DRAFT OF MINIMUM SEED CERTIFICATION STANDARDS FOR TISSUE CULTURE BASED SEED POTATO

I. Application and Amplification of General Seed Standards for tissue culture based potato seed (a) The general seed standards are applicable to tissue culture raised mini-tubers (G-0) multiplied through aeroponics or nethouse conditions and further multiplied in the field for four generations (G1-G4) as shown below: Stages of 'Tissue Culture Based Potato Production System' Mother Plant Multiplication through Tissue Culture (8-12) Cuttings/Subculture) G0 Aeroponics G0 Net House G1 Open Field G1 Open Field G2 Open Field G2 Open Field G3 Open Field G3 Open Field G4 Open Field G4 Open Field I. Eligibility requirements for tissue culture based mini-tubers (G-0) The mini-tubers to be eligible for certification shall be from a source meeting the following standards aeroponics or nethouse facilities. (i) Laboratory and aeroponics or nethouse facilities used for production of plantlets/micro-tubers or mini-tubers shall be maintained free of potato pests or vectors of potato pathogens. Failure to keep such pests under control may cause rejection of all lots maintained in the facility. All potting or growth media shall be sterile. Water to be used in a laboratory or aeroponics or nethouse operation should be free from impurities. (ii) Hygienic conditions like maintenance of air purity, sub culturing under laminar flow only, use of pure tissue grade chemicals for media preparation, proper autoclaving of cultured media, proper disposal of contamination etc. shall be strictly observed during micro propagation, potting, planting, irrigating, movement and use of equipment and other laboratory practices to guard against the spread of diseases or pests in the facilities used for seed multiplication. (iii) All micro propagation and aeroponics or nethouse facilities must be approved by authorised State or Central Govt agency, as per the standard/guidelines. These facilities must have a changing area between the double doors. (iv) The aeroponics facility used for mini-tuber production from micro-plants shall be approved by Central Potato research Institute, Shimla (v) The net house must be "insect proof" and be equipped with a double-door entrance, provision for footwear disinfection prior to entering the net house and insect proof ventilation screening on intakes and exhaust openings. The persons entering the aeroponics or nethouse facility should use Wellington boots (Plastic boots) and change lab-coat in the changing area to reduce the chances of inadvertent introduction of vector insects clinging to clothes. (vi) The material being initiated for producing mini-tubers must be of Notified variety and confirmed identity. It must be duly documented with respect to origin. (vii) The plants of a potato varieties being initiated for tissue culture should be tested in an accredited laboratory for freedom from the following PVA (Potato Virus A), PVS (Potato Virus S), PVM (Potato Virus M), PVY (Potato Virus Y), PVX (Potato Virus X), PLRV (Potato Leaf Role Virus), PALCV (Potato Apical Leaf Curl Virus), GBNV (Groundnut Bud Necrosis Virus), PSTVd (Potato Spindle Tuber Viroid) entophytic and Phytoplasma or epiphytic bacteria and fungi. Tests must be carried on a minimum of ten plantlets of each variety selected at random. For virus testing ELISA or an equivalent method should be used, for viroid RT-PCR should be used, and for fungi and bacteria light microscopy and culturing on media should be used. 2. Sources of seed (i) The tissue culture facility should use recognized aseptic initiation and propagation procedures (i.e. follow procedures and use equipment, which will maintain sterile conditions as per standard tissue culture norms). (ii) The initiating facility must maintain following information on each variety for review and audit by the competent authority once in a year: variety identification, date of initiation, origin and testing results from accredited laboratory. (iii) Tests must be carried out on a minimum of ten plantlets (lot size as per SOP of Department of Biotechnology, GOI) selected at random, for each variety by an accredited laboratory. No plant should contain PVA, PVS, PVM, PVY, PVX, PLRV, PALCV, GBNV, phytoplasma, PSTVd and other endophytic or epiphytic bacteria and fungi. (iv) Valid pathogen testing results are required prior to the initiation of micro tuber production cycle or planting of test tube plantlets in the aeroponics or nethouse facility. (v) Mini-tubers/micro-tubers shall be produced and multiplied from approved source in vitro plants as per the requirements. (vi) Aeroponics or nethouse produced mini-tubers must be certified as G0 by the Punjab State Seed Certification Authority. (vii) G0 shall be used for further production of G1 to G4 certified classes of seed as prescribed in proposed seed standards. The certificates shall be issued by the Punjab State Seed Certification Authority. II. Aeroponics or nethouse Environment Requirements (a) All micro propagation and aeroponics or nethouse facilities must meet the standards given above under eligibility requirements. (b) The soil used for mini-tuber production should not be infested with pathogen and pests of potato, particularly the following: • wart (Synchytrium endobioticum (Schilb.) Perc.) and or cyst forming nematodes; • brown rot (Ralstonia solanacearum (E.F. Sm.) E.F. Sm.) or non-cyst forming nematodes within the previous three years; • common scab (Streptomyces scabies (Thaxt.) Waks. & Henrici). • Black scurf (Rhizoctonia Solani Kuehn). (c) The nutrient medium used for aeroponics shall be prepared in clean water free from all potato pathogens. III. Inspection of Aeroponics or nethouse facility used for production of mini-tubers (G-0). a. The seed potato grower must notify the competent authority of his production plans 30 days prior to the recommended planting date. b. The crop must be grown from approved basic source in vitro plants which were produced, in an aseptic environment. c. The tissue plantlets in the aeroponics or nethouse facility will be planted at 15x15cm and 30x15 cm, respectively. d. A minimum of three inspections shall be made as follows: i. The first inspection shall be made at 30-35days to verify growing conditions, extent of disease infection and off types; ii. The second inspections shall be made at 60-65 days after planting to verify off types, disease infection if any and pathogen testing, on a representative sample, comprising of 1% of the plants with a minimum of 5 and a maximum of 25 plants sampled for each variety: iii. The third inspection shall be made immediately after haulms cutting/ destruction in the net house crop in order to verify that haulms have been cut/destroyed by the prescribed date and proper manner. iv. In case the crop is grown under aeroponics, third inspection can be after the crop has been finally terminated. v. Effective sanitation practices including insect and disease monitoring and prevention must be adhered to. vi. The aeroponics or nethouse must be free from all potato and solanaceous plant debris before planting. vii. No field-produced seed potatoes (including pathogen tested clonal selections), non-seed potatoes, nor any other solanaceous species of plants can be grown in the net house while used to produce minitubers. viii. Varieties must be separated by appropriate partitioning of net house to prevent varietal mixture. ix. If testing performed by an accredited laboratory reveals the presence of banned virus (es), fungus or bacteria, all the crops in the net house will be ineligible for multiplication and the entire material will be destroyed. x. In the eventuality of detection of insect (particularly aphids, thrips and white flies) vectors (for which yellow sticky traps should be put at least at three places in the net house) by competent Authority, the grower must provide post harvest test results to this authority. A representative sample, representing each variety grown in the net house must be post harvest tested and if the results are negative for PVA, PVS, PVM, PVY, PVX, PLRV GBNV, phytoplasma, and PALCV, the crop will be assigned G0 status or otherwise rejected. IV. Land Requirements A crop of seed potato (G1-G4) shall not be eligible for certification if grown on land infested with: (a) Wart (Synchytrium endobioticum (Schilb.) Perc. And or cyst forming nematodes; (b) Brown rot (Ralstonia solanacearum (E.F. Sm.) E.F. Sm.) Or non-cyst forming nematodes within the previous three years; (c) Common scab (Streptomyces scabies (Thaxt.) Waks. & Henrici). (d) Crop rotation schedule as per seed plot technique.