2019 Medicinal Cannabis Conference · 2019-04-02 · • In vitro propagation of plant parts, such...

© PharmOut 2019

2019 Medicinal Cannabis Conference

Plant Tissue Culture

Matthew Hayes- Horticulture Consultant26 March 2019

© PharmOut 2019

My back ground

Plant phenomics and physiology to quantify plant performance to abiotic stress.• Controlled environment research and breeding; new technology to automate. • Design & management of plant performance assays; plant production to tight

specifications; high throughput imaging & physiology to quantify performance.

SGWC: Bobwhite drought

0

20

40

60

80

100

120

21/08

/2012

23/08

/2012

25/08

/2012

27/08

/2012

29/08

/2012

31/08

/2012

2/09/2

012

4/09/2

012

6/09/2

012

8/09/2

012

10/09

/2012

12/09

/2012

14/09

/2012

16/09

/2012

18/09

/2012

20/09

/2012

22/09

/2012

24/09

/2012

SGW

C(%

)

Full waterDrought water

Chart1

41142411420000

41143411430.2297181650.2297181650.2297181650.229718165

41148411480.60118678930.60118678930.60118678930.6011867893

41150411500.76856349190.76856349190.76856349190.7685634919

41151411510.808235550.808235550.808235550.80823555

41155411553.5922117323.5922117321.59512473971.5951247397

41156411562.88901063772.88901063771.71894637251.7189463725

41157411572.74982317092.74982317091.46710279531.4671027953

41158411582.69608621732.69608621731.34796338521.3479633852

41162411622.63738962872.63738962870.91039166440.9103916644

41163411632.67676881562.67676881560.83318688980.8331868898

41164411642.94987127212.94987127210.87231534670.8723153467

41165411651.69087985271.69087985270.60456850280.6045685028

41169411693.86538742463.86538742460.73851719650.7385171965

41170411703.7527965683.7527965680.62020215980.6202021598

41171411713.07180614733.07180614730.71566314370.7156631437

41172411722.02934969852.02934969850.61418778450.6141877845

41176411761.83491515111.83491515110.37575261620.3757526162

41177411777.08555977117.08555977110.37528696780.3752869678

Full water

Drought water

SGWC(%)


100

100

98.1609932071

98.1609932071

84.8664171502

84.8664171502

78.2331642831

78.2331642831

73.6675882495

73.6675882495

75.2823802547

58.7759710168

84.9519532976

53.9228674984

79.8712855301

50.7069672

74.0991863631

46.9300841049

74.9749445609

36.8084589209

89.6144215717

33.1861028175

82.2373476564

30.6900501453

77.0901599603

27.8396149688

82.4659891289

22.1521633061

76.9017062006

20.748097656

71.633149376

19.4310982357

90.2418766095

18.2718215704

47.4413666197

12.9088936887

64.9574466156

11.5935658099

SGWC

8/21/12LICOR T1 50% SGWCT1 30%T2 20%T3 10%

Tub100%0%8/22/12diffSGWC8/27/12SGWC8/29/12SGWC8/30/12SGWC9/3/12SGWC9/4/12SGWC9/5/12SGWC9/6/12SGWC9/10/12SGWC9/11/12SGWC9/12/12SGWC9/13/12SGWC9/17/12SGWC9/18/12SGWC9/19/12SGWC9/20/12SGWC9/24/12SGWC9/25/12SGWC

110.1996.25510.1360.06398.40263691689.64285.87728194739.38379.31034482769.18174.18864097368.68661.63793103458.49756.8458417858.35353.19472616638.19549.18864097367.77138.43813387427.61634.50811359037.51932.0486815416

210.4656.25510.3770.08897.90973871739.88486.19952494069.61679.83372921629.41375.01187648469.35773.68171021389.78383.80047505949.59279.26365795729.33373.11163895499.30472.42280285049.78783.89548693599.45375.9619952494

310.1736.25510.110.06398.39203675349.57684.76263399699.30577.84583971419.13173.40479836658.62460.46452271578.43355.58958652378.30152.22052067388.15148.39203675347.73337.72332822877.58834.0224604397.49331.5977539561

410.296.25510.2250.06598.38909541519.68685.03097893439.4478.93432465929.25174.25030978939.19172.76332094189.56381.98265179689.39677.8438661719.17672.39157372999.24574.10161090469.89590.21065675349.60382.9739776952

