Chapt:er III

.9W'a-terialS and .9W'e-thods

3. 0 ::M.ateria{s anrf nzetfiorfs

The present study "Factors affecting somatic embryogenesis

and plant regeneration in Dendrocalamus asper and

Dendrocalamus tulda (Edible bamboo) sp." was carri ed out

at Department of Biotechnology, Sant Gadge Baba Amravati

University, Amravati. The original species of D. asper and D.

tulda were procured from Central Nursery, Maharashtra

Forest Department, Wadali, Amravati. These plants were

maintained in green house and used as source of explants.

The mother source plants D. asper and D. tulda were a lso

used as a source of explants from Wadali nursery.

3.1. Requirements

3.1.1 Chemicals

The deta il s of different chemicals used and collected from

different agencies to perform experiments are as under.

Sa lts of macro and microelements of analytical grade were

obtained from Loba chemie, Himedia, Sigma, Qualigens, Sd

fine, Merck. Etc.

Vitamins, Amino acids, Sucrose were obtained from Hi media

Laboratories Pvt. Ltd., Bombay.

Myo inositol, Thiamine HCI, Plant growth regulators (Auxin,

e.g. 2,4-D, NAA, IBA, IAA etc. and cytokinin e.g. Kinetin,

SAP) of cell culture tested grade were purchased from M/S

Sig ma Chemicals, Saint Louis, Missouri 63178 USA. and Hi

media Laboratories Pvt. Ltd . , Bombay.

Phytagel was purchased from (Hi media).

Bavisti n (Carbandezim: A systemic Fungicide) were obtained

from M/s BASF India Limited, Bombay.

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Streptomycium Sulphate (a n Antibiotic) was obtained from

M/S Hindusthan Pharmaceuticals, Pune .

Teepol (Detergent l iquid soap- Laboline) was procured from

M/S BDH Glaxo chemicals Limited, Bombay.

HgCl2 NaOCI, an explant sterilan t were used from Hi media. I

3.1.2 Glass wares

All the glasswares used for the experiment during the work were

'Borosilicate Laboratory grade' and were procured from M/s. Borosil

India Limited, Bombay.

3.2 Washing of Glass wares

Glasswares were soaked in 1: 10 Laboline (detergent)

so lu tion in d istilled water for two hours followed by rinsing

with tap water. Then they were soaked in lN HN03 for

overnight. On the next day the glasswares were washed 2-3

times w ith tap water and followed by double distilled water.

After washing they were dried in oven at 110°c. Finally the

glasswares were stored in dust proof cupboards until further

use.

3.3 Sterilization of glass wares

Test tubes and conical flasks were plugged with non ­

a bsorbent cotton wrapped in muslin cloth. Prior to use all

g lasswares were autoclave d at 1.06 kg/Sq.cm pressure at

approximately 121°c temperature for one hour. Autoclaved

glasswares were then dried in oven at 80-100°C for two

hours. Forceps, sca lpels, blades and scissors were f irst

autoclaved and then during inoculation sterilized by dipping

;n absolute alcohol and holding on flame alternately.

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3.4 Selection of culture media

The basal media deve loped by Murashlge a n d Skoog's ( 1962)

was s t andardized u si ng differen t co n cen tration s and

combllldllOn!:. of growth hormones for In vitro Cdllu::.

Inducti on and regeneration from tissues of D. asper ancJ D.

tulda.

3.S Adjuvant: t:o t:he basal medium

T h e MS rTIGdlum wds sur>pl emcnted with fo llowing ilOjuvant

t or Initiation of growth, est .. 1blis hni<"nt or cu ll1Jr<..·~,

rnctinl<'rh1nce d n d rnultlpllc dtlon of c ulturcs and

rnorphog<' n esls of th e cxp l ant.

• 2, 4 - Dich l orophenoxy acetic iH.ld (Auxin)

• Ndphthi:l l crie acet i c ncid (Auxin)

lndol hutyrlC dCld (Auxin)

3 - .lndo l acellc ac.id (/\uxln)

•

•

• • •

•

6 - Benzyl amino purine (Cyloklnin)

Klnetln (Cy t ok inln )

S u crose (Carbon source)

Thiamine HCI (Vitamin)

Myolnosito l (Vitamin)

Ascorbic Acid (Antiox idant)

3.6 composition of Media

The composit ion of MS basa l medium used for the present

study is given in Table 1. Concentrated so lution (stock

so lutions) conta ining different ingred ient s were prepared in

sterile double distilled water and stored in reagent bottles at

4°c not more than 15 days after preparation.

