Standard Operating Procedures

For Recognized Tissue Culture

Production Facility

_________________________________________________
(Name & Address of Recognized Tissue Culture Production Facility)

COPY
NO:___

National Certification System for Tissue Culture Raised Plants (NCS-TCP)

Department of Biotechnology, Government of India
New Delhi

October 2008

1
 SOPs for Tissue Culture Production Facility
Section-0 Table of Contents Page 1 of 1
October 2008

Table of Contents

Section Title Page

Number
Section-o Contents 2-2
Section-oo Control of Document 3-5
Section 1 Introduction (Scope/Purpose/Definitions/References) 6-8
Section 2 Organization structure and role & responsibilities 9-10
Section-3 Selection of Mother Plant/Establishment of mother stock 11-12
(nursery)
Section-4 Preparation of ex-plants 13-13
Section-5 Cleaning/Washing/Drying of glassware 14-14
Section-6 Preparation of media 15-18
Section-7 Inoculation 19-19
Section-8 Incubation of cultures 20-20
Section-9 Virus indexing and Genetic fidelity testing of tissue culture 21-24
raised plants
Section-10 Packing/labeling/dispatch of ex-agar plants 25-25
Section-11 Entry/Exit to tissue culture laboratory 26-26
Section-12 Maintenance of cleanliness & sterile conditions/servicing of 27-27
HEPA filters
Section-13 Monitoring of Microbial Contamination, Fumigation/disinfection 28-28
of facility
Section-14 Management of primary hardening facility (green house) 29-30
Section-15 Management of secondary hardening facility (shadenet house) 31-31
Section-16 Dispatch and monitoring of performance of tissue culture 32-33
raised plants
Section-17 Calibration of Measuring and Monitoring Equipments 34-35
Section-18 Document Management and Record Control 36-38
Section-19 Training of workers/staff 39-40
Section-20 Communication/Auditing/Review 41-45

2
 SOPs for Tissue Culture Production Facility
Section-00 Control of Document Page 1 of 2
October 2008

1. Document Issue and Revision:

This document issue and revison is controlled by the Document Approval Authority (Department of
Biotechnology) As and when a section of this document is revised, the revised section is issued in its
entirety together with a revision number, identifying the new issue status and the issue date of each
section. The revised sections are automatically issued to each of this document copy holders listed in
Section 2 of ‘Control of Document’:
Section Title Issue Date of Revision Date of
Number Issue / Number Rev/
approval Approval

October
10, 2008
Section-o Contents 1
Section-oo Control of Document 1
Section 1 Introduction 1
(Scope/Purpose/Definitions/References)
Section 2 Organization structure and role & 1
responsibilities
Section-3 Selection of Mother Plant/Establishment of 1
mother stock (nursery)
Section-4 Preparation of ex-plants 1
Section-5 Cleaning/Washing/Drying of glassware 1
Section-6 Preparation of media 1

Section-7 Inoculation 1
Section-8 Incubation of cultures 1
Section-9 Virus indexing and Genetic fidelity testing of 1
tissue culture raised plants
Section-10 Packing/labeling/dispatch of ex-agar plants 1
Section-11 Entry/Exit to tissue culture laboratory 1
Section-12 Maintenance of cleanliness and sterile 1
conditions/servicing of HEPA filters
Section-13 Monitoring of Microbial Contamination, 1
Fumigation/disinfection of facility
Section-14 Management of primary hardening facility 1
(green house)
Section-15 Management of secondary hardening facility 1
(shadenet house)
Section-16 Dispatch and monitoring of performance of 1
tissue culture raised plants
Section-17 Calibration of Measuring and Monitoring 1
Equipments
Section-18 Document Management & Record Control 1

Section-19 Training of workers/staff 1

Section-20 Communication/Auditing/Review 1 3
 SOPs for Tissue Culture Production Facility
Section-00 Control of Document Page 2 of 2
October 2008

Upon the receipt of a revised section, this document copy holder should remove the obsolete
section and replace it with revised section together with revision number. This issue status table
identifies the latest issue of all section of this manual which has been provided to all document copy
holders listed in section 2 of ‘Control of document’.

This document copy holders are responsible for ensuring all sections of this manual are at the
correct issue status prior to use. The document approval authority as evidenced by signature/date/stamp
of approving authority and document issuing authority as evidence by signature/date/stamp of issuing
authority will be placed on title page and approval of revision section indicated against the revision
number in the issue table above.

2. Document distribution
This document distribution and subsequent revisions distribution are controlled and issued by
the Document Issuing Authority (Accreditation Unit of the DBT established at Biotechnology
Consortium India Ltd).This document is issued to all those personnel responsible for various activities
and processes described in this document are undertaken.

This document is issued on a controlled copy basis. The only copies of this document that are permitted
are those held by the copy holders identified in the table below. This ensures that when changes to this
manual are made, all copy holders receive those changes. This manual is currently issued to the
following copy holders:

Document Copy Holder Contact Address

Copy No
Original Accreditation Unit (Biotech Anuvrat Bhawan, 5th Floor, 210, Deendayal Upadhyaya Marg,
Consortium India Ltd) New Delhi-02
1 Certification Agency Department of Biotechnology. Block-2, 7th Floor, CGO
Complex, New Delhi-03
2 Referral Centre under NCS-TCP Advanced Centre for Plant Virology, Division of Plant
Pathology, IARI, New Delhi-12
3 Referral Centre under NCS-TCP Centre for DNA Fingerprinting and Diagnostics, ECIL Road,
Nacharam, Hyderabad-500076
4 K. F. Bioplants Pvt. Ltd. S. No. 178, Kirtane Baug, Mundhawa Road, Magarpatta,
Hardaspur, Pune. Maharshtra-411036
5 Reliance Life Sciences Pvt. Ltd. R-282,TTC Area of MIDC, Thane Belapur Road, Rabale, Navi
Mumbai Maharashtra-400701
6 Shri Ramco Biotech 3rd Floor, Sabari Complex,24 Residency Road, Bangalore
Karnataka-560025
7 H.U Gugle Agro Biotech Co. Karmala Road, Jamkhed, Ahmednagar Maharashtra-413201
8 Pudumjee Plant Laboratories Ltd. Thergoan, Chinchwad, Pune Maharshtra-411033
9 Growmore Bioteh Ltd. 41-B, SIPCOT-II, Hosur Tamil Nadu-635109
10 Devleela Biotech Anand Vihar, Opposite Energy Park, VIP Road,Raipur
Chhatisgarh

