COLLEGE OF AGRICULTURE,

GWALIOR

ELP BIOTECHNOLOGY

Project report on: Planning to establish

commercial micropropagation to produce
1 lakh plants of Guava using tissue culture

Submitted by:
Shritama Bhunia
Roll no : 151A74
Batch: A (A-25)
 Objective of the project:
I. Setting of a laboratory for commercial micropropagation
for producing 1 lakh plants.
II. Production of Guava plants using suitable and feasible
tissue culture techniques.
III. Marketing of micropropagated guava plantlets in order to
obtain profit.

Introduction to Guava:
 Guava (Psidium guajava) is one of the important
commercial fruits in India. It is the fourth most important
fruit after mango, banana and citrus.
 The fruit is a good source of vitamin C, pectin, calcium
and phosphorus.
 The fruit is used for the preparation of processed
products like jams, jellies and nectar. Guava jelly puree
is very popular for its attractive purplish-red colour,
pleasant taste and aroma. Fruits can be preserved by
canning as halves or quarters, with or without seed core
(shells). Good quality salad can be prepared from the
shell of ripe fruits.
 Leaves of guava are used for curing diarrhoea and also
for dyeing and tanning.
 Health benefits of guava:
i) May Help Lower Blood Sugar Levels
ii) May Boost Heart Health
iii) May Benefit Your Digestive System
iv) May Aid Weight Loss
v) May Have an Anticancer Effect
vi) May Help Boost Your Immunity
 Major guava producing states:
Major guava producing states include Uttar Pradesh, Bihar,
West Bengal, Maharashtra, Chhattisgarh, Tamil Nadu,
Karnataka, Madhya Pradesh, Gujarat and Andhra Pradesh.

Need of tissue culture in Guava:

 Propagation through cuttings is extremely slow and
season specific.
 Late in bearing flowers and fruits when grown through
vegetative means.
 The cross pollination will results in loss of unique
characteristics which is not the case in micro-propagated
clonal plants.
 The occurrence of wide variability among the seedling
populations can be reduced and a uniform population of
clones can be ensured.

Tissue cultured Varieties :

 Allahabad safeda
 Lucknow 49
 Lalit
 Shweta
 L-49
 Suvarna pink guava
 Thai pink guava
 MICROPROPAGATION OF GUAVA

Materials and methods:

Explant source:
Nodal segments of healthy lateral branches of guava (Psidium
guajava L.) were collected from mature field-grown superior
trees. Nodal segments were chosen for the in vitro propagation
because shoot tips were too small and very difficult to be
sterilized or survived.

Sterilization:
Surface sterilization was done by immersing explants in 70%
ethanol (v/v) for one minute followed by 0.05% HgCl2
(mercuric chloride) solution containing few drops of Tween-20,
for two minutes followed by 3-4 washing using double distilled
water.
Then the explants were disinfected, using 2% (w/v) sodium
hypochlorite (NaOCl) for 10 minutes followed by 5-6 washing
with sterilized distilled water.
Finally, explants were left under aseptic conditions in laminar
air flow cabinet in an antioxidant solution (150 mg/l citric acid
and 100 mg/l ascorbic acid) until culture to overcome phenol
exudation.
Culture media and conditions:
Explants were cultured on Murashige and Skoog (MS) basal
medium.
Antioxidant agents viz. 150 mg/l citric acid and 100 mg/l
ascorbic acid and absorbent as 100 mg/l polyvinylpyrrolidone
(PVP) and 2 g/l activated charcoal (AC), were added to
establishment medium for reduce phenolic compounds.
Various concentrations and combinations of plant growth
regulators were added to the culture medium according to the
growth stage.
Cultures were incubated in a temperature of 25±2°C under dark
(for initiation and rooting stages) for one week.

Culture establishment:
Explants of guava cultured on MS medium supplemented with
6-benzyl adenine (BA), at different concentrations of (2.22, 4.40
and 8.90 µM) and indole-3-butyric acid (IBA) 0.98 µM in
addition MS medium without PGRs was used as a control. After
four weeks, sprouting percentage (%) and average shoot length
(cm) were recorded.

Shoot multiplication:
In this experiment, the in vitro established shoots were
cultured on MS medium supplemented with different
concentrations BAP and kinetin.
Rooting induction:
Elongated shoots derived from nodal explants, were cultured
on solid half strength MS medium containing IBA @ 10mg/l and
NAA.