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Separate stock solutions were prepared for each growth

reg ulators by dissolving it in a minimal quantity of the

appropriate solvents. The stock solutions of auxin ( lmg/ml)

were prepared by adding a few drops of 0.lN NaOH (3ml for

every 10 mg of auxin) to a weighed quantity of auxin an d

t hen making up the required volum e with sterile double

glass distilled water. The stock solutions of cytokinin were

pre pared by adding few drops of 0.1 N HCI (3ml for every 10

m g of cytokinin) to a weighed quantity of cytokinin and then

ma king up the required volume with sterile double glass

distilled water.

3. 7 Preparation of culture media

A more convenient and popular method, were to prepare a

se ries of stock solutions. To prepare Murashige and Skoog's

b asa l medium, four different stock solutions were prepdred.

a) Major salts (20X concentrated)

b) Minor salts (200X concentrated);

c) Iron ( 200X concentrated);

d) Organic nutrients except sucrose (200X concentrated).

:::o r the preparation of stock solutions (a)-(d) each

component were separately dissolved to the last parti cle

and then mixed with the others.

-~e sequence of steps involved in preparing a medium is as

fol lows :

1) Required quantities of Phytagel and sucrose were

weighed and dissolved in double distilled water, about

3/4 volume of the medium, by heating them in water

bath.

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2) Appropriate quantities of the various stock so lu tions,

including growth regulato rs and other special

supp lem e nts w e re added.

3) T h e final volume of the medium was m ade up with

double g lass di s tilled water.

4) Afte r mi x ing w e ll , the pH of t h e m e dium was adjusted

u s ing 0.1 N NaOH and/or 0. 1 N H C I .

5) Tl1 e n t h e m e dium was poured into t h e d es ired

cu ltured vesse l. 15 ml o f the m e dium wa s d is p e nde d In

a 25 X 150 mm c ulture tube, a nd about 50 m l In 150

ml f lask.

6) T ubes a nd co nica l flasks were plugged u sing cotton plugs

wrapp ed w ith muslin cloth and fina lly t ubes w e r e wrapped 0

with a luminum fo ils and autoclaved at 1 2 1 C temperature

a nd 1.06 Kg/Sq.cm pressure for 30 min. After t he steam wc:is

completely re leased, tubes and conica l f lasks were removed.

7) The test tube s la nts w e r e prepared. Th e s l a nt tubes

and flasks were stored in dust free area until further

use .

Table 1 : Stock solution for MS basa l medium

Stock solution I

Constituents

MgS0'4.7H20

K H 2PO.,

KN03

NH4N03

CaCl2.2H20

Stoc k Solution XX

H3l303

MnS0.,.4H,.O

ZnSO.,.lH70

Na2Mo 0 4 .2 112 0

cuso'1.sH7o

CoCl2 .6H;iO

Stock Solution XII

FeS04.7H20

Na2 EDTA.2H20

Stock Solution IV

Inositol

Th iamine HCI

Pyridoxine HCI

Nicotinic Acid

G lycine

Amount (Mg/I)

7400

3400

38000

33000

8800

1240

4460

1/20

50

5

5

5560

7460

20000

100

100

100

400

a . For preparation of stock solutlon III, Dissolved FeS04 . 7H 2 0 and Na:;iEDTA .2H 2 0 separatel y in 450 ml disllllcd wate r by healing and constant stirring. Mixed the two solutions, adjusted the pH to 5.5 and added double distilled water lo makeup the final volume to one liter and stored in umber color bottle b. To prepare one liter of media SO ml of stock solution I, 5 ml each of stock solution II, III, IV were taken.

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Table 2: Disinfectants used for sterili z ing plant material .

