA

concentration 3 and 5 mg/L then microrhizome in

vitro weight to achieve 0.93 gram/ microrhizome,

the different was not statistically significant

The microrhizome in vitro weight on BA

concentration (3, 5 and 7) the different was

statistically significant, the microrhizome in vitro average weight archive 0.55 gram/microrhizome

after 16 weeks cultured In general, the BA concentration not effect to the weight increase of

microrhizome in vitro

So, the experiment to supplemented 80 g/ L sucrose on MS medium with 1 mg/L NAA combine BA concentration 3 -7 mg/ L for

microrhizome in vitro weight was highest

Table 4 Effect of BA concentration and sucrose on up to weight (gram/microrhizome)

white turmeric in vitro after 16 weeks cultured

Sucrose

concentration (g/L)

(Sucrose)

F (BA x sucrose) **

Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1%; ns= the difference was not statistically significant

3.5 The diameter to increase of white turmeric

microrhizome

In the results on table 5, the BA concentration and

sucrose content to interact on the increase of

diameter of white turmeric in vitro microrhizome

The BA concentration to effect on the diameter

increase of rhizome in vitro In the BA

concentration 3 mg/L, the diameter of

microrhizome in vitro achieve highest was 8.10

mm, the difference was statistically significant on

1% as compared the BA concentration 7 mg/L,

but the difference was not statistically significant

than 5 mg/ L BA to achieve 7.8 mm

The sucrose content to effect on increase diameter

microrhizome in vitro The sucrose content in

80g/L to have diameter highest gain was 10.05

mm, the difference was statistically significant at

1% as compared the sucrose content other in the

culture medium

The result of table 5 suggest that the different was statistically significant about to increase diameter

microrhizome in vitro all experiment The diameter of microrhizome in vitro highest gain on

MS medium to supplement 3 mg/L BA and 80g/

L sucrose (13.7 mm), the difference was statistically significant at 1% as compared the experiment content other in the culture medium

Table 5 The effect of BA concentration and sucrose diameter to increase of microrhizome (mm) on white turmeric

in vitro after 16 weeks after cultured

Sucrose

concentration (g/L)

(Sucrose)

F (BA x sucrose) **

Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1%

In general, the experiments was supplemented 3 –

5 mg/ L BA with 80g/L sucrose in MS medium

culture to contain 1 mg/L NAA for the diameter

of microrhizome to biggest gain after 16 weeks

after cultured (Fig 3.)

In summary, the results of target about

microrhizome production in vitro suggest that the

microrhizome formation of white turmeric in in

vitro condition to effect of BA concentration and

sucrose content interact The white turmeric shoots were culture on MS medium to supplemented 80g/L BA combine 3 – 5 mg/L BA and 1 mg/L NAA then the time microrhizome induction was earlier, at the same time the

25

microrhizome in vitro formation rate, the number

of microrhizome production and microrhizome

diameter were highest When the BA

concentration to increase 7 mg/L combine 100

mg/L and 120 mg/L sucrose then the time

microrhizome induction, the microrhizome in

vitro formation rate, the number of microrhizome

production and microrhizome diameter were decreased

Fig 3 The microrhizome diameter of white turmeric shoot were growth on the medium supplemented 3-5 mg/L

BA and 80 g/L sucrose in 16 weeks after 9 (cultured)

The result of the experiment suggests that the BA

concentration and sucrose content to effect

interact on microrhizome in vitro formation On

the MS medium contain 1 mg/L NAA to

supplemented 80 g/L sucrose with 3-5 mg/L BA

for the time microrhizome induction, the

microrhizome formation rate, the number of

microrhizome productions, the weight and

diameter of microrhizome were highest It

suggests that this BA concentration to effect on

transport carbohydrate content in the white

turmeric shoots The result that similar of Nanak

(200), the number of microrhizome in vitro to

many formation on the medium supplemented

5mg/ L BA on C.aromatica Salisb

In Curcuma sp species, the shoots were rhizome

formation to view when the medium to supply

sucrose concentration was 60 – 90 g/L (Nayak

(2000); Sunitabala et al (2001) In addition, the

study about the formation zinger in vitro rhizome

of Sharma và Singh (1995), the sucrose

concentration was 75 g/L to effect in rhizome

formation Sedigeh et al (1998) to believe that the

sucrose from 6 – 8% (w/v) was the most

important ingredient for formation in vitro tuber

on Diascoria composita It suggests that the study

topic results concluded that, in the study to show the medium MS supplemented 80 g/L sucrose for

the number of microrhizome in vitro was most The number of microrhizome in vitro was shortest

on the MS medium contain 20 g/L and 120 g/L sucrose The sucrose was role to promote formation organ store in plant When the sucrose concentrate from 20 g/L to 80 g/L to show the

microrhizome in vitro formation to rate, the

number microrhizome, the weight microrhizome and diameter microrhizome are increase It suggests that the show of carbon content in sucrose and the white turmeric ingredients contain carbohydrates and sucrose The results of the experiment showed the sucrose concentration in the medium to effect on the formation

microrhizome in in vitro condition

In summary, the results showed the plant growth regulator was role on cell division, dimensions of shoot germination and the effect on size of

rhizome The sucrose was effect in the growth

process of microrhizome in vitro, the development

of baby rhizome when the sucrose was highest,

the sucrose supplemented nutrient to growth

rhizome and rhizome induction, the rhizome was

formation and development

3.6 The Acclimatization of white turmeric in

vitro

The white turmeric rhizome in vitro were

harvested on the medium rhizome production,

clean wash agar and nursery on rice husk ash The

moisture was provided by water spray twice a day (humid 70 – 80%) The temperature was 28 – 30

0C in the nursery step The results showed, after 2 weeks plant on rice husk ash all the white

turmeric rhizome in vitro show shoots, leaves and

roots ( Fig 5A) After 30 days planted on pot to

show the white turmeric from rhizomes in vitro

the root system was developed and deep in the ground, the nursery plant was developed with 3 –

4 leaves, the plant was moved out off the pot and planted in black plastic (Fig 5B)

A B

Fig 5 The white turmeric rhizome in vitro to show shoot after 2 weeks cultured (A); the white turmeric from

microrhizome in vitro (B) after 30 days planted into pot

4 CONCLUSION

The medium microrhizome production in vitro

was MS medium supplemented 3 mg/ L or 5

mg/L BA combine with 80 g/L sucrose

5 RECOMMENDATION

Continue evaluation of the growth and

development of rhizome in vitro on nursery stage

of the white turmeric

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