Acclimatization:
The rooted shoots were removed from the medium, washed in
running tap water to remove all traces of phytagel and dipped
carefully for 5 min in 1% Benlate (systemic fungicide) and
transferred to 6 cm diameter plastic pots filled with a mixture of
sand, garden soil and peat moss at equal volume. Initially,
plantlets were covered with a polyethylene bags and
transferred to the greenhouse at (26 ± 2 C° 70 – 80% relative
humidity).
After eight weeks, polyethylene bags were eliminated gradually
from pots for compatible hardening. The plantlets were
irrigated with 1/10 MS medium every five days. Finally, they
were transferred outside the greenhouse in field conditions.
TISSUE CULTURE PLANT FOR COMMERCIAL
MICROPROPAGATION OF 1 LAKH GUAVA
PLANTS
Requirement of tissue culture project:
 Land
 Explant
 Source of Technology
 Laboratory
 Culture media
 Green house
 Electricity
 Water
 Raw materials
 Skilled manpower
 Equipments
Autoclave, pH meter, Laminar air flow, Distillation unit, Analytical
balance, Magnetic stirrers, Refrigerator, Hot air oven, Computer system, Air
conditioners with stabilizers, Microscopes, Bottle washing unit, Hatches,
Tubelights, Dissecting kits.

Civil Structures of Laboratory:

 Culture room
 Staff room
 Incubation room
 Washing room
 General instrumentation room
 Chemical storage room
 Media preparation room
 Sterilization room
 Toilets
Man power : For managing laboratory and production,
following staff should be present-
 General manager
 Technical staff
 Cleaner
 Guards
 Sales manager

Location:
The tissue culture laboratory should be preferably located in a
moderated climate condition having uninterrupted supply of
water and power. The tissue culture operations have to be
carried out under controlled conditions of temperature.
Extreme climatic condition adds to the cost of maintenance.

PROJECT COST:
FIXED ASSET REQUIREMENT
S. No. Head Cost ( Rs. In lakhs)
1. Land 5.00
2. Land development 5.60
3. Building 35.20
4. Utilities 16.00
5. Equipment 69.40
6. Green and shade house 30.00
7. Miscellaneous fixed asset 2.75

Total 163.95
1. Land:
Approximate 5 acres land should be adequate for setting up a TC
unit with the above capacity. Cost of land is assumed at Rs. 5.00
Lakhs
2. Building and civil works:
The building of about 8800 sq.ft includes class 1000 clean
rooms and areas with comfort AC for laboratory, growth rooms
and office space. The following facilities would be required in the
building.
a) Storage room for chemicals
b) Washing and Media preparation room
c) Sterilization room
d) Inoculation room
e) Culture room
The total cost is estimated at Rs. 35.20 lakhs @ Rs. 400/sft.
3. Green house:
A green house of 7500 sq.ft. and a shade house of 80,000 sq.ft.
have been assumed at a cost of Rs. 22.00 lakhs and 8.00 lakhs
(total Rs. 30 lakhs) respectively. The greenhouse should be
provided with heating equipment, fans and cooling systems.
4. Equipment: Major equipment and instruments required for the
plant are as follows- Autoclave, Laminar air flow cabinet
,Equipment for sterilization, Electronic weighing balance, Water
distillation apparatus, Air handling units, Refrigerator, Air
conditioners, Stereo microscope, Digital pH meter, Shelves/racks,
Green house material.
WORKING CAPITAL REQUIREMENT:

(I) Raw material

The basic inputs for the production of micropropagated

plantlets include meristems of elite and disease free plants,
ready to use culture medium, sucrose and agar.
(II) Manpower
The unit with the proposed capacity may need 40-50
people at various positions including managerial,
supervisory, skilled and unskilled
(III) Recurring expenses (per month)
S.no. Head (Rs.lakhs)
1. Raw Material 2.50
2. Manpower 2.41
3. Utilities (power, water) 0.45
4. Contingencies (marketing,
office expense, repair etc) 0.40
Total 5.76

Recurring expenses (per annum) Rs. 69.12 lakhs

CAPITAL INVESTMENT:
Head (Rs. lakhs)
Fixed assets 163.95
Technology knowhow 15.00
Working Capital (3 months) 17.28
Total 196.23

MEANS OF FINANCE:
Particulars (Rs. in Lakhs)
1. Debt 117.73
2. Equity 78.50
Total 196.23
Debt: Equity - 3:2
Interest - 16%
FINANCIAL ANALYSIS :

I. Cost of Production Rs.(lakhs)

Recurring cost (per annum) 69.12
Depreciation (@10%) 17.87
Interest (@16% Pa) 18.83
Total Rs.105.82 lakhs

II. Turnover Rs. (lakh)

Average selling price - Rs. 30 per plant
Total no. of plants - 1 lakh
Total turnover Rs. 300 lakhs

III. Profitability Rs. (lakh)

Net profit - Rs. 194.18 lakhs
% Profit on sales - 49%
IRR - 26%
Return on investment - 53%