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Tissue Culture Guide
These are the protocols that we use for virus cleanup and culture maintenance.
Sterilize 15PSI for 15 min
Potato
Media
| Cleanup | Propagation | Tuberization | Storage | |
| M&S Strength | 1 | |||
| Agar (g) | 8 | |||
| BAP (mg) | 3 | |||
| GA3 (mg) | 0.2 | |||
| Inositol (mg) | 100 | |||
| Kinetin (mg) | 1 | |||
| Mannitol (g) | 0 | |||
| NAA (mg) | 0.1 | |||
| NaH2PO4 (mg) | 0 | |||
| Ribavirin (mg) | 35 | |||
| Sorbitol (g) | 0 | |||
| Sucrose (g) | 30 | |||
| Thiamin (mg) | 30 | |||
| Chlormequat Cl (mg) | 0 | |||
| Ca-Panto (mg) | 0 |
Cleanup
Store tubers at 37C in a humid incubator for five weeks.
Store tubers at 20C until sprouting begins.
Detach six sprouts from heat treated tubers and surface sterilize in 4% sodium hypochlorite solution with Tween for 5 min.
Culture three sprout meristems each in two deep Petri dishes on M&S media, 3.0mg BAP, 0.2mg GA3 adjusted to pH 6.0 with NaOH, supplemented with 35mg/L ribavirin for ten weeks.
Transfer surviving plantlets onto standard M&S propagation medium in flip top vessels until at least two inches tall (about eight weeks).
Select the two best looking plantlets.
Divide each plantlet three ways, reserving two backups.
Once the third plantlet has rooted well, transfer to a screened pot. Treat with an all-purpose insecticide.
When large enough, take foliage samples and ELISA test for AMV, APLV, CMV, PapMV, PLRV, PVA, PVS, PVT, PVX, Potyviruses.
If all test clean, send on to Agdia for PCR group tests.
If those come back clean, transplant to larger pot and grow under tuberization conditions.
Use the tubers as foundation stock for the next field planting, reserving ten tubers to produce the next generation of minitubers.
Propagation
Storage
M&S, 40g sucrose, 20g manitol
Tuberization
Medium: M&S, 10mg BA, 8% sucrose
20 C temp, 8 hours of light
Ulluco
Media
| Cleanup | Propagation | Tuberization | Storage | |
| M&S Strength | ||||
| Agar (g) | ||||
| BAP (mg) | ||||
| GA3 (mg) | ||||
| Inositol (mg) | ||||
| Kinetin (mg) | ||||
| Mannitol (g) | ||||
| NAA (mg) | ||||
| NaH2PO4 (mg) | ||||
| Ribavirin (mg) | ||||
| Sorbitol (g) | ||||
| Sucrose (g) | ||||
| Thiamin (mg) | ||||
| Chlormequat Cl (mg) | ||||
| Ca-Panto (mg) |
Cleanup
Store tubers at 37C in a humid incubator for five weeks.
Store tubers at 20C until sprouting begins.
Detach six sprouts from heat treated tubers and surface sterilize in 4% sodium hypochlorite solution with Tween for 20 min.
Culture three sprout meristems each in two deep Petri dishes on M&S media, Inositol 100mg, Thiamine HCl 30mg, Sucrose 30g, Monosodium phosphate 221mg adjusted to pH 6.0 with NaOH, supplemented with 50mg/L ribavirin for ten weeks.
Transfer surviving plantlets onto standard M&S propagation medium in flip top vessels until at least two inches tall (about eight weeks).
Propagation
For shoot production: M&S with 0.1mg NAA, 0.1mg BA, 0.01mg GA3, 3% sucrose
For rooted plantlet production: M&S with 0.3mg NAA, 0.1mg BA, 0.01mg GA3, 20 mg thiamine, 3% sucrose
Storage
M&S plus 30g mannitol, 90g sucrose, 30g sorbitol, ABA 3mg
Tuberization
Medium: M&S with 0.3mg NAA, 0.1mg BA, 0.01mg GA3, 3% sucrose
Oca
Media
| Cleanup | Propagation | Tuberization | Storage | |
| M&S Strength | ||||
| Agar (g) | ||||
| BAP (mg) | ||||
| GA3 (mg) | ||||
| Inositol (mg) | ||||
| Kinetin (mg) | ||||
| Mannitol (g) | ||||
| NAA (mg) | ||||
| NaH2PO4 (mg) | ||||
| Ribavirin (mg) | ||||
| Sorbitol (g) | ||||
| Sucrose (g) | ||||
| Thiamin (mg) | ||||
| Chlormequat Cl (mg) | ||||
| Ca-Panto (mg) |
Cleanup
Store tubers at 37C in a humid incubator for five weeks.