510.3876.25510.3080.07998.08809293329.78285.35818005819.49578.41239109399.30473.78993223628.759.17231364968.51954.79186834468.37651.33107454028.20647.21684414337.76636.56824782197.6132.79283639887.50430.2274927396

610.2276.25510.1670.0698.48942598199.6585.47331319239.38678.82678751269.2174.39577039279.45980.6646525689.71187.00906344419.581.6968781479.27275.95669687819.13472.48237663659.83690.15609264859.58983.93756294069.32777.34138972819.47180.96676737169.26875.85599194369.09271.42497482389.76788.41893252778.12547.07955689839.15873.0866062437

710.1966.25510.1080.08897.76706419699.5784.11570667349.31377.59451915769.13373.02715046948.59459.35041867558.38554.04719614318.25150.64704389758.146.81552905357.70636.81806648067.56733.29104288257.48131.10885562047.37728.46993148957.15322.78609489987.09221.2382643999

810.1856.25510.1260.05998.49872773549.61985.59796437669.37279.31297709929.22175.47073791359.39679.92366412219.73988.65139949119.5483.58778625959.31677.88804071259.24976.18320610699.7288.16793893139.50182.59541984739.26576.59033078889.46781.73027989829.27276.7684478372

910.2166.25510.130.08697.82883110339.59584.32214087359.33377.70764958349.15573.21383489028.5457.68745266358.3452.6382226718.21749.53294622578.07145.84700833127.70336.55642514527.57733.37541024997.46430.52259530427.36528.0232264587.1622.84776571577.09921.3077505687.05720.24741226967.00618.95985862166.78313.329967186.73112.017167382

1010.1046.25510.0270.07797.99948038459.51984.80124707729.26778.25409197199.10974.14912964419.20776.69524551839.55485.71057417519.36280.72226552359.13574.8246297749.34180.17666926479.83793.06313328149.57786.30813198239.32779.81293842569.66688.62042088859.44982.9825928813

1110.1746.25510.1050.06998.23934677219.57784.7665220729.33778.64251084469.15373.94743557038.56658.96912477679.27176.95840775719.07872.03368206188.86766.64965552448.26751.3396274568.09446.92523602967.93742.91911201847.79139.19367185517.45130.5179892837.36228.2470017862

1210.2046.25510.1350.06998.25272220829.58684.3504684739.33778.04507470259.14373.1324385929.27376.42441124349.68986.95872372759.47281.46366168659.21474.9303621179.32877.81716890359.88591.9220055719.56583.81868827559.30977.33603443919.63385.54064320089.38679.28589516339.20974.80374778429.90592.4284629028.20749.43023550278.62860.0911623196

1310.1116.25510.0430.06898.23651452289.5184.41390041499.24177.43775933619.05472.58817427398.46857.39107883828.29552.90456431548.1849.92219917018.05446.65456431547.67436.79979253117.53533.19502074697.4430.73132780087.34628.29356846477.11822.38070539427.06621.0321576763

1410.1976.25510.1170.0897.9705733139.58684.50025367839.30977.47336377479.12672.83105022839.06971.38508371399.47381.6336884839.25576.1035007619.03970.62404870629.16773.87113140549.85791.37493658049.54783.51090816849.28776.91527143589.35878.71638762059.13473.0339928978.96268.670725529.79889.87823439888.06145.81430745818.68761.6945712836

1510.3166.25510.2390.07798.10391529189.69184.60970204389.40277.49322826899.22573.13469588778.59557.62127554798.39952.79487810888.28750.03693671518.13746.34326520567.735.58236887477.55331.96257079547.45329.50012312247.36427.30854469347.11821.25092341797.06719.99507510477.02318.91159812856.97717.7788721996.76712.60773208576.71411.302634819

1610.2116.25510.1330.07898.02831142579.60784.73205257849.35378.31142568259.17673.83720930239.25175.73306370079.72787.76541961589.50982.25480283119.29976.94641051579.21574.82305358959.75288.39737108199.44680.66228513659.20474.54499494449.38979.22143579379.16273.4833164813

1710.3216.25510.2380.08397.95868175119.67884.185932129.39777.27496310879.21672.82341367448.5656.68962124948.3651.77078209548.23848.77029021158.08444.9827840637.71835.98130841127.5732.34136743737.46629.78357107727.35727.10280373837.12921.49532710287.07520.16724053127.03319.13428430896.9918.07673389086.77512.78898180036.72111.4608952287