4
11 Labland Biotech Pvt Limited. 8th K.M., K.R.S. Main Road, Mysore Karnatka-570016
12 Nirmeeti Biotech Gut No. 167/170 at post Kurali, talk Khed, Pune Maharshtra-
410501
13 Technico Agri Sciences Ltd. SCO 162-163, First Floor Sector 9-C, Madhya Marg
Chandigarh-160009
14 Jain Irrigation Systems Limited. Agripark, Jain Hills, PO Box 72, Shirsoli Road,Jalgaon
Maharashtra-
15 L.J.International Ltd. Plot No. 66, KINFRA Export Promotion Industrial Parks Ltd,
Kusumagiri PO., Kochi Kerala-682030
16 Micropropagation Technology Park Darbari Seth Block, India Habitat Centre, Lodhi Road, Delhi-
(TERI) 110003
17 Ajeet Seeds Ltd. Gut No. 233, At. Post Chitegaon, Tq. Paithan, Aurangabad
Maharashtra-431105
18 Rise N Shine Biotech Pvt. Ltd Dattaprabha, Ganeshwadi, Theur, Pune-411000, Mharashtra
19 Aryave Biotech Private Limited Plot No.149, Sajjanpur Peeli, Shyampur Post, Najibabad Road,
NH-74, Haridwar-249408, Uttrakhand
20 A. G. Bioteck Laboratories (India) Bachupally (Post), Quitbullabur (M), Ranga Reddy, Hyderabad
Limited. (AP)-500072
21 Anantha Biotechnologies 1-792A, Opp. D.P.O, Ram Nagar Extension, Ananthapur (AP)-
515001
22 Shaili Biotech (P) Ltd. 401, Sarthik Square, Sarkhej Gandhinagar Highway, Near
GNFC Tower, Bodekdev, Ahemadabad Gujarat
23 Aditya Biotech Lab & Research Nandanvan Road, Chandinidih, Raipur, Chhatisgarh
Pvt. Ltd.
24 SPIC Agro Biotech Centre Chitraichavadi, Puluvapatty Post, Siruvani Road, Coimbatore-
641101, Tamil Nadu
25 Cadila Pharmaceutical Ltd. (Agro Tissue Culture Lab, 755 Prakurtifarm, Tal: Dascori, Vill:
Division) Hirapur, Ahemdabad Gujarat-382435

5
 SOPs for Tissue Culture Production Facility
Section-1 Introduction Page 1 of 3
Ovtober 2008

1.1. Scope:

This document provides guidance and describes the standard operating procedures (SOPs) for tissue
culture production facility involving various steps of production of tissue culture plants.

1.2. Purpose

The purpose of this document is to facilitate adoption of standard operating procedures by all the
recognized tissue culture production facilities under National Certification System for Tissue Culture
Raised Plants established by the Department of Biotechnology, Ministry of Science & Technology for
undertaking certification of tissue culture production facility in accordance with the guidelines
established by the Department of Biotechnology (certification agency) in exercise of the powers
conferred under section 8 of the Seeds Act, 1966.

1.3. Definitions & Terms:

Accredited Test A test laboratory accredited by the Department of Biotechnology

Laboratory for virus/quality (genetic fidelity) testing of tissue culture raised
plants and certification.
Acclimatization It is a physiological adaptation of plants to climate or environment
such as, light, humidity, temperature, etc.
Recognized tissue A tissue culture production facility recognized by the Department
culture production of Biotechnology for quality production of tissue culture plants.
facility
Clone A progeny of plant derived through vegetative propagation having
identical genetic make-up with that of parent plant.
Controlled Documents formally identified. These documents are registered,
document: maintained and their change, as well as, their implementation is
regulated.
Controlled Record: A record that requires to be kept and maintained under safeguard
for future reference
Culture medium It is a liquid or gelatinous substance containing nutrients for the
growth of explants.
Corrective action Action to eliminate the cause of a detected non-conformity.

6
 SOPs for Tissue Culture Production Facility
Section-1 Introduction Page 2 of 3
October 2008

Data Quantified information in documents.

Document: Procedures, work instructions, references, specifications or
regulatory material for the administration of the system.
Explant An explant is any portion of the plant that will be used to initiate
the culture.

Hardened plant In-vitro derived plants which have developed good root and stem
system to grow in the field conditions and ready for field
plantation.
Inoculation Transferring of sterilsed explants on to the nutrient media to in a
culture tube/bottle.
Incubation Maintenance of inoculated explants in bottle/tube in an
environment of controlled conditions of temperature, light, humidity
and nutrients.
Internal Audit Independent activity to verify, through an exam and evaluation of
objective evidence, if the processes and elements applicable to
the quality system have been developed, documented and
implemented.
Internal document Document generated outside the limits of the administrative
system for example: a regulatory document that is referred to a
procedure or work instruction.
Micropropagation It is the practice of rapidly multiplying a large number of progeny
plants from a desired plant using modern plant tissue culture
methods.
Mother plant A plant which acts as a source of material for multiplication by
micropropagation.
NCS-TCP National Certification System for Tissue Culture Raised Plants
(NCS-TCP) established by the Department of Biotechnology,
Ministry of Science & Technology.
Non-Conformity Any situation that differs from standard procedures, guidelines or
regulations
Objective evidence Data supporting the existence or verify something.
Plantlet A baby plant produced in vitro on an auxenic culture medium from
a meristematic plant tissue.

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 SOPs for Tissue Culture Production Facility
Section-1 Introduction Page 3 of 3
October 2008

Plant Tissue Culture Plant tissue culture is a technique of culturing plant cell, tissue or
organ in artificial, controlled and aseptic conditions. It mainly
covers micropropogation, organogenesis and somatic
embryogenesis.
Pest Any species, strain or biotype of plant, or pathogenic agent,
injurious to plants or plant products.
Procedure Document that describes, “Who does the job”, “when”, “where”,
and “why”.
Protocol or work A written instruction to carry out a specific task or activity or job.
instruction
Record: Document (electronic or print), product or sample statement,
which will confirm that a procedure (or part of the procedure) has
been carried out.
Somaclonal It is the term used to describe the variation seen in plants that
variation have been produced by plant tissue culture. Chromosomal
rearrangements are a major source of this variation.
Stock culture Tissue culture derived from mother plant

Standard Operating Standard operating procedures (SOPs) are sets of written

Procedures (SOPs) instructions that document the routine or repetitive activity followed
by an organization. The development and use of SOPs are an
integral part of a successful quality system as it provides
individuals with the information to perform a job properly.
Virus Indexing: Testing of the plants for known viruses and ensuing their
elimination before micropropagation.

1.4. References:

Guidelines for recognization of tissue culture production facilities (2006),

Department of Biotechnology, New Delhi.

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 SOPs for Tissue Culture Production Facility
Section-2 Organization structure & Responsibilities Page 1 of 2
October 2008

1.1. Organization structure:

The Organisational structure and job titles may vary with different tissue culture production
facility depending on level of production. However the following organisation structure of
tissue culture production facility is considered to be a minimum for recognition of tissue
culture production facility.

