1

TRAINING REPORT

On

Micropropagation techniques in chrysanthemum species

Submitted in partial fulfillment
requirement for the award of the degree of
Bachelor of Technology (Bio -Technology Engineering)
of Kurukshetra University,
Kurukshetra

By:-
DEEPANSHI BANSAL
1507610

Department of Bio -Technology

N.C. College of Engineering, Israna
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INTRODUCTION
German plant physiologist Gottlieb is known as the father of tissue culture,

who coined the term ‘totipotency’ at the first time. All the in vitro methods of plant

propagation rely on the phenomenon of totipotency of plant cell, which has the

capacity to regenerate into a complete plant.

Tissue culture and in vitro culture are two broadly used term for

micropropagation, which basically include aseptic culture of various plant parts.

All the biological principles of micropropagation technique are based on the

phenomenon of totipotency of plant cell, which has the capacity to regenerate

into a full-fledged plant.

Clonal - true - to - type propagation of plants by tissue and cell culture

method is called as in vitro propagation which is considered as the most efficient

and commercial method of plant propagation more precisely called as

micropropagation. This method refers to the production of plants from very small

plant parts, tissue or cells grown in a test tube or other containers under

controlled nutritional environmental and aseptic conditions.

Since the discovery of micropropagation, while attempting short tip culture

in orchid by Prof. G. Morel (1960). This technique has been demonstrated in over

5000 plants, however, commercial success has been achieved in only over 500

plants. Most of which are the ornamental and herbaceous plants.

Chrysanthemum morifoleum is a perennial flowering plant belongs to

family Asteracae, is a semi-hardy herbaceous, annual or evergreen shrubs. The

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name chrysanthemum owes its origin to J.P. de Tournefort and in Greek signifies

“golden flower” chrysanthemum name was derived from the Greek words

Chrysos (gold) and anthemom (a flower) by Linneaus in 1753. There are 38

genera and 20 species in chrysanthemum. In the literature five major genera and

only two species have been defined now remain in the chrysanthemum genus in

the strict sence. The widely grown cultivator called florists chrysanthemum or

garden chrysanthemum previously classified as Chrysanthemum morifoleum

Ramat. It is one of the three most important flower crops of the world, which is

extensively used as pot plant, decorative green plant and cut flower production.

Furthermore, Chrysanthemum cinerariefolium and Chrysanthemum

coccineum are grown commercially for the extraction of pyrethrum.

Chrysanthemum can be propagated by seed (annual chrysanthemum),

suckers, stem or shoot tip cuttings under field conditions. The plant has the

capability of regeneration in vitro from leaf, shoot tip, node, internode, petal etc.

Chrysanthemum is commercially a very important flower crop. There is

always a demand for large number of homogenous true-to-type plant material. In

the global export market, the plant material of a specific variety must be

propagated in a very short period.

For rapid propagation of chrysanthemum tissue culture techniques can be

utilized for successful regeneration of various plant parts. Considering the above

facts, the present project entitled “Micropropagation technique in chrysanthemum

species” was undertaken at the Central Tissue Culture Laboratory, NRC

 4

Biotechnology, Lal Bahadur Shastry Building, Indian Agricultural Research

Institute, New Delhi.

The objectives of present investigation are as follows :

1) To standardize the surface sterilization process for nodal segment.

2) To study the culture medium for rapid shoot proliferation

3) To study the effect GA3 or micro-shoot elongation

4) To study the rooting process

5) To study the hardening process of micro plant

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REVIEW OF LITERATURE

Haberlandt (1902) was the first person who attempted plant tissue culture.

Although the attempt made by him to culture isolated cell was unsuccessful, but

his views provided basis for the present day tissue culture.

With discovery of auxins (Kogl et al., 1934) and cytokinins (Miller et al.,

1956), Skoog and Miller (1957) explained the action of growth regulators in

morphogenesis in relation to tissue culture. They discovered that adventitious

shoots or roots could be induced through manipulation of auxin and cytokinin

ratio. A higher levels of cytokinin combined with low concentration of auxin

developed adventitious shoots from tobacco callus. Contrasting to this, high

auxin and low levels of cytokinin induce rooting.

On the basis of experiments conducted in Cymbidium orchids, Morel

(1960) showed the possibility of virus-elimination and multiple shoot production

through tissue culture. The method was later designated as ‘meristem culture’ or

'meri-cloning' which produce the plants in large numbers.

The research work of Murashige (1974), Street (1977), Thorpe (1981),

Bonga and Durzan (1982), Dodds and Roberts (1982) and Dixon (1985) has

significantly contributed to the present day tissue culture.

Murashige and Skoog (1962) medium is being extensively used since the

first ever endeavour of micropropagation in various species.

Murashige (1974) proposed a three stage protocol for micropropagation of

plants. However, it is now agreed that there are five critical stages for successful
 6

micropropagation. According to Debergh and Read (1991), the five stages are:

Stage 0: the preparatory stage; stage I: culture establishment and initiation of

culture by inducing the growth of shoots in vitro from pre-existing meristems in

shoot tips or axillary buds; Stage II: multiplication of microshoots through a series

of subcultures, shoots are proliferated and maintained; Stage-III: elongation, root

induction and development; Stage IV: hardening and transfer of microplants to

green house (ambient conditions).

Chrysanthemum is a perennial ornamental plant. Pathogens may persist

endogenously in the plant cell, which may ooze from the vascular tissue years

after the initial explant was cultured. The external contaminants like dust etc. can

easily be removed from the explant by washing in running tap water for 30-90

(De Fossard, 1976; Jones et al., 1979). Hasegawa (1979), Davies (1980).

Khosh-khui and Sink (1982) and Sauer et al. (1986) used sodium hypochlorite (3-

12%) as chemical surface sterilants for the establishment of axenic cultures.