1810.1776.25510.1120.06598.34268230499.56984.49770525249.29477.48597654269.11172.81998980119.01170.27027027039.43481.05558388589.23275.90515043359.00970.21927587979.11472.8964813879.75989.3421723619.40780.3671596124

AVG100AVG98.1609932071AVG84.866417150278.233164283173.6675882495DW58.7759710168DW53.9228674984DW50.7069672DW46.9300841049DW36.8084589209DW33.1861028175DW30.6900501453DW27.8396149688DW22.1521633061DW20.748097656DW19.4310982357DW18.2718215704DW12.9088936887DW11.5935658099

STDEV0STDEV0.229718165STDEV0.60118678930.76856349190.808235551.59512473971.71894637251.46710279531.34796338520.91039166440.83318688980.87231534670.60456850280.73851719650.62020215980.71566314370.61418778450.37575261620.3752869678

0%

16.281FW75.2823802547FW84.9519532976FW79.8712855301FW74.0991863631FW74.9749445609FW89.6144215717FW82.2373476564FW77.0901599603FW82.4659891289FW76.9017062006FW71.633149376FW90.2418766095FW47.4413666197FW64.9574466156

26.2573.5922117322.88901063772.74982317092.69608621732.63738962872.67676881562.94987127211.69087985273.86538742463.7527965683.07180614732.02934969851.83491515117.0855597711

36.227

6.255LICOR done todayavg DW31.2913094124

STDEV DW0.9484794973

SGWC

0000

0.2297181650.2297181650.2297181650.229718165

0.60118678930.60118678930.60118678930.6011867893

0.76856349190.76856349190.76856349190.7685634919

0.808235550.808235550.808235550.80823555

3.5922117323.5922117321.59512473971.5951247397

2.88901063772.88901063771.71894637251.7189463725

2.74982317092.74982317091.46710279531.4671027953

2.69608621732.69608621731.34796338521.3479633852

2.63738962872.63738962870.91039166440.9103916644

2.67676881562.67676881560.83318688980.8331868898

2.94987127212.94987127210.87231534670.8723153467

1.69087985271.69087985270.60456850280.6045685028

3.86538742463.86538742460.73851719650.7385171965

3.7527965683.7527965680.62020215980.6202021598

3.07180614733.07180614730.71566314370.7156631437

2.02934969852.02934969850.61418778450.6141877845

1.83491515111.83491515110.37575261620.3757526162

7.08555977117.08555977110.37528696780.3752869678

Full water

Drought water

SGWC(%)


Biomass

PlantFWDWFW:DWFW-DWFW AVGDW AVGPlantFWDWFW:DWFW-DWFW AVGDW AVGPlantFWDWFW:DWFW-DWFW AVGDW AVGPlantFWDWFW:DWFW-DWFW AVGDW AVGPlantFWDWFW:DWFW-DWFW AVGDW AVGPlantFWDWFW:DWFW-DWFW AVGDW AVG

1012.20930.27518.03089785531.93422012.6420.3048.69078947372.3383011.88920.23777.94783340341.65154012.3110.26988.56560415122.04125011.93810.24427.93652743651.69396018.45350.99688.48063804177.4567

1022.07190.2568.0933593751.81592023.25820.36398.953558672.89433021.72750.2158.03488372091.51254022.4640.28798.55852726642.17615021.70380.21118.07105637141.49276028.0950.93128.69308419247.1638

1031.41830.16798.44729005361.25042032.62520.29958.76527545912.32573032.02410.24858.14527162981.77564031.8030.2058.79512195121.5985031.39440.17687.88687782811.21766038.14250.93918.67053561927.2034T 30%T 20%T 10%

1041.57850.19268.19574247141.38592042.80250.31698.84348374882.48563041.55290.19527.95543032791.35774041.66730.19978.34902353531.46765041.58250.20017.90854572711.38246048.66581.01788.51424641387.648Drought tub 1Drought tub 2Drought tub 3Full water 1Full water 2Full water 3

1052.08920.24828.41740531831.8412052.47560.27698.9404117012.19873051.6910.21147.99905392621.47964051.59710.18118.81888459411.4165052.43080.30517.9672238612.12576058.11950.95388.51279094157.1657