Organizational Chart of Tissue Culture Production Facility

Facility
Manager/ In-charge

Manager Manager Manager Manager

(R&D/QM) (Production) (GH/SH) (Marketing)

Technician Supervisor Supervisor Supervisor Supervisor Field

(R&D) (QC) (Media Lab) (TCP) (GH/SH) Officer

1.2. Responsibilities

1.2.1. Facility Manager/ In-charge:

The Facility Manager/ In-charge of tissue culture production facility will be responsible for
overall management of tissue culture production facility and the responsibilities include:

• recruitment of technical/administrative personnel

• approval of purchase of equipments and quality chemicals
• adoption of standard operating procedures (SOPs) for tissue culture production
facilities
• planning resources for development of tissue culture production facility
• review of implementation of quality system and procedures
• contracting tissue culture production

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 SOPs for Tissue Culture Production Facility
Section-2 Organization structure and Responsibilities Page 2 of 2
October 2008

1.2.2. Manager (R&D/QM):

The Manager (R&D/QM) is responsible for:

• research and development/standardization/validation of tissue culture protocols for the
initiation of new plant species
• selection of mother plants and maintenance of pest-free mother stock (nursery)/ stock
cultures
• maintenance of appropriate records related to selection of mother plants/stock
cultures
• quality management and internal auditing of activities related to tissue culture
production
• implementing SOPs
• organizing virus-indexing and quality (genetic fidelity) testing of tissue culture plants
• calibration of measuring/monitoring equipments

1.2.3. Production Manager

The Production Manager is responsible for:

• planning, execution and supervision of tissue culture production activities
• maintenance of tissue culture multiplication records
• overall maintenance of cleanliness and sterile conditions of tissue culture laboratory
facilities

1.2.4. Green House Manager

The Green house manager is responsible for:

• management of tissue culture hardening facilities (greenhouse) as well as nursery
(secondary hardening) raised under shade net facility
• maintenance of appropriate records related to green house/shade net facilities
• evaluation of field performance of tissue culture plants in the field

1.2.5. Marketing Manager:

The Marketing manager is responsible for:

• planning, execution and supervision of marketing/shipment of tissue culture plants
• providing package of practices for growing tissue culture plants in farmer’s field
• demonstration of tissue culture technology to the farmers for improvement of
agriculture production
• receiving customer’s feed back.

10
 SOPs for Tissue Culture Production Facility
Section-3 Selection of Mother Plants/Establishment of Page 1-2 of 2
mother stock (nursery)
October 2008

3.1. Selection of Mother Plants:

3.1.1. The selection criteria employed should be limited to a few important phenotypic traits
estimated to have a relatively high heritability (the ability of the parents to transmit
their characteristics to the progeny) such as stem straightness, branching and
flowering habits

3.1.2. The information collected about the mother plant will be recorded in format prescribed
in Annexure-3A.

3.1.3. The selected mother plants will be healthy and free from pest and diseases.

3.1.4. The selected mother plants will be appropriately labelled giving Ref No/Date, Name of
plant species (common/scientific name)/variety, plant parts, location, name of the
collector

3.1.5. The selected mother plants will be appropriately packed in card board cartons and
transported to tissue culture production facilities with in the same date of collection.

3.1.6. If the selected mother plants are of foreign origin, the same will be imported subject to
existing phytosanitary regulations covered under PQ Order, 2003 and amendments
issued there under.

3.2. Establishment of mother nursery:

3.2.1. The selected mother plants (elite clones with proven yield potential and improved
agronomic characteristics/horticulture traits) will be thoroughly screened before
planting in a mother nursery in isolated area and /or under protected condition (such
as glasshouse) at the tissue culture production facility, where appropriate.

3.2.2. Each plant will be appropriately labeled giving accession number, plant
species/variety, date of planting and the particulars will be recorded in a mother stock
register maintained by the facility.

3.2.3. Each plant will be tested especially virus-free before planting in mother nursery and
maintained in virus-free condtion until used in tissue culture production.

11
 Annexure-3A

Information Sheet on Selection of Mother Plant

1. Ref. No/Date
2. Plant species (common/scientific name)
3. Variety
4. Name of the owner/ contact person
(Telephone/fax/mobile/mailing address)
5. Geographic location (latitude/longitude /
altitude) from which mother plant collected
6. Climate/soil type/topography of the area:
7. Location address (Village/Mandal/Taluk/
District/State)
8. Survey No/Field/Plot No
9. Total planted area
10. Source of material for raising the planting
11. Month/year of planting & Age of the crop
12. Type of cultivation Open field/protected area (green house/glass
house growing)
12. Description of plant parts collected
13. Description of phenotypic characteristics of
the plant:
a. Plant Height a.
b. Girth of plant b.
c. Growth habit c.
d. Flowering type d..
e. Fruiting type e.
f. Yield f.
g.. phenotypic markers g.
h. Any other Characteristics
h.
(______________________)
(Specify)

14. Month/year of collection

15. Any information on pest/disease status
observed
16. Signature/Name/Designation of collecting
person

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 SOPs for Tissue Culture Production Facility
Section-4 Preparation of Ex-plants Page 1 of 1
October 2008

4.1. The ex-plants for initiation of tissue culture will be taken only from mother stock (nursery)
tested and maintained in virus-free condition.

4.2. The explants taken for tissue culture will be healthy and disease-free and the cutting
tools used for excising plants will be appropriately disinfected with 70% alcohol and also
the hands will be cleaned and washed thouroughly with suitable detergent.

4.3. The working surface will be appropriately disinfected with 70% alcohol or sterile filter
paper pads used for excising plants

4.4. The explants will be sterilized by keeping in appropriate sterilent solution such as 0.1% -
0.2% mercuric chloride solution for 5-20 minutes and wash it 3-4 times in sterile distilled
water before inoculating to culture media depending on the types of tissue

4.5. In the case of meristem tissue culture plants, the ex-plants will be cut 1.0 to 1.5 mm
close to the shoot-tip with the help of sterile scalpel and planted on culture media with
the help of sterile forceps (tweezers).

4.6. The culture bottles will be appropriately labeled giving information about accession
number, plant species/variety, date of transfer & name of technician.

4.7. The culture bottles will be incubated in growth rooms under controlled
temperature/humidity/conditions for proliferation of shoot buds.

4.8. The proliferated shoot buds in clumps are individually transferred to another nutrient
medium for shoot elongation and root development.

4.9. The stock cultures thus obtained will be maintained in a refrigerator and the particulars
of stock cultures maintained will be recorded in a stock culture register.

4.10. The stock cultures will be utilized for further multiplication for obtaining desired number
of plantlets.

13
 SOPs for Tissue Culture Production Facility
Section-5 Cleaning/washing/drying of glassware Page 1 of 1
October 2008

5.1. All the glassware used for tissue culture work will be thoroughly cleaned/washed by
trained workers under the supervision of media laboratory supervisior.

5.2. The cleaning/washing will be done either manually or through automatic washing
machine.

5.3. The water used for cleaning and washing will be of potable quality and mixed with a
detergent such as teepal.

5.4. All the contaminated culture bottles will be autoclaved before washing with detergent
and the agar collected into a suitable container and disposed by composting in a pit
covered with soil.

5.5. After cleaning/washing the glassware will be rinsed with clean water to remove the
detergent.

5.6. After rinsing the glassware such as bottles or jars will be kept in inverted position on
drying stand.

5.7. The glassware such as Petri dishes or pipettes will be sterilised in hot air oven by
wrapping in a clean kraft paper or keeping in suitable air-tight container at 160 0C for a
minimum period of 1 hour.

5.8. The scalpels and forceps used will be wrapping in a clean kraft paper and sterilised in a
hot air oven at 160 0C for a minimum period of 1 hour.

14
.

SOPs for Tissue Culture Production Facility

Section-6 Preparation of culture media & Autoclaving Page 1 of 4
October 2008

6.1. Preparation of culture media:

6.1.1. All the preparation and autoclaving of media will be done under the direct supervision
of media laboratory supervisor.

6.1.2. The laboratory chemicals of either analytical or laboratory grade, as the case may be
will be used in preparing the media. The agar used for preparation of media will be of
TC grade.