Other sterilents like calcium hypochlorite @ 5.0 per cent and mercuric chloride @

0.1 per cent have also been utilized by several researchers (Dubois et al., 1988;

Vijaya et al., 1986; Choudhary, 1989; Bhat, 1990; Prasad, 1995). The

conventional antimicrobial compounds, viz., 8-hydroxy quinoline sulphate (8-

HQS) (Machado et al., 1991) and 8-hydroxy quinole citrate (8-HQC) (Read and

Yang, 1990) were also found to be successful as it established 50-90 per cent

cultures free from contamination. Pre-culture dipping of axillary buds in a solution

with carbendazim (0.1%) and 8-HQC (200 ppm) for 3-4 h reduced both bacterial

as well as fungal contaminations (Prasad, 1995).

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Chrysanthemum morifoleum Ramat is a perennial flowering plant belongs

to family Asteracae, semi-hardy, herbaceous, perennial shrub. Chrysanthemum

name was derived from the Greek words Chrysos (gold) and anthemom (a

flower) by Linneaus in 1753. There are 38 genera and 200 species in

chrysanthemum. In the literature five major genera and only two species have

been defined, now remain in the chrysanthemum genus in the strict sence. The

widely grown cultivars called florists chrysanthemum or garden chrysanthemum

previously classified as Chrysanthemum morifoleum Ramat.

Chrysanthemum is commercially propagated by division of suckers, tip

and stem cuttings. In vitro regeneration has been reported for several cultivars of

chrysanthemum species using micropropagation techniques. Successful

regeneration has been reported using different explants like receptacle (Hill

1968), shoot tip (Ben-Jaacov and Langhans, 1972), pedicel (Roest and

Bokelmann, 1975), petal segment (Bush et al., 1976), leaf (Slusarkiewicz et al.,

1981; Bhattacharya et al. 1990; Kaul et al., 1990), node (Lu et al., 1990). The

available literature on micropropagation with special reference to chrysanthemum

species is summarized as follows.

MS medium supplemented with different concentrations of cytokinins, i.e.

BAP and kinetin, auxins i.e. IAA, NAA and IBA individually or in combinations are

extensively used for plant regeneration in chrysanthemum. Fujii and Shimizu

(1990) regenerated the plants from achenes and petals in Chrysanthemum

coccineum. They cultured achenes and petals of coccineum on MS and White

Medium supplemented with BAP and NAA or 2,4-D. Without being transferred,
 8

shoots were formed directly and immediately after callus formation on the surface

of the achene walls and from the cut ends of the petals. High concentrations of

BA and NAA supported the callus induction and shoot formation, however, higher

concentrations of 2,4-D inhibited shoot formation. Shoots obtained from both the

explants formed roots when transferred to hormone-free medium and they could

be transplanted to soil form further growth. The regenerated plants contained as

much pyrethrins as the original plants.

Direct plant regeneration was obtained by Lu et al. (1990) from fresh

chrysanthemum (Chrysanthemum morifolium Ramat cv. Royal Purple) stem

segments cultured on Murashige and Skoog’s (1962) basal medium

supplemented with 6-benzylaminopurine (BAP, 0.5-2.0 mg/l) and -

napthaleneacetic acid (NAA, 0.2-2.0 mg/l).

Smith (1991) carried out the investigation on chrysanthemum, rose and

grapevine with the object of achieving thresholds of hardness that would allow

micropropagated plantlets to be transferred to soil without acclimatization.

The inorganic formulations of 14 common plant tissue culture basal media

were examined by Owen and Miller (1992) from the primary literature. In

accuracies and errors were foundfor molecular formulae. Chemical hydrations

and molar equivalences for fe/EDTA complexation. A comparison with published

basal medium formulations from 6 commercial suppliers unconvered additional

inaccuracies, modifications and errors.

Chrysanthemum (Denduanthema,grands flora) was micropropagated on

murashighe and Skoog (MS) medium using 0.0, 035, 0.7 or 1.05% agar and pH
 9

adjusted to 4.6, 5.0, 5.4, 5.8, 6.2, 6.6 or 7.0.Different agar concentration and pH

and levels did not affect in vitro development of D. morifolium. No difference were

observed in short length and the number of shoot and leaves. However, in order

to achieve the best medium consistency and make the explant inoculation

process more efficient 0 medium containing 0.7% agar and pH adjusted to 5.8 is

recommended (Revista Ceres, 1996).

After sterilization with HgCl2 at 0.1, 0.2, 0.3, 0.4 or 1.0% (w/v), shoot

explants of Chrysanthemum morifolium were cultured on MS medium

supplemented with benzyladenine (BA) at 0.0, 0.2, 0.5, 0.8, 1.0, 1.2 or 1.5 mg/l.

An inverse relationship was observed between HgCl 2 concentration and the

percentage of contaminated cultures. However, bud growth only occurred when

explants were sterilized with 0.5% HgCl 2. While callus induction occurred on

explants cultured on MS medium supplemented with 0.5-1.2 mg/l BA good callus

was only produced at 1.2 mg/l BA. Time required for bud sprouting ranged from

16 (BA at 1.2 mg/l) to 19 days (BA at 1.5 mg/litre) (Haq et al., 1998).

Chakrabarty et al. (1999) isolated and multiplied the sectorial type of

chimeral mutants through direct regeneration from florets of chrysanthemum

(Chrysanthemum morifolium Moret) they standardized an efficient technique for

direct shoot regeneration from individual florets of chrysanthemum cultivar Colchi

Bahar. The regeneration potential varied with the developmental stages of the

flower head. The highest percentage of shoot formation and more shoots per

responding explant were observed on florets taken from Stage II flower heads.