1061.55510.18678.32940546331.368420603061.88290.23567.99193548391.64734062.57890.298.89275862072.28895062.22120.27877.96986006461.94256061.86910.21128.8499053031.6579

1071.51950.18688.13436830841.33272073.30460.37428.83110636022.93043071.74510.20848.37380038391.53674072.52830.2918.68831615122.23735071.73110.21847.92628205131.51276076.67450.77168.65020736135.9029

1081.67810.20668.12245885771.47152082.37060.26358.99658444022.10713081.77860.22148.03342366761.55724082.05450.24088.53197674421.81375081.78910.22897.81607688951.56026087.38250.82818.91498611286.5544

1091.53780.18188.45874587461.3562093.48840.38229.12715855573.10623091.81280.22278.14009878761.59014092.83310.32188.80391547552.51135091.69030.21857.73592677351.47186098.83771.01058.7458683827.8272

1101.73830.21228.19180018851.52612102.9090.31759.16220472442.59153101.34810.16548.15054413541.18274105102.12690.27287.79655425221.85416108.08780.90868.90138674887.1792

1111.92070.2388.07016806721.68272112.71130.29749.11667787492.41393111.56730.19358.09974160211.37384111.61910.18568.72359913791.43355111.80890.23197.80034497631.5776119.75611.16558.37074217078.5906

1121.54630.19288.02022821581.35352121.95340.22128.83092224231.73223121.20080.1527.91.04884121.60490.19978.03655483221.40525121.65780.20987.90181124881.4486127.09950.82388.61798980346.2757

AVG1.73858333330.21205833338.20932250411.526525AVG2.77643636360.31065454558.93256120462.2603AVG1.6850250.20898.06433475571.476125AVG2.09647272730.24294545458.61493476911.8535272727AVG1.8395750.2330257.89309062341.60655AVG0.87983333338.66019842426.7187916667

STDEV0.26621754210.0341256890.16254152760.2323512941STDEV0.44877394370.04877206910.15623737380.8076907745STDEV0.23464748460.02869209580.12810166220.2061575572STDEV0.46194237970.05077563120.24937622370.4115143645STDEV0.29243979060.03655195870.09291034020.2560011204STDEV1.97920483580.2351877290.17282259561.7447691009


7012.1180.30746.89004554331.81068012.3790.26029.14296694852.11889012.1760.57343.79490756891.602610013.8390.42199.09931263333.417111012.530.30048.42210386152.229612017.30690.86878.41130424776.4382

7023.280.48196.80639136752.79818023.5640.38379.28850664583.18039022.06770.53843.84045319471.529310024.9560.53079.33860938384.425311023.4390.40978.39394679033.029312027.90710.87859.00068298247.0286

7031.9770.25287.82041139241.72428030.6970.07998.72340425530.61719031.61780.38854.16422136421.229310034.3610.47229.2354934353.888811033.260.39598.23440262692.864112038.57290.94789.04505169877.6251

7042.1070.29167.22565157751.81548042.7660.3278.45871559632.4399041.51950.36844.12459283391.151110044.2320.48088.80199667223.751211041.4470.1967.38265306121.25112049.6451.0968.80018248188.549

7052.690.37897.09949854842.31118054.5960.49169.34906427994.10449051.96060.5073.8670611441.453610054.2420.44739.48356807513.794711054.0750.49938.16142599643.575712059.02110.99339.08194905878.0278

7062.8660.38827.38279237512.47788064.3140.43639.88769195513.87779061.62920.41993.87997142181.209310064.0170.41789.6146481573.599211061.5220.19987.61761761761.322212067.41410.81419.10711214846.6

7072.4610.32917.47797022182.13198073.9220.4219.31591448933.5019072.00720.51623.88841534291.49110073.4670.35879.66545860053.108311073.2810.38978.4192968952.891312076.38310.70269.0849701115.6805

7082.4530.33917.23385432032.11398082.8320.31748.92249527412.51469081.88890.46474.06477297181.424210083.30.36169.12610619472.938411083.4410.44547.72563987432.995612087.91250.86499.1484564697.0476

7092.6650.37557.09720372842.28958096.660.71019.37896070985.94999092.07780.55383.75189599131.52410093.9370.40589.70182355843.531211094.3890.5458.05321100923.844120910.96581.21439.03055258179.7515

7101.680.22747.38786279681.45268104.6060.48519.49494949494.12099102.29690.583.96017241381.716910102.1630.24428.85749385751.918811103.8990.47458.21707060063.424512107.62250.82759.21148036256.795

