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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. 164 PUNJAB GOVT. GAZ. (EXTRA), NOVEMBER 6, 2020 (KRTK 15, 1942 SAKA) (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. 165 PUNJAB GOVT. GAZ. (EXTRA), NOVEMBER 6, 2020 (KRTK 15, 1942 SAKA) 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. 166 PUNJAB GOVT. GAZ. (EXTRA), NOVEMBER 6, 2020 (KRTK 15, 1942 SAKA) V. Field Inspection A minimum of four inspections shall be made as follows: (a) the first inspection shall be made about 30-35 days after planting the mini-tubers to verify isolation, offtypes and the extent of disease infection with specific reference to mild and severe mosaics, leaf roll, PACLV, GBNV, phytoplasma, brown rot late blight and other relevant factors; (b) the second inspection shall be made about 60-65 days after planting the mini-tubers to check isolation, off types and extent of disease infection with specific reference to mild and severe mosaics, leaf roll, yellow, brown rot and other relevant factors; (c) the third inspection shall be made immediately after haulms cutting/ destruction in order to verify that haulms have been cut/destroyed by the prescribed date and in proper manner; (d) the fourth inspection shall be made about 10 days after haulms cutting/destruction and before harvesting in order to verify that no re-growth of haulms has taken place. VI. Seed Standards A. Standards for mini-tuber (G-0) production in the aeroponics/ nethouse facility (1) General requirements (i) Isolation: Minimum 1 meter between the different varieties grown in net house, so as to avoid mechanical admixture. In aeroponics facility separate grow boxes shall be used for each variety and two varieties cannot be grown in the same grow box. (ii) All micro propagation and aeroponics or nethouse facilities must be approved by competent authority set up by the state Govt., as per the standards given above under eligibility requirements. 167 PUNJAB GOVT. GAZ. (EXTRA), NOVEMBER 6, 2020 (KRTK 15, 1942 SAKA) (2) Specific requirements Maximum permissible limits Factor Maximum permissible limit *Off type 0.001% *Plants showing symptoms of mild 0.001% mosaic *Plants showing symptoms of severe Nil mosaic, leaf roll, PACLV, GBNV, phytoplasma and apical leaf curl **Plants infected with brown rot Nil (Ralstonia solanacearum) * At the time of crop termination **Maximum permitted at final inspection in the net house raised crop (3) Seed Standards for mini-tuber Factor Permissible limit Weight of minituber (minimum) 1.0 g (minimum) Germination (minimum) 90% (minimum) Varietal purity (percent plants) 99% (minimum) Virus infestation (percent plants) 0.001% (maximum) B. Field standards for G1 to G4 Crop raised out of Potato Tissue Culture based mini-tuber. (1) General requirements Isolation: The fields of seed potato shall be isolated from the contaminants shown in column 1 of the Table below by the distances specified in columns 2-6 of the said Table Containments Minimum distance (meters) G1 G2 G3 G4 Fields of other varieties 5 5 5 5 Fields of same variety not conforming 5 5 5 5 to requirement for certification 168 PUNJAB GOVT. GAZ. (EXTRA), NOVEMBER 6, 2020 (KRTK 15, 1942 SAKA) (2) Specific requirements Factor Maximum Permissible limits G1 G2 G3 G4 Off types (%)(1st and 2nd 0% 0% 0.05% 0.05% inspection) Plants showing symptoms of mild 0% 0% 1.0% 2.0% mosaic (1st and 2nd inspection) Plants showing symptoms of 0% 0% 0.5% 0.75% severe mosaic, leaf roll, PACLV, GBNV, phytoplasma (1st and 2nd inspection) *Total virus 0% 0% 1.0% 2.0% Plants infected by brown rot None None None None (syn Bacterial wilt) Ralstonia solanacearum (1st and 2nd inspection) **Regrowth of plants after 0% 0% 0.5% 0.5% destruction of haulms (4th inspection) *Of the two inspections, the higher virus percentage will be considered for the purpose of the specified limits of tolerance. **Standards for re-growth after destruction of haulms shall be met at fourth inspection to be conducted about 10 days after haulms cutting. Note: (i) All off types and diseased plants should be rouged out along with the tubers and destroyed. (ii) Gaps in the seed plot should not be more than 10.0%. (iii) Haulms must be destroyed as close to the ground as possible before the date specified by the Certification Agency. Failure to destroy haulms in time shall render the crop liable for rejection. 169 PUNJAB GOVT. GAZ. (EXTRA), NOVEMBER 6, 2020 (KRTK 15, 1942 SAKA) C. Seed Standards (G1 to G4) (1) Size and weight Specification Size Mean length and two widths Corresponding at the middle of tuber weight Seed Size 30-55 mm 25-125 g Large size Above 55 mm Above 125 g Note: (i) The size of tuber will be decided either on the basis of mean of two widths of a tuber at the middle and that of length or on the basis of corresponding weight of tuber. (ii) In a seed lot, tubers not conforming to specific size of seed shall not exceed more than 5.0% (by number). (iii) The seed material shall be reasonably clean, healthy, firm and shall conform to the characteristics of the variety. The tubers not conforming to the varietal characteristics shall not exceed 0.050% for G1 and G2 class of seed and 0.10% (by number) for G3, G4 and G4 class of seed. (iv) Cut, bruised, unshapy, cracked tubers or those damaged by insects, slugs or worms shall not exceed more than 1.0% (by weight). (v) Greenish pigmentation on tubers will not be a disqualification for certification. (2) Maximum tolerance limit of tubers showing visible symptoms caused by the diseases mentioned below will be as follows: Disease Maximum Permissible limits (By number) G1 G2 G3 G4 Late blight (Phytophthora infestations 0.1% 0.1% 0.5% 1.0% (Mont.) de Bary), dry rot (Fusarium caeruleum (Lib.) Sacc.) or Charcoal rot (Macrophomina phaseoli (Tassi) G. Goidanich). 170 PUNJAB GOVT. GAZ. (EXTRA), NOVEMBER 6, 2020 (KRTK 15, 1942 SAKA) Wet rot (Scierotium rolfsii) Sacc.) None None None None *Common scab (Streptomyces 1.0% 1.0% 2.0% 3.0% scabies (Thaxt) Waks. & Henrici) Brown rot (Ralstonia solanacearum 0.0 0.0 0.0 0.0 (E.F. Sm.) E.F. Sm.) **Black scurf (Rhizoctonia solani 2.0% 3.0% 5.0% 5.0% Kuehn.) Total diseases 3.1% 4.1% 7.5% 9.0% *Even if a single tuber infected with common scab is detected in a seed lot, the entire seed lot shall be treated with approved chemical before seed lot is declared fit for certification. Seed lots having infected tubers more than the prescribed limits will not be certified even after treatment. **(i) A tuber carrying 5% or above scuffed surface will be considered as one infected unit. (ii) Seed lots having black scurf infection more than the prescribed limits (5-10%) could be certified after treatment with approved chemical/fungicide. (iii) For all diseases, the higher disease percentage will be considered for the purpose of the specified limits of tolerance.