Direct shoot formation was confirmed by scanning electron microscopy and

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histological observations. The technique has been tested on 16 other cultivars in

the medium containing 0.2 mg/l NAA and 1 mg/l BAP. Gamma ray of 1.5 and 2.0

kRad induced yellow and white floret colour sectors in Maghi (Original colour –

mauve) have been established in pure form using this technique. The

regenerated plants were rooted in vitro and successfully transferred to soil,

where they grew vigorously and flowered true to the floret colour type.

Logical advancing of regeneration protocols as an end result of response

to screening and use of alternate explants is reported by Annadana et al. (2000).

Stem explants screened on medium 1 (MS with 1 mg/l BAP and 0.1 mg/l IAA)

produced 4 responses and were classified as Group 1 : more than 1.6 shoots per

explant; Group 2: less than 1.6 shoots per explant; Group 3 only callus and

Group 4: no response. To regenerate groups 2, 3 and 4 specific media were

developed (media 2 a,b, media 3a,b and media 4 a-i). Media 2 and 4 had

changes in hormonal combination compared to medium 1, while medium 3 had

additional changes in vitamins. Nineteen out of thirty seven cvs. tested were

regenerated on stem explants, based on the response to screening technique.

Cultivars recalcitrant on stem explant even after exposure to modified media

were reinvestigated on leaf. High concentration hormone pulse as induction

(medium 5) followed by a modified regeneration medium (medium 6d) ended in

successful regeneration of leaf explants. Four out of the 14 cultivars recalcitrant

on stem were successfully regenerated using leaf explants. Response based

advancing of regeneration protocols in addition to use of alternate explants for

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recalcitrant cultivars resulted in efficient regeneration of 23 of the 37 cvs on

assessment.

Kumari and Varghese (2003) reported that sterilized explants receptacle

of young floral bud of two chrysanthemum cultivars namely, Snow Ball and Miss

Universe, dissected under aseptic condition were inoculated on solidified MS

basal medium, supplemented with various concentrations of growth regulators

viz. BAP (0.5-2.0 mg/l) and NAA (0.1-0.5 mg/l) alone and in various

combinations. Callus initiation took place after 10-12 days of inoculation. Initially

the callus was soft and fragile and subsequently it converted into leafy callus

after 20 days of first callus initiation. Later on it started forming shoots after 25

days. Shoots formed in this way were tiny. These were made to elongate on MS

medium containing GA3 (5.0-10.0 mg/l). Rooting was initiated on the same

medium after 10-12 days. The regenerated plantlets (6-7 cm long) were

transferred to sterilized sand and subsequently to soil.

Promotion of elongation in microshoots makes them ready for the

induction of in vitro / ex vitro adventitious rooting (Bhat, 1990). Successful

hardening of plantlets and their subsequent performance under ambient

conditions depends mainly upon the root system. Rooting can not be produced in

a medium enriched with cytokinins and 2,4-dichlorophenoxyacetic acid, whereas

indole-3-acetic acid (IAA) and naphthaleneacetic acid (NAA) used alone or in

combination induced rooting. Transfer of elongated microshoots to MS medium

devoid of auxins also produces rooting, but the roots are thin, long and

comparatively inferior than those induced by auxins.

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Salt strength in basal medium is very important with regards to early and

quality rooting. In some cases, the salt strength reduction may eliminate the need

of auxins essentially required for rooting. Good rooting system can be produced

on quarter to half-strength MS medium containing sucrose 40-60 g per litre.

Elongated microshoots of chrysanthemum cultivar Royal Purple of 1.5-2.0

cm tall transferred to hormone free half-strength MS medium supplemented with

3% sucrose rooted very well (Lu et al., 1990; Fujii and Shimizu, 1990).

Chakrabarty et al. (1999) induced the rooting on to microshoots in half

strength of MS medium supplemented with 0.1 mg/l NAA, 1.5% sucrose and 0.8

bacto-agar. The regenerated plants with well established roots transferred to

potting compost containing a mixture of sand, soil and manure (1:1:1) and kept in

a moist chamber with 80-90% relative humidity for 10 d-4 transfer to glasshouse

conditions showed successful establishment.

The microshoots regenerated from callus observed to be very tiny by

Kumari and Varghese (2003). They transferred these microshoots to MS medium

containing GA3 (5-10 mg/l) a sufficient amount of elongation could be observed in

the shoots. Rooting was initiated on same medium after 10-12 days.

The rooted plantlets obtained through tissue culture can not be transferred

directly to glasshouse or open field conditions. It is essential to acclimatize the

plantlets gradually from the environment provided for them in tissue culture

vessels characterized with high humidity, low light level and optimum

temperature to the natural conditions with low relative humidity, high light

intensity and more variable temperature (Wainwright, 1988).

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The tissue culture produced plants possess leaves incapable of significant

photosynthesis, stomata unable to close and minimum cuticular wax layer on the

surface (Grout and Aston, 1978).

The high salt level supplemented to the growing medium, particularly

potassium, accumulates on the guard cells in the stomata, is responsible for the

stomata being fully opened (Short et al., 1981). Gradual reduction in the strength

of the salt level may be useful for lowering the potassium level in the leaves.

During the hardening of plantlets, three major factors i.e., relative humidity,

temperature and light intensity are to be controlled. Hu and Wang (1983)

suggested that a high humidity should be maintained during the acclimatization

of the plantlets. For this purpose, three methods of controlling relative humidity

viz., polythene tent, misting and fogging have been suggested in the literature. In

polythene tent, it is possible to create high humidity and to take an advantage of

carbon dioxide enrichment during hardening (Lakso et al., 1986).