Store tubers at 20C until sprouting begins.
Detach six sprouts from heat treated tubers and surface sterilize in 4% sodium hypochlorite solution with Tween for 20 min.
Culture three sprout meristems each in two deep Petri dishes on M&S media, Inositol 100mg, Thiamine HCl 30mg, Sucrose 30g, Monosodium phosphate 221mg adjusted to pH 6.0 with NaOH, supplemented with 50mg/L ribavirin for ten weeks.
Transfer surviving plantlets onto standard M&S propagation medium in flip top vessels until at least two inches tall (about eight weeks).
Propagation
Shoot production: M&S 1mg BA, 1mg NAA, 5mg Kin
Rooted plantlets: 0.5mg/l GA3 on liquid
Storage
+ 0.5% activated charcoal
Tuberization
Mashua
Media
| Cleanup | Propagation | Tuberization | Storage | |
| M&S Strength | ||||
| Agar (g) | ||||
| BAP (mg) | ||||
| GA3 (mg) | ||||
| Inositol (mg) | ||||
| Kinetin (mg) | ||||
| Mannitol (g) | ||||
| NAA (mg) | ||||
| NaH2PO4 (mg) | ||||
| Ribavirin (mg) | ||||
| Sorbitol (g) | ||||
| Sucrose (g) | ||||
| Thiamin (mg) | ||||
| Chlormequat Cl (mg) | ||||
| Ca-Panto (mg) |
Cleanup
Store tubers at 37C in a humid incubator for five weeks.
Store tubers at 20C until sprouting begins.
Detach six sprouts from heat treated tubers and surface sterilize in 4% sodium hypochlorite solution with Tween for 20 min.
Culture three sprout meristems each in two deep Petri dishes on M&S medium with 2mg 2,4-D, 0.5mg kinetin
Transfer surviving plantlets onto standard M&S propagation medium in flip top vessels until at least two inches tall (about eight weeks).
Propagation
For shoot production M&S with 5mg BAP, 1 mg K
For rooted plantlets, M&S with 1mg NAA,
Storage
Tuberization
M&S with 2mg NAA, 3mg BAP, 3mg K, 6% sucrose
Yacon
Media
| Cleanup | Propagation | Tuberization | Storage | |
| M&S Strength | ||||
| Agar (g) | ||||
| BAP (mg) | ||||
| GA3 (mg) | ||||
| Inositol (mg) | ||||
| Kinetin (mg) | ||||
| Mannitol (g) | ||||
| NAA (mg) | ||||
| NaH2PO4 (mg) | ||||
| Ribavirin (mg) | ||||
| Sorbitol (g) | ||||
| Sucrose (g) | ||||
| Thiamin (mg) | ||||
| Chlormequat Cl (mg) | ||||
| Ca-Panto (mg) |
Cleanup
Store tubers at 37C in a humid incubator for five weeks.
Store tubers at 20C until sprouting begins.
Detach six sprouts from heat treated tubers and surface sterilize in 4% sodium hypochlorite solution with Tween for 20 min.
Culture three sprout meristems each in two deep Petri dishes on M&S media, Inositol 100mg, Thiamine HCl 30g, Sucrose 30g, Monosodium phosphate 221mg adjusted to pH 6.0 with NaOH, supplemented with 50mg/L ribavirin for ten weeks.
Transfer surviving plantlets onto standard M&S propagation medium in flip top vessels until at least two inches tall (about eight weeks).
Propagation
Shoot growth: M&S, 30g sucrose, 100mg myo-inositol, 5mg BAP, 1mg thiamine
Rooting: 1/2 M&S, 3% sucrose, 0.3mg NAA
Storage
1/2 MS, 10g mannitol