6.1.3. The water used for preparation of media will be double glass distilled, RO or
demineralised.

6.1.4. The stock solutions of macronutrients, micronutrients, vitamins & amino acids will be
prepared in distilled water (Annexure-6A) and labeled appropriately indicating name,
strength of solution and date of preparation and stored in refrigerator at 4 C. However
the stock solutions of auxins such as IAA (Indole Acetic Acid), IBA (Indole Butyric
Acid), NAA (Napthol Acetic Acid) and cytokinins (6-benzylaminopurine (BAP) are
prepared separately by dissolving the required quantity of hormones in little quantity of
1 N NaOH and finally made up to required strength (1 mM or 10 mM) by adding
distilled water.

6.1.5. In the first instance the required quantity of sucrose (30 g/l) and agar (8 g/l) weighed
and added to three fourth of required quantity of water and heated on a hot plate with
magnetic stirrer until the both ingredients are dissolved followed by addition of
required quantities of stock solution of macronutrients, micronutrients, vitamines,
amino acids and harmones and the rest of distilled water is added to make the final
volume of media.

6.1.6. The pH of media will be adjusted between 5.6-5.8 using 01 N HCl solution with the
help of pH meter.

6.1.7. The medium will then be distributed uniformly into culture bottles or jars with the help
of automatic media dispensor or manually through a fixed volume dispensing pipette
as the case may be and will be capped tightly.

6.1.8. The culture bottles will be appropriately labelled indicating name of media, batch
number and date of preparation and the particulars of the same will be entered in a
media register (Annexure-6B).

15
 SOPs for Tissue Culture Production Facility
Section-6 Preparation of culture media/autoclaving/ Page 2-4 of 4
storage
October 2008

6.2. Autoclaving:

6.2.1. The media will be autoclaved at 1kg/cm2 (i.e., 15 lbs/sq. inch) for 15-20 min at 121 0C
in an autoclave The culture bottles after autoclaving will immediately be removed to
media storage room in clean area to avoid contamination.

6.3. Filter sterilisation:

6.3.1. If any heat labile chemicals are used in preparation of meida, the stock solutions of
heat labile chemicals will be filter sterilised through a syntex filter with pore size of
0.22 µm and the filtersterilized solution will be dispensed under aseptic conditions to
autoclaved media after cooling to 36 0C. The syntex filters will be sterilized by
autoclaving before use.

6.4. Storage of Culture Media.

6.4.1. After autoclaving the culture media will be stored for 3 days in storage room in clean
area (with sterility level of class 100,000) and observed for microbial contamination
before issue for inoculation. If any microbial contamination detected, the contaminated
bottles will be removed for autoclaving immediately and the particulars of
contamination will be recorded for each batch of media prepared in the media register.

16
 Annexure-6A
Stock solutions for Nutrient Medium

Stock solution Constituents Quantity (mg/litre) Volume of stock

solution
Stock Solution-I MgSO4. 7H2O 7400 1000 ml
(20X) KH2PO4 3400
KNO3 38000
NH4NO3 33000
CaCl2. 6H2O 8800
Stock Solution-II H3BO3 1240 1000 ml
(200X) MnSO4.4H2O 4460
ZnSO4. 7H2O 1720
Na2MoO4.2H2O 50
CuSO4. 5H2O 5
CoCl2. 6H2O 5
Stock Solution-III* FeSO4. 7H2O 5560 1000 ml
(200X) Na2.EDTA. 2H2O 7460
Stock Solution-IV Inositol 20000 1000 ml
(200X) Thiamine HCl 100
Pyridoxine HCl 100
Nicotinic Acid 100
Glycine 400
*For preparation of stock solution-III, dissolve separately in 450 ml of distilled water by heating and constant
stirring. Mix the two solutions, adjust pH to 5.5 and add distilled water to make up the final volume to one litre.

17
 Annexure-6A

Media Register
Batch Date of Quantity No of Prepared Particulars of No of Issued
No. Preparation (in litres) bottles by contamination bottles to/Date
prepared if any issued

18
 SOPs for Tissue Culture Production Facility
Section-7 Inoculation of tissue cultures Page 1 of 1
October 2008

7.1. All the inoculation of tissue cultures will be carried out by trained workers (operators)
in inoculation room under a laminar air flow cabinets fitted with HEPA filters and U.V.
germicidal lamps.

7.2. The operators will wear the clean lab coats, hair cap and the face mask covering nose
and mouth, while carrying out inoculation of cultures to prevent microbial
contamination.

7.3. The inoculation room will be maintained at a minimum sterility level of class 100,000
with positive pressure to prevent microbial contamination.

7.4. The tissue culture multiplication will be carried out as per the production plan
established by the facility. Only one clone/genotype will be handled for sub-culturing
at any one by an operator. The particulars of stock cultures issued for multiplication
will be entered in a computer using pre-installed software for production monitoring.

7.5. The operator each time will use sterilised scalpel to excise the tissue after placing it on
sterilised pads and transfer the excised tissue to culture media with the help of sterile
scalpel. The culture bottle will be capped tightly and wrapped with parafilm at the
margin of the lid. If test tubes are used, they will be covered with a clean sterile cotton
plug and wrapped with a parafilm to facilitate free exchange of air

7.6. The operator will use glass-beed steriliser for sterilising scalpel and forceps and the
working bench will be disinfected at the end of each transfer.

7.7. The technician at the end of completion of transfer of each clone/genotype will label
each culture bottle or jar indicating the stock culture number, plant species/variety,
multiplication cycle number and the week of transfer and name of the technician.

7.8. The technician will complete the job sheet indicating the number of bottles transferred
at the end of complete transfer of each clone/genotype.

7.9. At the end of complete transfer of each clone/genotype, the production supervisor will
enter the particulars of culture transfers in the computer using pre-installed software
for production monitoring.

19
 SOPs for Tissue Culture Production Facility
Section-8 Incubation of tissue cultures Page 1 of 1
October 2008

8.1. The culture bottles or jars are kept in trays and incubated on storage racks each frame
fitted with four daylight flourescent tubes to provide light intensity of 1200 lux/m2. An
automatic time control switch is installed to maintain desired photoperiods in growth
room, which will very with different plant species. Also dark room facilities will be
required for certain species. The storage racks will be of either fixed type or movable
type and the latter one are space shaving.

8.3. The temperature of growth room will be measured with the help of temperature sensors
connected to thermostatic control and the temperature maintained in growth rooms will
be continuously monitored with the help of data loggers. The temperature of incubation
will very between temperate and tropical plant species, which will be set with the help of
thermostatic control.

8.4. The growth rooms will be maintained at a minimum sterility level of class 100,000 and
with positive pressure to prevent microbial contamination.

8.5. The production supervisor will make observation on growth of cultures and
contaminations at weekly intervals and the particulars will be recorded in a growth room
register indicating accession number of clone/genotype, plant species/variety,
multiplication cycle number, medium type, date of inoculation, tray number and date of
observation and observation made. The contaminated cultures, if any, will be
immediately removed for autoclaving after making entry in the register.