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MATERIALS AND METHODS

The present project was undertaken to learn the micropropagation

techniques in chrysanthemum

3.1 PLANT MATERIAL

Chrysanthemum cv. Yellow Bunglow was selected as experimental

material. The variety has shining, yellow coloured blooms (Plate 1) produced on

long stems. Two to three years old, vigorous, disease and pest free plants of

chrysanthemum cv. Yellow Banglow were selected as mother/stock plants for the

experiment.

3.2 MATERIALS

3.2.1 Glassware: The glassware, i.e., beakers, glass bottles with polypropylene

lids, conical flasks, pipettes, test tubes, petri plates, etc. were of borosilicate

glass from Borosil/Corning.

3.2.2 Chemicals: All the chemicals used for the preparation of basal media were

procured from Hi- Media Laboratories Pvt. Limited, Mumbai, India and vitamins

and plant growth regulators from ‘Sigma’, Sigma Chemicals Co., St. Louis, USA.

’
3.2.3 Culture media: Murashige and Skoog s medium (1962) is used in present

project. The composition of this medium is presented in Table 1.

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Table 1: Composition of Murashige & Skoog (1962) medium.

Sl. Chemical Quantity

-l
No. (1x ) (mg/l)

A. MACRO
1. NH4NO3 1650.0
2. KNO3 1900.0
3. MgSO4.7H20 370.0
4. KH2PO4 170.0

B. CALCIUM
1. CaCl2.2H20 440.0

C. MICRO
1. H3BO3 6.20
2. MnSO4.4H20 22.3
3. ZnSO4.7H20 8.6
4. Na2MoO4.2H20 0.25

5. KI 0.83
D. IRON-Sodium EDTA
1. FeSO4.7H2O 27.8
2. Na2.EDTA.2H2O 37.3

E. Copper-Cobalt
1. CuSO4.5H20 0.025
2. CoCl2.6H20 0.025

F. ORGANIC
1. meso-Inositol 100
2. Nicotinic acid 0.5
3. Thiamine-HCl 0.1
4. Pyridoxine-HCl 0.5
5. Glycine 2.0

3.2.4 Explant: Axillary buds were employed as explants.

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3.3 METHODS

3.3.1 Cleaning of glassware: The glassware was steam sterilized and then

cleaned with detergent (Teepol @ 0.1%) and thoroughly washed with running tap

water, rinsed twice with double-distilled water and dried in a hot air oven for 2 h.

3.3.2 Preparation of stock solution: The stock solutions of various chemicals

were prepared in autoclaved double distilled water and stored in sterile reagent

bottles in a refrigerator after filtering the contents. Benzylaminopurine (BAP),

gibberellic acid (GA3) and naphthaleneacetic acid (NAA) were dissolved first in

1N KOH solution and then, the required amount of sterile double distilled water

was added to make the required concentration of the stock solution. Fresh

solutions of organic salts and vitamins were used in the culture media.

3.3.3 Preparation of culture media: Double-distilled water was used for the

preparation of medium. The required amounts of macro- and micronutrients,

organic salts, vitamins, growth regulators and sucrose were added to the double

distilled water kept in a vessel. The final volume was made in a graduated

cylinder/beaker by adding double distilled water. The pH of the solution was

adjusted to 5.7- 5.8 using either 0.1 N HCl or 0.1N NKOH. For solidification of the

medium, agar powder (Tissue culture grade; agar-agar type) @ 0.8% w/v was

added to the luke warm solution and then, boiled for proper dissolving and

melting of agar powder. Then the medium was poured immediately in glass

vessels. After plugging, the vessels were covered with aluminium foil.

3.3.4 Autoclaving: The prepared medium, glassware, forceps, scalpels, cotton

flakes, tissue papers, glass plates, double distilled water in air tight bottles
 17

0
wrapped/covered with aluminium foil were carefully autoclaved at 121 C for 20

2
min. in a vertical autoclave device at 1.05 kg/cm (15 psi) and kept in UV hood till

inoculation.

3.3.5 Explant: Axillary buds from the middle portion of current season flowering

shoots were selected and cut during cooler parts of the day. Cut shoots were

transported in moist condition to the laboratory and axillary buds were isolated

with a sterilized secatuer.

3.3.6 Surface sterilization of explant: The collected explants were washed with

a solution containing 3-4 drops of liquid detergent Teepol. Thereafter, the

detergent was completely drained out from the explant by 3-4 washings with

vigorous shaking by hand. The mouth of the flask was covered with a piece of

cheese cloth and then, kept in running tap water for 30 min. to remove the

microbial load and dust particles adhering to the surface of the explant. For

surface sterilization of explant, two distinct strategies as indicated below were

employed.

S. Treatment No. Details of treatment

No.
1. T-1 Mercuric chloride (0.1%) for 3 min.
2. T-2 Carbendazim (0.1%)+ 8-HQC (200ppm) for 2 h
followed by mercuric chloride (0.1%) for 3 min.
The explants were then, cultured on MS medium supplemented with 3.0

mg BAP, 0.01 mg NAA and 0.5 mg GA3 per liter

3.3.7 Inoculation: Before inoculation, the laminar air-flow chamber was

fumigated carefully by putting small amount of KMnO 4 and few drops of

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formaldehyde solution (35%) in a petri plate and closing the door immediately for

atleast 12 h. The working table of laminar airflow chamber was wiped thoroughly

with ethanol before use. The material required for inoculation was steam

sterilized. The hands were cleaned with 70% ethanol. Then, the individual

explant was inoculated in culture medium. Forceps and scalpels were flame

sterilized before each inoculation.

3.3.8 Incubation of cultures: The cultures were incubated in culture room and

o
provided with a photoperiod of 16/8 h light/dark at 25±2 C temperature

maintained by automatic photoperiod and temperature control devices.