Sr . No . Disinfectants Concentration Duration of

( % ) treatme nt

(Min) ,__

1 Benzalkonium Chloride 0.01 - 0.1 5 - 20.

2 Bromine water 1 - 2 2 - 20 - 3 Calcium hypochloride 9 - 10 5 - 30

4 Ethyl alcohol 75 - 95 30 sec

5 Hydrogen p e roxid e 3 - 1 2 5 - 15 - 6 Mercuric chlorid e 0.1 - 1 . 0 2 - 10 -

7 Silver nitrate 1 5 - 3 0

8 Sodium hypoc hlorid e 0 . 5 - 5 5 - 3 0 -

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3.8 Transfer area for aseptic manipulation

All the aseptic manipulations like expla nt preparation,

surface sterilization, inoculation as wel l as their subculturing

operations were carried out i n the lamina r airflow hood . The

working surface wa s c leaned u s in g 0 .5 °10 D e ttol so luti on

( v IV) a nd fin a 11 y wiped with 9 0 °/o a I c oho I. The door of t h e

laminar flow ben c h was c losed and the working a r ea wa s

expos ed to ultra violet ligh t for 3 0 minutes before each

exp e riments.

Tho s urgical Instruments were c lea ned and wrapped In brown

paper /aluminum f o ll an d autoc l aved. Fiiter pap e r of

required s i zes w e r e c u t and kept In th e petriplates and

wrapped in br-own pap e r . All these materi a l s were autoc laved

f o llowing usual procedure a nd s t ore d In dust free area.

Du ring the co u rsc of ex plant prep a rallon, s u rface

s terilization, lnoc vl a llon a nd other aseptic manipulation, all

the s urgica l instruments viz. forceps, sca lpe l, n eed le and

blade were deeped Into Lh e 90°/o a l cohol an d fl a med Lo

s t e rili ze before e very operation. Th e sta ndard s Lerll e

te c hniqu es were fo l lowed as s ugges t e d by Street ( 1977) for

inoc ul ation and s u bcu lLurln g of explants.

3.9 Selection, isolation and preparation of explants

D e ndrocalamus asper and Dendrocalamus tulda plants were

regu larly Irrigated and ma i ntained In the green house. Th e

p lant tissues such as l eaf bid, shoots t i p, nod e seg m e nts and

root segments were se lected from healthy plants. The

iso lated plant tissues were washed with tap water and then

double glass distilled water . The explants were cut in proper

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size in the petriplate and were washed with double glass

distilled water. These exp lants were ready for surface

sterilization .

3.10 Sterilization of Explant

The surface sterilization of explants was performed on laminar

airflow. The leaf, shoot tip, node bud segments and roots were

washed three times with tap water and then with double glass

distilled water. Then the ex plants were ready for surface

sterilization. The different concentrations with different incubation

period of HgCl2 were chosen for the surface sterilization of leaf bid,

shoot tip, node bud segments and root segments.

A. S urface steri lizatio n of Leaf bid, s hoot tip, node bud segm e nts

a nd root segme nts.

The diffe r e nt con centrations of m e r c uric c hloride w ere u sed for the

s urface sleri llz ation of explants, leaf bid, s hoot tip, node bud

segments a nd root segments. The leaf bid, shoot tip, node bud

seg m e nts a nd root segments were s u spended in 0.1 to 1 .0 °/o HgCl2

(W/V) solution for 1 min to 10 min tim e duration. These treated

explants was hed thrice in b eaker containing sterile double dis tilled

water. Afte r washing, these explants w ere blotted by sterile blotting

paper. Then explants inoculated aseptically on MS medium

supplemented with various concentrations of auxin and cytokinin.

The observations were recorded after 3 -4 days, of inoculation in

terms of their survival and occurrence of contamination.

3.11 Incubation room

All the inoculated cultures of D. asper and D. tulda w e re

incubated in culture room. The t e mperature wa s maintai n e d

36

0 at 27 + 2 C. The cultures were kept in light for 16 hours

(1000- 4000 lux) and 8 hour dark respective ly.

Out of a l l the four different explants selected and tested for

their growth responses in various combinations of MS media

only new sprout nodal exp lants showed the response.

Therefore it was continued for further study.

3 . 12 Induc tion a nd growth of c a llus

The explant new sprout bud segment were c u ltu red on MS

media fortified with different concentrations of auxin and

c ytoklnln. Ten exp lants and s lants per treatment were taken,

which were replicated thrice. Days required for induction of

ca llus were observed.

Physical appearances of callus were noted after 30 days.