20
 SOPs for Tissue Culture Production Facility
Section-9 Virus/Quality (genetic fidelity) testing of Page 1 of 4
tissue culture raised plants
October 2008

9.1. The recognized tissue culture production facility will submit an application for
virus/quality (genetic fidelity) testing and certification of tissue culture raised plants
(Annexure-9A) to the Director/HOD of accredited test laboratory sufficiently in advance
and get it registered after payment of prescribed fees notified by the Department of
Biotechnology from time to time.

9.2. The recognized tissue culture production facility will submit samples for testing as per
the guidelines provided by the accredited test laboratory or permit drawl of samples
through the authorised representative of accredited test laboratory, as the case may
be.

9.3. The sample collected will be blotted dry to remove excess moisture before packing.
The sample will be placed in between paper towels, packed in self sealing/zip-lock
polythene bags of appropriate size. The sample will be affixed with a label (Annexure-
3C) and kept in a ventilated card board box and /or thermocool box for forwardal to
accredited test laboratory.

9.4. The packing box will be marked on top of the box with the address of accredited test
laboratory with appropriate instructions such as “Handle with care/Tissue Culture
Plants/Rush Delivery” and either couriered or delivered in person to the concerned
accredited test laboratory within 24 hrs period.

9.5. A separate record of samples referred for virus testing/quality (genetic fidelity) testing
and the report of results of testing received from accredited testing laboratory will be
maintained.

9.6. Where a facility undertakes in-house testing for virus and or/ quality (genetic fidelity),
it should be carried out as per the protocols approved/validated by the referral
laboratory recognised by the Department of Biotechnology under national certification
system for tissue culture raised plants.

9.7. In case of clones/genotypes or stock cultures, the virus testing will involve hundred
percent testing of each individual clones/genotypes or the stock cultures just prior to
initiation for tissue culture multiplication.
9.8. However in case of ex-agar plants/tissue culture hardened plants appropriate samples
will be drawn for each batch of production and separately for each plant
species/variety for virus/quality (genetic fidelity) testing just prior to dispatch for
shipment/marketing.

21
 SOPs for Tissue Culture Production Facility
Section-9 Virus indexing and quality (genetic fidelity) Page 2-4 of 4
testing of tissue cultures of plants
October 2008

9.9. If the test results for virus testing proved positive the affected clones/genotype or stock
culture will be rejected for tissue culture multiplication and the same will be destroyed
as per the direction of Accredited Test Laboratory.

9.10. If the test results of batch testing proved positive for virus and or/ the results of quality
(genetic fidelity) testing exceeded the tolerance limits prescribed by the crop specific
standards established by the Department of Biotechnology, the entire batch will be
rejected for shipment/marketing and the same will be destroyed as per the direction of
Accredited Test Laboratory.

22
 Annexure-9A

Application for Virus/Quality (genetic fidelity) Testing & Certification of Tissue

Culture Raised Plants

1. Name/Location Address of the Recognized tissue culture

production facility:
2. Certificate No./date of issue/validity:
3. Name of authorised person and his contact details
(Telephone/ Fax/Mobile/E-Mail):
4. Details of tissue culture plants required to be sampled:
Plant species Variety Accession/ Batch No of Tests Category of tissue culture
Batch No size packages required material*
for
[ ] Clonal material
[ ] Invitro-culture
[ ] Ex-agar washed plants
[ ] Primary hardening plants
[ ] Secondary hardening
plants
*Tick out in appropriate box
5. Purpose of testing/certification*: [ ] Import Quarantine requirements
[ ] Mother stock for initiation
[ ] Domestic sale & distribution
[ ] Phytosanitary certification
* Tick out in appropriate box
6. Particulars of payment of testing fees:
Amount in Rs:
Demand Draft/Banker’s Cheque No./Date of Issue:
Bank Name/Branch:
7. Date by which sampling:
8. Date by which certification is requested:
9. Any additional information:
Declaration
I/we hereby declare that the information furnished above is complete and correct to the best of my/our
knowledge and belief. I/we will meet the TA/DA of technical personnel deputed for sampling in addition
to the testing fees as indicated above and provide necessary facilities for sampling.
Date:_________________
Place:________________ __________________________________
(Signature/Name/Stamp of Applicant/Date)
For Office (Testing Facility) Use Application Reg. No.________/Date________
Check list Status Scrutinized by Action by TCPF Applicant Response
Application complete Yes No
Payment of Fees Yes No
Final Action Taken:
_________________________________
(Signature/Name of Director or HOD of
Accredited Test laboratory/Date)

23
 Annexure-9B

Sampling Label

Sample No*:
(12 digit code)
Application Reg. No/Date:
Plant Species/Variety:

Lot/Accession No:
Lot size:
Tissue Culture Production Facility:
Date of Sampling:
Sample size/No of samples drawn:
Samples drawn by:
______________________________
(Signature/Name/Designation Stamp/Date
of Authorised person of/by Accredited Test
laboratory)

In the presence of: ______________________________

(Signature/Name of authorized person from
Tissue Culture Production Facility)
* Sample Number will be assigned by Accredited test laboratory and maintained
throughout certification

24
 SOPs for Tissue Culture Production Facility
Section-10 Packing/labeling/dispatch of ex-agar plants Page 1 of 1
October 2008

10.1. The plantlets after attaining good growth will be removed from culture bottles or jars by
trained workers and gently washed in a stream of potable water to remove the agar
sticking on to the roots.

10.2. The plantlets after washing will be blotted dry to remove excess moisture and graded
by placing on the glass surface using the standard chart of the plant species/variety
as back ground and the plantlets which does not confirm to the required standard will
be discarded.

10.3. The grading and virus testing of ex-agar plantlets will be carried out as per the
certification standards for tissue culture plants established by the Department of
Biotechnolgy for domestic purpose and or/as per the requirements specified by the
importer in respect of export consignments, where applicable.

10.4. The ex-agar plants after grading will be wrapped in tissue paper and placed in
thermocool boxes kept in a cardboard carton for overseas shipment. The package will
be sealed and labelled giving information about the Name of tissue culture production
facility; plant species/variety; no of plantlets; date of packing and the address of
consignee. The packages will carry appropriate instruction such as “HANDLE WITH
CARE-TISSUE CULTURE PLANTS”.

10.5. The sealed packages will be stored in cold storage room at appropriate temperature
until shipment.

25
 SOPs for Tissue Culture Production Facility
Section-11 Entry/Exit Control to tissue culture laboratory Page 1 of 1
October 2008

11.1. Entry Control:

11.1.1. The entry into tissue culture laboratory will be restricted/regulated through a double
door entry.

11.1.2. All the persons, who are authorised entry, will remove their chapals/shoes including
shocks and leave them at shoe stand and wear the sanitary sandals and wash their
hands and feet with soap before the entry.

11.1.3. All the persons will change cloths at the dress changing area and wear clean
sanitised laboratory overcoat, head cap and face mask covering the nose and
mouth. They will preferably pass through an air shower for 30 seconds or
alternatively through an air curtain. They will dip their hands in basin filled with
disinfectant and feet in foot bath filled with disinfectant before entering into the
sterile areas of tissue culture production facility.

11.1.4. The persons entering will not be permitted to carry out any articles such as writing
aids/mobiles/wallets etc in side the facility with out passing through UV sterilizer. No
smoking/chewing or eatables will be permitted inside the facility. Suitable lockers will
be provided to the staff/workers for storing their belongings at the entry.