CULTURE ESTABLISHMENT (STAGE-I)

The surface sterilized axillary buds were cultured on MS medium

supplemented with BAP (3.0mg/l),NAA(0.01mg/l) and GA 3 (0.5mg/l) under

aseptic conditions and kept for incubation in the culture room.

SHOOT PROLIFERATION (STAGE-II)

Standardization of growth regulators

Growth regulators viz. BAP and NAA were used in combinations. To find out

the optimum combination for growth regulator(s) and multiple shoot formation,

the sprouted shoots were cultured on the medium with following growth regulator

combinations.

S. Treatmen Concentrations (mg/l)

No. t No.
BAP + NAA
1. T0 0.0 + 0.0
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2. T1 1.0 + 0.01
3. T2 1.0 + 0.1
4. T3 2.0 + 0.01
5. T4 2.0 + 0.1
6. T5 3.0 + 0.01

7. T6 3.0 + 0.1

SHOOT ELONGATION (STAGE III)

Standardization of GA3 concentration

Different GA3 concentrations of were tested to standardize the optimum

dose for elongation of microshoots as indicated below :

S.No. Treatment No. Details

1. T-0 Control
2. T-1 GA3 (0.5 mg/l)
3. T-2 GA3 (1.0 mg/l)

RHIZOGENESIS (STAGE-IV)
Standardization of auxins and their concentrations

For obtaining successful and quicker root initiation, auxin i.e., NAA in

different concentrations was added to the MS medium (1/2 strength ). A constant

dose of sucrose i.e., 60 g per liter was supplied to all the treatments.

The different treatment combinations are as follows:

S.No. Treatment No. Auxin Concentration (mg/l)

1. T-0 control -
2. T-1 NAA 0.5
3. T-2 NAA 1.0
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ACCLIMATIZATION (STAGE-V)

Aseptically, the microplants were transferred to autoclaved glass bottles

with polypropylene screw lid containing agropeat supplemented with half-strength

of MS liquid medium devoid of calcium, organic, growth regulators and sucrose

components. The pH of the medium was adjusted to 5.7-5.8. The cultures were

kept in culture rooms for one week. The relative humidity inside the glass bottle

was gradually reduced by unscrewing and finally, removing the lid.

TRANSFER OF PLANTLETS TO FIELD CONDITIONS

The hardened plantlets were transferred to the earthen pots (12” size)

filled with FYM: sand: garden soil (2: 1/2: 1/2) and supplemented with one full tea

spoon of bone meal and 20 g neem cake per pot and kept under field conditions.

Observations recorded

The observations recorded are as follows:

CULTURE ESTABLISHMENT

(i) Per cent survival: It was worked out by calculating the number of axillary

buds that remained green and showed growth initiation, out of the total

number of axillary buds inoculated.

(ii) Duration required for sprouting of axillary buds (days): The number of

days taken for sprouting of buds was recorded by visually observing the

cultures, and counting the days from the date of inoculation to date on

which sprouting starts.

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(iii) Contamination-free cultures (%): It was worked out by counting the

infected cultures with bacteria or fungus or both out of the total number of

buds inoculated.

(iv) General growth: The visual observations were recorded on colour of

culture i.e., yellowish-green, light green or dark green and comparative

growth of the cultures such as stunted or normal.

SHOOT PROLIFERATION

(i) Average number of multiple shoots per sprout: The number of multiple

shoots formed per sprout after 20 days of transfer to proliferation medium

was recorded.

(ii) Culture appearance: It was recorded by visually observing the colour and

growth of cultures.

(iii) Visual observations: The visual observations were recorded on colour and

comparative growth of the cultures.

SHOOT ELONGATION

(i) Shoot length (cm): It was measured after 20 days of cultures on

elongation medium with the help of a scale.

RHIZOGENESIS

(i) Per cent rooting: The number of microshoots rooted out of total number of

microshoots cultured on rooting medium was counted and rooting

percentage was worked out.

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(ii) Duration required for root initiation (days): It was calculated by noting

the day on which the first root appeared on the microshoots after they were

placed in the rooting medium.

(iii) Number of roots per shoots: The number of primary roots formed after 20

days of cultures on rooting medium was counted.

ACCLIMATIZATION

(i) Per cent survival: Percentage of plantlets survived after transfer to ambient

conditions was worked out by counting the plantlets that survived out of the

total plants transferred for hardening after 20 days.

STATISTICAL METHODS

The experiments were conducted in completely randomized design (CRD)

was followed for the layout of the experiment. Each treatment was replicated 3

times and each replication constituted of minimum ten units.

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EXPERIMENTAL FINDINGS

Experimental findings pertaining to the project entitled “Micropropagation

techniques in Chrysanthemum species” are presented in this chapter.

4.1 CULTURE ESTABLISHMENT (STAGE-I)

The axillary bud explant of chrysanthemum cv. Yellow Banglow were

subjected to various surface sterilization treatments to establish the

contamination free cultures. Data are presented in Table 2. Significant

differences were observed among the treatments tried.

Table 2 : Effect of various surface sterilization treatments on

establishment of axillary bud explant of chrysanthemum cv. Yellow

Banglow cultured on MS medium supplemented with BAP (3.0 mg/l) + NAA

(0.01 mg/l) + GA3 (0.5 mg/l)

Treatmen Treatment Survival Sproutin Contamination Duration

t No details g required
for
sprouting
(days)
T-1 Mercuric 41.22 80.37 15.78 9.62
chloride (0.1%) (39.93) (63.72) (23.42)
for 3 min.
T-2 Carbandazim 93.48 92.25 6.66 6.40
(0.2%) + 8-HQC (75.23) (73.89) (15.00)
(200 ppm) for 2
h followed by
mercuric
chloride (0.1%)
for 3 min.
 24

4.1.1 Survival percentage of axillary buds

The cultures infected with fungus and bacteria did not survive for long

period and turned black and died necrotic. On the other hand, those free from

infection, remained green and considered as surviving cultures. Highest survival

(93.48%) was recorded when the explant were treated with carbendazim (0.2%)

+ 8-HQC (200 ppm) for 2 h followed by dipping in mercuric chloride (0.1%) for 3

min whereas, the axillary buds exhibited more contamination rate when treated

with mercuric chloride (0.1% for 3 min.) (Table 2).