Ca llus growth was measured in terms of fresh weight a nd

dry w e ight after 30 days. The n ew sprout node bud seg ment

showed the better respon se for ca llus induction. Cal l u s was

taken on filter paper and washed 2-3 times with steril e

double glass distil led water, to remove the remaining media

from ca l lus. The ca lli were soaked on butter paper and fresh

weights were d etermi ned after harvesting the tissue on

butter paper. This ca lli were again used for dry weight

determination. For dry weight, ca l lu s ti ssue were dried for 0

overnight in oven at 65 C for 12- 18 hours to remove

complete water. Then the dry weight were taken. The data

was recorded and analyzed statistically for all the

observations.

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3.13 Somatic embryogenesis and multiple shoot

induction

The fresh calli were transferred to different modified MS

media fortified with different concentrations of auxin and

cytokinin either single or in combination, for the induction of

somatic embryos. The different physical and chemical factors

were studied responsible for somatic embryogenesis. The

cu ltures were kept in 16 hours light ( 1000- 4000 lux) and 8 0 0 0

hours dark at the temperature 23 C to 27 C + 2 C. The

embryo formations were observed after incubation of callus.

Note d down the days r eq uired for e mbryo formation as well

as the induction of multiple s hoots. Th e s ubc ulturing of the

multipl e shoots were performed on the same medium . The average number of s hoots were recorded and analyzed statica lly.

Wh e n the height of multipl e s hoots attained 5- 7 cm, t h e shoots were Isolated and transferred on rooting medium . The who le exp e rim e nt was repeated thri ce .

3.14 :Isolation of shoots for root: induction

The Individual shoots were Inoculated on rooting m edia. The

isolate d shoots were washed thoroughly with sterile glass

doubl e distilled wate r and transferred for rooting on full and

half strength MS medium su pplemented with different

concentrations of auxin and cytokinin. The cultures were

kept under 16 hours light (3000-3200 lux) and 8 hours dark.

In individual shoot, root initiation was observed after 10

days of shoot transfer. Each treatment was repeated thrice

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and the observations for average numbers of roots we re

record ed aft er 15 days .

3.15 Hardening of in vitro raised bamboo

T h e in v itro m ature p la ntl e t s w e r e about 7 c m t a ll , whic h

we r e r eady for t h e harde n i ng . Th e p la n t le t s w e r e re m o v e d

f ro m t h e m e d ium a n d r oots w e r e was h e d gen t l y w i t h steri le

dou b le g l a ss dist i ll e d w a l e r to r e move th e excess m e d ia

f r o m t h e r oots. I n v it ro g r o wn r oo t s y stem s a ppea r e d to b e

v e ry d e lica t e, th e r e f o r e ca r e w as t a k e n t o r e t a in a ll th e fibe r

roots Intac t to th e pl a n t l c t s. T h ese p la n t le t s w e r e

tra n s f e rre d into di f f e r e n t harde ning m e dia co n s lllute d s u c h

as 1 ) S o ll : Sa n d, 2 ) Soll : Sa nd : Cow du n g : 3 ) So ll : Sand :

C ow dung : V e rmic ulite.

Tli e tempe r ature, humidity a n d p ti o t o p e rt od we r e mainta in e d

duri n g h a r d e ning th e In vit ro grow n p l an llets . A f te r 1 0 - 15

days , t h e pl a n t tets w e r e tra n s f e rre d Into p o lyth e n e b a g s

with h a rd e n m e dia . T h e n t h e p la n t s we r e tra n s f e rre d t o

green h o u se . Thro ugh o ut th e h a rd e nin g procedure th e

s urv iva l rate o f t h e pla n t was measure d . O b se r vat i o n s o f

s u ccess f u ll y grown p l a n t le t s w e r e t a k e n a f ter 15 a nd 30

days a nd data w e r e s t at is ti c a ll y a n a l y zed.

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3.16 Statistical analysis Ten aliquots of each treatment w ere used for recording

observations. The data obtained was statistically analyzed as per procedure by Panse and Sukhatme (1958) using the formula as follows.

Whe r e

L Xi X =

n

x = mean

S um of ' I' observa tions

=J L (Xi - x )2

S. D. n - I

Whe r e X i observa tions ( I = 1 , 2 , 3 , ...... ... n )

s. D . = S tandard D e vi a tion .

S . D . 5 . E. = \r.;-

Whe re S. E. = Standard Error.

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