11.1.5. The facility will display signs such as “NO ENTRY WITH OUT PERMISSION” at the
entry and also display step by step entry protocols for the guidance and compliance
by all persons entering the sterile areas of tissue culture production facility.

11.2. Exit control:

11.2.1. All the persons will leave the laboratory overcoat, head cap and face mask at the
dress changing area in disposing bin and leave the sanitary sandals and wear their
own cloths. They will collect their belongingings, if any before leaving the place.

11.2.2. The used overcoats, head cap and face mask will be removed for washing and
autoclaving.

26
 SOPs for Tissue Culture Production Facility
Section-12 Maintenance of cleanliness and sterile conditions/ Page 1 of 1
servicing and changing of HEPA filters
October 2008

12.1. Maintenance of cleanliness and sterile conditions of the facility:

12.1.1. The sterile areas of facility will be cleaned with vaccum cleaner and the floor area will
be mopped with disinfectant daily.

12.1.1. The waste collected in the disposal bins inside the clean room areas of tissue culture
laboratory will be removed daily.

12.1.2. The glass pans of the windows/doors will be wiped with clean chamoised cloth or wet
sponge periodically to remove the dust, if any collected.

12.1.3. The inoculation room, media storage room and growth room will be maintained at a
minimum sterility level of class 100,000 and provided with positive pressure.

12.1.4. The tissue culture facility will be either provided with centrally air-conditioned plant
providing ducted cool-air filtered through HEPA filters (0.22 µm) @ two air changes
per min or with individual air-handling units provided with HEPA filters (0.22 µm)
feeding specific areas of the facility.

12.1.5. The inlets of receiving air will be located at the top side or at shelf level and the out
lets of exhausting air will be located close to the floor level on opposite side to
facilitate uniform air circulation and minimize contamination.

12.2. Servicing and Changing of HEPA filters:

12.2.1. The HEPA filters fitted on air-conditioning ducts or air handling units will be serviced
at least six monthly intervals by a qualified service engineer and if found
damaged/clogged the filters will be replaced by new ones.

12.2.2. The HEPA filters fitted in laminar air flow cabinets will be serviced as and when
necessary and if found damaged/clogged will be replaced by new ones.

12.2.3. The clogging of HEPA filter will be revealed by drop in pressure of manometer fitted
on to laminar air flow cabinet and the damage of filters will be revealed by physical
examination of the filter.

12.2.4. The facility will ensure that the servicing of air-conditioning equipments and changing
of HEPA filters are covered under annual maintenance contract and strict schedule
is followed for in-house maintenance and a record will be maintained for this
purpose.

27
 SOPs for Tissue Culture Production Facility
Section-13 Monitoring of Microbial Contamination/ Page 1 of 1
Disinfection of facility
October 2008

13.1. Monitoring microbial contamination

13.1.1. The microbial contamination will be monitored in the sterile areas of the facility by
exposing agar plates at periodic intervals and the same will be incubated for 72
hours at 25+2 0C and examined for microbial colonies. A record of periodical
monitoring of microbial contamination will be maintained indicating date/time of
monitoring, area monitored, no of plates exposed, date/time of observation and no
of microbial colonies encountered and colony types (fungal or bacterial) and the
action taken.

13.1.2. Besides the above, counting of dust particles through a particle counter at regular
intervals either manually or through an automatic particle counter to ensure required
sterility levels are maintained with in the tissue culture laboratory facility and a
record of particle counts carried out at periodic intervals will be maintained.

13.2. Disinfection of facility

13.2.1. The facility will be disinfected with 4% formalin with an exposure period of at least
24-48 hours usually at monthly intervals or as and when necessary depending on
microbial counts/ level of contamination.

13.2.2. The disinfected area will be thoroughly aerated and checked before allowing any
entry of workers/staff inside the facility.

13.2.2. At the end of each day’s work, the U.V. germicidal lamps fitted inside the laminar air
cabinets will be switched-on overnight to disinfect the laminar air flow cabinet and
also the dress changing area.

28
 SOPs for Tissue Culture Production Facility
Section-14 Management of primary hardening facility (green Page 1-2 of 2
house)
October 2008

14.1. The green house facility will have controlled micro-climatic conditions such as relative
humidity, temperature, light intensity and air circulation. It will have controlled misting
arrangements.

14.2. The green house facility will have double door entry and vector-proofing of all external
openings (covering with a screen of 40-60 meshes per linear inch) to the facility in
order to prevent entry of insect vectors such as aphids, leaf hoppers, whiteflies and
thrips.

14.3. The ex-agar washed and graded plantlets will be transplanted in pro-trays (micro-pots)
in soil less peat mix growing medium.

14.4. Each batch of the plantlets will be appropriately labelled indicating plant species/
variety, Accession Number of clone/genotype, batch of production, no of plant lets,
date of transplanting and the particulars will be recorded in green house register
(Annexure-14A).

14.5. The pro-trays will be held under specially designed poly tunnels on benches above the
ground. If protrays held on the ground, the soil or sand floors will be covered with
impermeable sheet to avoid soil or sand coming in direct contact with protrays. The
poly tunnels provided with adjustable openings to provide optimum microclimatic
conditions and to facilitate examination of plantlets.

14.6. The plantlets will be monitored regularly for their growth and presence of any
infestation/infection will be recorded in green house register.

14.7. The dead plantlets will be promptly removed to avoid attack by saprophytic fungi and
the plantlet mortalities will be recorded in green house register.

14.8. Vector monitoring will be carried out at periodic intervals by placing yellow sticky cards
close to the micropots at the rate of one sticky card for every 10 m2 space inside the
facility and the same will be recorded indicating date/time, pest species traped,
average number/trap, action taken and name signature of supervisor.

14.9. Any kind of treatment given to the plant such as application of fertilizers or micro-
nutrient sprays or application of pesticides will be recorded in green house register
indicating date/time of application, fertiliser or other chemicals applied, dosage rate
and if pesticides applied (including botanicals and microbial pesticides), pest against
which applied and name/sign of applicator.

29
 Annexure-14A

Green House Register

S. Plant Batch Date of No. of Record of operation Record of observation Total No of Name/
species/ No. trans- Plantlets Date Operation carried out Date Findings (Mortalities/pest plantlets Sign of
No. variety planting & diseases) died/discorded GHS

30
 SOPs for Tissue Culture Production Facility
Section-15 Management of secondary hardening facility Page 1 of 1
(shadenet house)
October 2008