4.1.2 Sprouting of axillary buds

Similar trends was also seen in respect of bud sprouting (Table 2). A few

axillary buds ramained green and survived for long period without sprouting.

Such type of buds did not produce micro-shoots, rather turned black and later got

degenerated. Most of the axillary buds (Plate 2) sprouted well and produced

multiple shoots. Maximum sprouting of buds was observed when explant were

treated with carbendazim (0.1%) + 8 HQC (200 ppm) for 2h followed by mercuric

chloride (0.1%) for 3 min. A highest value of 92.25 per cent was recorded in this

treatment(T-2) as compared to 80.37% in T-1 (Table 2).

4.1.3 Contamination

The treatment with carbendazim (0.1%) + 8 HQC (200 ppm) for 2h

followed by mercuric chloride (0.1%) for 3 min. reduced the microbial infection

from 12.31 to 6.66 per cent (Table 2).

 25

4.1.4 Duration for sprouting

It is evident from table 2 that the axillary buds cultured after treating with

carbendazim (0.1%) + 8-HQC (200 ppm) for 3 h followed by mercuric chloride

(0.1%) for 3 min. (T-1) significantly reduced the duration required for bud

sprouting (6.40 days) as compared to those treated with mercuric chloride (0.1%)

for 3 min. (9.62 days).

4. 2 SHOOT PROLIFERATION (STAGE-II)

Sub-culturing of sprouted explants on MS medium supplemented with

various concentrations of different growth regulators i.e. BAP and NAA indicated

significant response in respect of shoot proliferation and growth of cultures

(Table 3).

Table 3 : Effect of different concentrations of BAP and NAA supplemented

to MS medium on shoot proliferation in chrysanthemum cv. Yellow
Banglow after 15 days of first subculture

Treatment Treatment Average Callus Culture

No. BAP+NAA number of formation appearance
micro
shoots/shoot
(mg/l)
T-0 0.0 + 0.0 1.25 - Poor
T-1 1.0 + 0.01 2.03 - Poor

T-2 1.0 + 0.1 1.82 + Poor

T-3 2.0 + 0.1 2.57 + Poor

T-4 2.0 + 0.1 2.51 - Good

T-5 3.0 + 0.1 3.27 - Good

T-6 3.0 + 0.01 4.43 - Excellent

+ = Callus formation; ++ = More callus formation; – = No callus formation

 26

4. 2.1 Average shoot proliferation

Use of BAP in combination with NAA significantly improved the

proliferation rate over the control (Table 3). Data presented in Table 3 shows that

3.0 mg/l BAP in combination with 0.01 mg/l NAA (T-6) resulted in highest shoot

proliferation (4.43 shootlets/explant) after 15 days of first sub-culturing as

compared to control and the other combinations of BAP and NAA (Plate 3).

4. 2.2 Callus induction

Visual observations of cultures indicated that the higher concentration of

NAA induced callus formation (Table 3). Dark-green cultures with normal growth

were recorded in T-6 as compared to the other treatments.

4. 2.3 General growth of shootlets

Excellent growth with dark green leaves was observed in the cultures

growing on MS medium supplemented with 3.0 mg/l BAP in combination with

0.01 mg/l NAA (T-6). Sufficient number of good quality microshoots were

obtained after 3-4 sub-cultures in this medium.

4. 3 SHOOT ELONGATION (STAGE-III)

The proliferated microshoots were thin and small. To make them strong,

longer with more and bigger size of leaves and suitable for induction of

rhizogenesis, they were transferred to elongation medium.

4.3.1 Effect of gibberellic acid

Effect of different concentrations of GA 3 i.e. 0.0, 0.5 and 1.0 mg/l was also

studied and the data of the experiment are given in Table 4. Incorporation of GA 3
 27

in various concentrations in MS medium significantly increased the length of

shoots over the control.

Table 4 : Effect of different levels of gibberellic acid on elongation of

microshoots of chrysanthemum cv. Yellow Banglow cultured on MS
medium

Treatmen GA3 Initial Shoot Shoot Increase Visual

t No. (mg/l) shoot length length in shoot observations
lengt after 20 after 40 length
h days of days of
inoculatio inoculatio
n n
G-0 Control 1.17 1.33 1.53 0.37 Poor growth

G-1 0.5 1.27 3.63 5.67 4.41 Excellent

G-2 1.0 1.27 3.68 7.80 6.53 Very long

It is evident from the Table 4 that supplementation of 0.5 mg/l GA 3 into

MS medium induced optimum increase (4.41 cm), healthy and stout growth in

microshoots after 20 days (Plate 4). The shoots obtained in the treatment with

1.0 mg/l GA3 showed maximum increase in length (6.53 cm) but the shoots were

lanky.

Application of GA3 improved the appearance of the cultures showing dark

green leaves and thick stem whereas, the control plants were very poor in growth

with yellowish-green leaves. Among the various concentrations of GA 3, 0.5 mg/l

GA3 registered excellent cultures with good thickness and optimum length of
 28

microshoots. However, very long and thin shoots with light-green leaves were

obtained in 1.0 mg/l GA3 treatment.

4. 4 ROOT INDUCTION (STAGE-IV)

4. 4.1 Auxin concentration

Although, half-strength MS medium devoid of any auxin induced 68.49%

rooting of the micro shoots in 12.67 days, however, the quality of roots observed

was inferior and unsuitable for successful hardening of the plantlets. For

accelerating the in vitro rooting process and improving the quality of roots as well

as shoots, different levels of NAA were added in the MS medium (1/2 strength)

and data collected in respect of per cent rooting, duration required for root

initiation, number of roots/shoot, length of longest root, quality of roots along with

colour, etc. are presented in Table 5. A perusal of data given in the Table 5

reveals the superiority of NAA (0.5mg/l) over the higher concentration in all

respects (Plate 5).

Table 5 : Effect of various auxins and their concentrations on in vitro

induction of rooting in chrysanthemum cv. Yellow Banglow on
MS medium* (half strength)

Treatment Auxin (mg/l) Rooting (%) Duration for No. of primary

No. root initiation roots/shoot
(days)
T-0 Control 79.00 19.37 5.73
(62.72)
T-1 NAA (0.5) 88.66 6.78 11.77
(70.36)
T-2 NAA (1.0) 76.78 8.56 7.17
(61.21)
 29

* Supplemented with 60 g/l sucrose

Values in parenthesis indicate arc sin transformed
4. 4.2. Per cent rooting

The effect of auxins on in vitro rooting was found to be significant among

the different treatments and control. Out of the different treatments tried 0.5 mg/l

NAA induced maximum rooting (88.66%), followed by control (79.00%) as

compared to 1.0 mg/l NAA (76.78%).

4.4.3 Duration required for root initiation

Perusal of data presented in Table 5 reveals that earliest root initiation

was recorded in the micro-shoots treated with 0.5 mg/l NAA (6.78 days) followed

by 1.0 mg/l NAA (8.56 days) compared to control days.

4.4.4 Number of roots

A reference to Table12 again indicated that microshoots proliferated on

MS medium fortified with 0.5 mg/l NAA (11.77/shoot) produced maximum primary

roots followed by 1.0 mg/l NAA (7,17/shoot) as compared to control (5.73/shoot).

4. 5 ACCLIMATISATION (STAGE-V)

4. 5.1 Per cent plantlet survival

A highest survival of plantlets (86.89%) was recorded after 40 days of

transplanting in glass jars for hardening (Plate 6).

 30

DISCUSSION

The present project entitled " Micropropagation techniques in chrysanthemum

species" was carried out to learn the micropropagation techniques. In the

following paragraphs the results obtained in the different experiments have

discussed in the light of available literature and scientific wisdom.

5.1 CULTURE ESTABLISHMENT (STAGE-I)

Chrysanthemum morifolium is a vegetatively propagated perennial

ornamental plant. The first step on initiating in vitro culture is to successfully

control the fungal as well as bacterial contaminations. The general surface

sterilization procedure i.e. dipping in 0.1% mercuric chloride for 3 min. failed to

control the microbial infection in the explant

Hence, two procedures of surface sterilization were tried in the present

study. The treatment of nodal segments with carbendazim (0.2%) + 8-HQC (200

ppm) for 2 h followed by mercuric chloride (0.1%) for 3 min. (T-2) was found to

be the best in respect of explant survival (93.48%), bud sprouting (92.25%) and

reduced contamination (6.66%) over the other treatments. Carbendazim is a

common systemic fungicide, which effectively controls different latent fungal

contaminations under field conditions. Similarly, 8-Hydroxy finnoline citrate has

bactericidal action, generally used for extending the vase-life of cut flowers.

Earlier, Prasad (1995) had also used these two chemicals in combination to

control the microbial contamination in axillary bud explant.

 31

5.2 SHOOT PROLIFERATION (STAGE-II)

The shoot proliferation medium comprised of different concentrations of

BAP and in different combinations. The best proliferation with well differentiated

microshoots was achieved when the cultures were transferred to MS medium

fortified with 3.0 mg/l BAP and NAA 0.01 mg/l. The treatments recorded 4.0 to

st
4.43 microshoots per explant after 15 to 20 days of 1 sub-culture. The shoot

proliferation in tissue culture is largely due to the action of BAP. Optimum dose of

BAP enhances axillary branching and multiple shoot formation. An increase in

NAA concentration beyond an optimal concentration induced more callus

formation and retarded the axillary bud sprouting. In general, the methods, which

avoid callus formation and stimulate axillary branching, is considered to be ideal.

The present results lends support from the previous work done in the

micropropagation of chrysanthemum (Lu et al.,1990; Chakrabarty et al.,1999;

Annadana et al.,2000; Kumari and Varghese,2003) who had regenerated the

different explants of chrysanthemum using various concentrations and

combinations of BAP and NAA in vitro. It is well established that optimal ratio of

cytokinin : auxin cause the dormant meristematic zone existing in the axillary

node regions to show shoot organeses.

5. 3 SHOOT ELONGATION (STAGE-III)

The microshoots multiplying on a proliferation medium were observed to

be very thin, small, with few very small leaves and hence, were not suitable for in

vitro rooting. For root induction, all the physiological manipulation may be
 32

performed on separate, study and medium long microshoots with well developed

leaves (Anderson, 1980).

The appropriate concentration of gibberellic acid, was found to be 0.5 mg/l

to which aided information of ideal size of elongate shoots. The shoots were

found to be of medium length having well-developed dark-green leaves.

Although, the microshoots proliferated on MS medium supplemented with 1.0

mg/l GA3 were longer, but were found to be unsuitable for root induction due to

weak and lanky stem with poor strength. The cluster of microshoots, which had

short shoots, developed into easily separable shoots, which survived individually.

Gibberellins are known for inducing stem elongation in a number of crops.