15.1. The shade net facility will have double door entry with all the sides covered with
insect-proof screen of 40-60 meshes for linear inch with top covered with polythene
sheet against rain protection during monsoon period.
15.2. If soil is used it will be treated by vapour heat at 80 0C for I hour and only fully
decomposed organic manure will be used and mixed with soil before treatment for
raising tissue cultured plants.
15.3. The plants will be raised individually in poly-bags or other suitable containers on
benches or raised beds on the ground. The soil floors are covered by an impermeable
plastic film to prevent plants coming in direct contact with soil and weed growth.
15.4. Each batch of the plants will be appropriately labeled indicating plant species/variety,
accession Number of clone/genotype, batch of production, date of transplanting and
the particulars will be recorded in shadenet house register, which is maintained in
similar way as that of green house register.
15.5. The shadenet house will have drip irrigation and or fertigation arrangements for
watering individual plants in poly bags or other containers.
15.6. The plants will be monitored regularly for the presence of any pest/diseases and if any
plants are suspected of virus symptoms, the affected plants will be immediately
segregated and isolated and tested for the presence of virus before the same will be
destroyed and the results will be recorded in the shadenet house register.
15.7. The dead plant will be promptly removed to avoid attack by saprophytic fungi and the
seedling mortalities will be recorded in the shadenet house register.
15.8. Vector monitoring will be carried out at periodic intervals by placing yellow sticky cards
close to the pots at the rate of one sticky card for every 10 m2 space inside the facility
and the same will be recorded indicating date/time, pest species traped, average
number/trap, action taken and name signature of supervisor.
15.9. Any kind of treatment given to the plant such as application of fertilizers or micro-
nutrient sprays or application of pesticides will be recorded in shadenet house register
indicating date/time of application, fertiliser or other chemicals applied, dosage rate
and if any pesticides applied (including botanicals and microbial pesticides), pest
against which it is applied and name/sign of applicator.

31
 SOPs for Tissue Culture Production Facility
Section-16 Dispatch/Monitoring Performance of Tissue Page 1-2 of 2
Culture Plants
October 2008

16.1. The hardened plants will be checked physically for their health (freedom for diseases)
and size (plantable) as per specified in certification standards laid down by the
Department of Biotechnology.

16.2. The hardened plants in poly bags will be either dispatched in ventilated plastic crates/
cardboard cartons in suitable trucks to the concerned growers or farmers for direct
planting or else the plants will be removed from poly bags, washed free from soil,
wrapped in tissue paper, and packed in ventilated cardboard cartons.

16.3. Each package will be affixed with a certification label (Annexure-16A) as prescribed
under the certification standard for tissue culture plants established by the Department
of Biotechnology. Duly signed/stamped certification labels will be provided by the
Accredited Test Laboratory (ATL) to the Tissue Culture Production Facility at the time
of issue of certificate of quality of tissue culture raised plants for affixing on the
packages. Colour of the label shall be diagonally yellow No. 356 (IS 5-1978) and
opaline green (IS No. 275). ATL will issue 10 labels with Certificate of Quality and
issuance of other label might be suitably charged.

16.4. The Tissue Culture Facility will maintain up to date record of the deployment of the
labels in terms of the size of packages, number of plants per package, the name and
contact details of the consignee etc.

16.5. A small handout or printed leaf let will be placed in each package giving relevant
information about after-care of tissue culture plants such as planting, irrigation
scheduling, fertigation, agronomy and plant protection etc., for the benefit of
farmers/growers.

16.6. The Tissue Culture Facility will maintain database of customers and will contact them
from time to time to check on various aspects (such as conditions of plants,
packaging, initial mortality in the field etc.)

16.7. The Tissue Culture Facility will undertake visit to the plantation site of their customer
facing problems with the dispatched material and extend technical support
troubleshooting.

32
Annexure: 16 A

Label

Certified Tissue Culture Raised Quality Plants

__________________ Accreditation No. of the
NCS-TCP logo (Emblem) ______________________ Accredited Test
(Name and Address of Accredited Test Laboratory:
Laboratory)
Certificate No. Bar-coding

Date of issue

Botanical Name (Common name):

Variety:

Lot/Batch No:

Batch size:

Number of Plants/ Packages:

Stages of Tissue Culture Plant Ex-agar plantlets

Primary hardened plants

Secondary hardened plants

Name of Tissue Culture Production Facility:

Certification No. and validity of Certificate of

Recognition

Name of the contact person

Address with phone number:

:
Name/Sign/Stamp of Certifying Authority (ATL) _______________
with date

33
 SOPs for Tissue Culture Production Facility
Section-17 Calibration of Measuring & Monitoring Page 1-2 of 2
Equipments
October 2008

17.1. The Manager (R&D/QM) will be responsible checking and verification of measuring
and monitoring equipment and their calibration.

17.2. The laboratory analyst (QM) will identify and document measuring/monitoring
equipment to provide the necessary accuracy of the measurement. It is required to
identify and record each critical equipment using a serial number, lab number/code
and or model number and register in a logbook.

17.3. The laboratory analyst (QM) will internally calibrate equipments in accordance with
written instructions and tolerances provided along with supply of equipment. He will
maintain records of calibrated equipment with the information of frequency, conditions,
tolerances, method and current status in the format prescribed in Annexure-14A. The
calibrated equipment will be labeled with a sticker indicating the status of calibration.

17.4. The equipment that requires external calibration will be calibrated by an authorised
agency for which annual maintenance/service contract will be entered by the quality
manager and ask for a certificate of proof of calibration and file it for future record.

17.5. Equipment that does not require calibration will be labeled with a “Not calibrated”
sticker. The list of equipments will be verified and updated at least annually

17.6. The Manager (R&D/QM) will ensure that the equipments are maintained, stored and
handled to preserve their accuracy and protect from damage and deterioration and
proper maintenance of record of the calibrated equipment.

34
 Annexure-17A

Calibration Record Format

Name of the Equipment/ Make/ Model:

Date of purchase:

Supplied by:

Location of equipment:

Equipment used for:

Identification number:

Tolerance (if applicable)

conditions:
Frequency of calibration:

Method of calibration:

Remarks:

Details of Calibration:

date time condition adjust initial

1
2
3
4
5
6
7
8
9
10

35
 SOPs for Tissue Culture Production Facility
Section-18 Document Management & Record Control Page 1 of 3
October 2008

18. 1. Document management:

18.1.1. The Recognized tissue culture production facility will adopt the standard formats for
documentation of information related to tissue culture production as prescribed in
the Standard Operating Procedures for Tissue Culture Production Facility
established herewith by the Department of Biotechnology or else will establish their
own SOPs in line with these established by the Department of Biotechnology. If the
SOPs established by the recognized tissue culture production facility deviate from
the one established by the Department of Biotechnolgy, they will be required to be
technically justified a copy of the same will be made available to the Accreditation
Unit of Department of Biotechnology for record.

18.1.2. The tissue culture production facility will not make any changes to the SOPs
established by the Department of Biotechnology as this being issued as controlled
document

18.1.3. Any request for changes in the document (modification/deletion/addition/revision) will

be made through a document change application (Annexure-15A). The Accreditation
unit of Department of Biotechnolgy will evaluate the changes and recommend for its
revision or reject changes or suggest modifications. The revised document will be
approved by the Department of Biotechnolgy.

18.1.5. The Accreditation unit of Department of Biotechnolgy will issue controlled copies of
revised document to all copy holders.

18.1.6. The tissue culture production facility will ensure discarding of ‘OBSOLETE’ document
and replacement with revised document

18.1.7. The facility will make available photocopies of controlled document of SOPs to all
those technical personnel with production responsibilities for necessary compliance.

18.2. Record Control:

18.2.1. The facility will maintain appropriate records as per the formats prescribed herewith
for selection of mother plants from field, mother stock (nursery); stock cultures;
production of tissue culture plants including hardening.

18.2.2. The facility will maintain records related to calibration of equipments and annual
maintenance contract of air-conditioned and other equipments.