The elongation in stems is not due to increased formation of nodes and

internodes but results from rapid elongation of internodal cells which is due to

both cell division followed by cell elongation (Krishnamoorthy, 1981). The

application of gibberellic acid into shoot elongation medium resulted in rapid

growth of shootlets probably due to the increased activity of the endogenous

auxin in the presence of GA3 (Brian and Hemming, 1955; Nitch, 1955 and

Michiewiez, 1962). The shoot elongation stage is also considered as the

preparatory stage for root induction.

5. 4 RHIZOGENESIS (STAGE-IV)

For successful root induction onto elongated shoots, strength of basal

medium salts, sucrose level and supplementation of auxins are considered to be

important. It is well known that rooting in certain plant species, may occur
 33

ideally when the overall salt strength of the medium is reduced. In some cases,

the salt strength reduction may eliminate the need for auxin(s) for rooting. The

results of the present findings on Induction of rooting with out auxin (in control

microshoots) are in conformity with the previous work done by Fujii and

Shimizu(1990) who reported that good rooting in the in vtro raised shoots of

chrysanthemum could be achieved on hormone free MS medium.

Although, a high rooting percentage was recorded when the shoots were

placed on half-strength of MS with 60 g/l sucrose devoid of auxin, but the quality

of roots was in general very poor. The roots were invariably very long, thin,

unbranched and turning black in colour, which not only affect the ex vitro survival

but also the overall performance of the in vitro raised plantlets.

The rooting of microshoots could be significantly improved after addition of

auxin into the rooting medium. The highest rooting (88.66%) and more number of

roots (11.77) in shortest duration (6.78 days) were obtained after placing the

shoots on half-strength MS medium supplemented with 0.5 mg/l NAA and 60g/l

sucrose. Like any morphogenetic process, rooting is an energy intensive process

and thus a higher level of carbon source is required. Thus, high sugar level

alongwwith low auxin were very effective. These results are in conformity with

those of Chakrabarty et al.(1999).

5. 5 HARDENING (STAGE-V)

The preparation for hardening-off and planting out of the microplants starts

from shoot elongation and rooting stages of plant propagation by tissue culture

(Bhojwani and Razdan, 1983). During short elongation stage, the addition of
 34

gibberellic acid in the medium resulted in rapid shoot growth in terms of length

and thickness, and formation of well developed leaves, which made them sturdy

and ready for root induction followed by acclimatization. Furthermore, reduction

in salt levels in the MS medium particularly macronutrients including potassium

during in vitro rhizogenesis, utilizes and prevents the accumulation of salt

complex by guard cells in the stomata. Therefore, the plantlets on transfer to the

soil show high water contents in leaves due to minimum water loss through

stomata (Prasad, 1995).

Furthermore, the in vitro derived plantlets have an another characteristic

feature i.e. the epicuticular waxy layer is poorly developed. This leads to

uncontrolled foliar water loss when the plants are taken out from the culture

vessels. However, when the plants are kept at high humidity conditions, they

synthesize more epicuticular wax, which enhances the survival success during

acclimatization (Short et al., 1981).

For obtaining the high success during the plantlet acclimatization, the two

strategies were employed in the present investigation. The microplants

transferred to glass jars with polypropylene lids each filled with sterile agropeat

moistened with half-strength MS medium (devoid of growth regulators, calcium,

organics and sucrose) recorded 86.89 per cent survival. The plantlets

acclimatized in glass jars received less open space but appropriate relative

humidity. Gradual loosening the lids of glass jars at regular interval enabled the

moisture to escape. The ex vitro plantlet mortality may be due to the fact that the

microshoots developed in vitro have several anatomical abnormalities like poor

 35

cuticle development, less pallisade cells, more air space, poor vascular bundles

etc. Besides, the in vitro plants have, poorly developed epicuticular waxes, more

stomata per unit area and raised guard cells with wide open opening, which

result in more transpiration losses and less survival of plantlets (Prasad, 1995).
 36

SUMMARY

The present investigation "Micropropagation tequeniques in

Chrysathemum species)" was undertaken at the Central Tissue Culture

Laboratory, Lal Bahadur Shantry Building, Indian Agricultural Research Institute,

New Delhi. The objectives of the project are as follows;

The objectives of present investigation are as follows :

1. To standardize the surface sterilization process for nodal segment.

2. To study the culture medium for rapid shoot proliferation

3. To study the effect GA3 or micro-shoot elongation

4. To study the rooting process

5. To study the hardening process of micro plant

The salient results of the present investigation are summarized hereunder:

6.1 CULTURE ESTABLISHMENT

The treatment of nodal explant with carbendazim (0.2%) + 8-HQC (200

ppm) for 2 h followed by mercuric chloride (0.1%) for 3 min. resulted in highest

survival (93.48%) and sprouting (92.25%) of explant with minimal the microbial

infection (6.66%).

6.2 SHOOT PROLIFERATION

Murashige and Skoog medium fortified with 3.0 mg/l BAP and 0.01 mg/l

NAA GA3 mg/l was found to be superior than other treatment combinations with

regard to proliferation of number of microshoots per culture (4.43/explant).

 37

6. 3 SHOOT ELONGATION

Murashige and Skoog medium supplemented with 0.5 mg/l GA 3 was

found to be appropriate dose to obtain reasonably long (4.41 cm) microshoots.

6.4 RHIZOGENESIS

Half-strength MS (1/2 MS) medium containing 60 g/l sucrose and 0.5 mg/l

NAA observed to be the ideal medium compared to other treatments. It recorded

the highest rooting (88.66%) more number of roots (11.77) within 6.78 days of

culture.

6.5 ACCLIMATIZATION

For acclimatization, rooted plantlets transferred to glass jars with

polypropylene lid each filled with sterile agro-peat supplemented with half-

strength MS (1/2 strength) resulted in 86.89 per cent survival of plantlets.

 38

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