36
 SOPs for Tissue Culture Production Facility
Section-18 Document Management & Record Control Page 2-3 of 3
October 2008

18.2.3. The facility will maintain a record of tissue culture plants referred for virus/quality
testing by accredited test laboratory and the test reports and certification of plants in
a single folder. The records related to virus testing/quality testing will be maintained
for at least one year.

18.2.5. The records related to clonal selection and maintenance of mother stock (nursery)
will be maintained for a period of five years or until such time the stock is maintained
to facilitate tracing back of lineage of tissue culture plants to the mother stock.

37
 Annexure-15A

Document Change Application

CHANGE REQUESTED BY APPLICATION DATE:

DOCUMENT TITLE

CHANGE REQUESTED

REASON

RECOMMENDATION (SELECT ONE)

REJECT (reason)

ACCEPT WITH CHANGES (explain)

ACCEPT

IF ACCEPTED SUGGESTED DATE VALID SINCE

IF TRAINING PROPOSED DATE

NAME/SIGNATURE OF MANAGER (R&D/QM); DATE

AUTHORIZATION BY THE HEAD OF TISSUE CULTURE DATE

PRODUCTION FACILITY:

38
 SOPs for Tissue Culture Production Facility
Section-19 Training of workers/staff Page 1 of 1
October 2008

19.1. Manager (R&D/QM) in consultation with section in-charges (managers) will evaluate
the technical personnel to determine training needs.

19.2. Manager (R&D/QM) in consultation with section in-charges (managers) will identify
internal/external training needs after taking into account the resources available and
prepare training programme and request the approval of head of tissue culture
production facility for organising the training.

19.3. Manager (R&D/QM) and/or section in-charges (managers) will ensure that each
technical staff/workers will receive operational training for implementation of SOPs.
Besides this, the laboratory technicians will be given hands-on training in tissue
culture propagation and further training will be given whenever new tissue culture
protocols are developed. The green house workers will be given specific training in
management of tissue culture hardening facility (greenhouse) and management of
secondary hardening facility (shade net facility). The laboratory analysts attached to
the quality laboratory will be given training in virus testing/quality (genetic fidelity)
testing and quality management

19.4. Manager (R&D/QM) will designate the training experts who will be responsible for
preparing and conducting training. If it is external training, he will identify the training
institute for conducting the training and recommend to the Managing Director or Head
of tissue culture production facility for approval.

19.5. Manager (R&D/QM) will distribute training certificates for those who are qualified in the
evaluation tests and maintain record of training in respect of each individual staff
giving information about the name/designation of staff, type of training
(internal/external), area of training, period of training/dates, name/address of training
Institute & remarks, if any

39
 SOPs for Tissue Culture Production Facility
Section-20 Technical Auditing (Internal/External)/Review Page 1 of 6
October 2008

20.1. Communication:

20.1.1. The Accreditation Unit of Department of Biotechnology will timely communicate to

Recognized Tissue Culture Production Units regarding information on:

- crop specific tissue culture standards

- changes to guidelines for recognition of tissue culture production facilities
- list of Accredited Testing Laboratories
- any other relevant information

20.1.2. The tissue culture laboratories will ensure timely report any changes to contact
addresses for updating mailing list

20.2. Auditing:

20.2.1. The Accreditation Unit of Department of Biotechnolgy (DBT) will establish an auditing
panel of technical experts for technical auditing of recognized tissue culture
production facilities to ensure that the tissue culture processing activities are
performed as per the standard operating procedures established by the concerned
tissue culture production facility, which are approved by the Certification Agency.
20.2.2. The Accreditation Unit of DBT will establish a schedule of technical audit and
nominate at least two experts from the auditing panel for carrying out the technical
audit of activities of recognized tissue culture production facility and intimate the
concerned experts at least 10 days in advance, to facilitate making travel
arrangements. The concerned recognized tissue culture production facility will be
intimated at least one week in advance of the proposed visit.

20.2.3. The nominated experts will have adequate training/experience in auditing quality
system and procedures and activities of tissue culture production. The scheduled
audits will be carried out once in every year.

40
 SOPs for Tissue Culture Production Facility
Section-20 Communication/Auditing/Review Page 2 of 6
October 2008

20.2.4. The scheduled technical audit will involve verification of records and the procedures
actually followed both for selection of mother plants, maintenance of pest-free
mother stock (nursery), aseptic production of tissue culture plantlets in vitro,
virus/quality (genetic fidelity) testing, management of tissue culture hardening
facilities both primary (greenhouse) and secondary (shade house) and testing skill
competency of the staff involved in tissue culture production and management of
tissue culture hardening facilities and compliance with regulatory requirements.

20.2.5. At the end of each technical audit, an audit report in prescribed format (Annxure-
20A) will be prepared and forwarded to the accreditation unit by the nominated
experts. The audit report will indicate non-conformities based on objective evidence,
corrective/preventive action to be taken and time schedules by which the measures
will be implemented to improve the functioning.

20.2.6. The Accreditation Unit of DBT will go through the technical audit report submitted by
the nominated experts (auditors) and communicate corrective/preventive actions to
be to the concerned tissue culture production facility for implementation at the
earliest.

20.2.7. The Recognized tissue culture production facility will be re-inspected by an expert to
ensure corrective/preventive actions are implemented and a follow-up report of
surveillance auditing (Annexure-20B) will be submitted to Accreditation Unit.

20.3. Review:

20.3.1. The Apex Committee established by the Department of Biotechnology (Certification

Agency) will periodically review the effectiveness of all aspects of the Recognition of
tissue culture production facility in consultation with all the representatives of
recognized tissue culture production facility and implement changes to the system if
required.

20.3.2. Such review meetings will be held biannually to discuss the issues and implement
action plans for improving efficiency and quality production.

41
 Annexture-20A

Audit (Scheduled) Report.

1. Name & Address of recognized tissue

culture production facility

2. 2.1. Head of Tissue culture production

facility (Name & Designation/
contact number/fax/e-mail ):

2.2. Sectional Incharges ( managers)

2.3. Supervisory staff

3. Auditing related to the period of
4. Date (s) of Auditing: From: To:

5. List of Records Audited/Documents

verified:

6. Audited by (Name & Designation):

7. Details of Auditing reported:

7.1 General Comments:

42
7.2. Specific non-conformities observed:
S. Type of non-conformity observed Frequency Corrective Action/ preventive
No. measures to be taken
.

8. (a) Signature/Name Designation of (b) Signature/Name/Designation of Auditors with

Auditee (Head of Tissue Culture date
Production Facility with date

______________________ 1._____________________2.____________________

43
 Annexure-20B
Audit (Surveillance) Report
1 Name & Address of Accredited tissue
culture production facility

2. Date of auditing (surveillance):

3. Audited by (Name/Designation):

4. Details of audit (surveillance) carried out:

S. Type of non-conformity/ observation Corrective Action / prevent Remarks of
No. made by previous audit measures undertaken Auditor

_______________________ ___________________________
Head of Tissue Culture Unit (Auditor)

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Document developed on behalf of Department of Biotechnology (DBT) by Accreditation
Unit (NCS-TCP) at Biotech Consortium India Limited (BCIL),
New Delhi

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