7113.0010.42167.11812144212.57948113.630.3899.33161953733.2419111.76120.44024.00090867791.32110113.7950.39769.54476861173.397411114.1170.50178.20609926253.615312118.93440.98959.029206677.9449

7122.0950.2867.32517482521.8098123.2250.38068.47346295322.84449121.95440.46574.19669315011.488710123.6720.42078.72830995963.2513111212128.1580.91988.86931941737.2382

AVG2.44941666670.33995833337.23874817822.1094583333AVG3.599250.39015833339.14731267833.2090916667AVG1.91310.48468333333.96117217291.4284166667AVG3.831750.4132759.26646576163.418475AVG3.21818181820.39612727278.07576978142.8220545455AVG8.32028333330.92641666678.98502235247.3938666667



13012.1970.31416.99458771091.882914018.23650.97938.41059940777.257215012.10660.57573.65919749871.530916016.7010.79218.45979043055.908917012.36190.60143.92733621551.760518012.47350.29378.42185903982.1798

13021.780.25826.89388071261.5218140211.22931.29138.6961201899.93815021.9620.52333.74928339381.438716024.8110.53099.06197023924.280117021.78180.45783.89209261691.32418022.59340.31118.33622629382.2823

13032.3890.32937.25478287282.059714038.5581.00418.52305547267.553915031.73650.44763.87957998211.2889160317031.68040.43693.84618905931.243518033.15510.37318.4564459932.782

13042.5850.34617.46893961282.238914046.17550.70468.76454726095.470915041.6170.39794.06383513451.219116044.250.49188.64172427823.758217041.49220.39793.75018848961.094318042.35010.27878.43236454972.0714

13050.9720.14226.8354430380.8298140513.33811.54248.647627074711.795715051.99210.53093.75230740251.461216055.2670.60168.75498670214.665417051.71010.42554.01903642771.284618052.82950.32258.77364341092.507

13061.8180.25837.03832752611.559714068.16980.95138.58803742257.218515061.92790.51043.77723354231.417516064.7150.52718.94517169424.187917061.64320.41133.99513736931.231918062.83310.32038.84514517642.5128

13073.3740.4767.08823529412.89814078.72371.0198.56104023557.704715072.19350.55963.91976411721.633916075.3360.58919.05788490924.746917072.22860.60733.66968549321.621318072.5990.29698.75378915462.3021

13081.6230.22967.0688153311.393414086.30880.75168.39382650355.557215082.01050.51613.89556287541.494416085.5960.64158.72330475454.954517082.09620.56343.72062477811.532818082.1440.24958.59318637271.8945

13093.1060.44247.020795662.6636140912.2931.37778.922842418510.915315092.06190.53793.83324037931.52416095.4280.60269.00763358784.825417092.23430.53864.14834756781.695718093.89450.43818.889522943.4564

13102.2350.31327.13601532571.9218141014.38831.64078.769610532112.747615102.04680.52883.87065052951.51816105.4690.58459.35671514114.884517101.24720.31193.99871753770.935318103.10080.34958.87210300432.7513

13112.8460.39377.22885445772.452314112.48470.27888.91212338592.205915111.85750.49263.77080795781.364916112.8510.33688.46496437052.514217111.78280.46083.86892361111.32218112.46010.29228.41923340182.1679

13122.2790.31557.22345483361.963514126.89110.8028.5923940156.089115120.93650.23224.0331610680.704316124.2570.49198.65419800773.765117121.94730.5263.70209125481.421318122.65110.31168.50802310652.3395

AVG2.2670.31821666677.10434436461.9487833333AVG8.89973333331.02856666678.64848532657.8711666667AVG1.87073333330.487753.85038532341.3829833333AVG4.9710.56271818188.8298494654.4082818182AVG1.85050.47823333333.87819753511.3722666667AVG2.75701666670.31976666678.60846187032.43725


32.0486815416

75.9619952494

31.5977539561

82.9739776952

30.2274927396

80.3671596124

Biomass

0.0341256890.034125689

0.04877206910.0487720691

0.02869209580.0286920958

0.05077563120.0507756312

0.03655195870.0365519587

0.04905192670.0490519267

Dry weight

Growth rates

0.23235129410.2323512941

0.80769077450.8076907745

0.20615755720.2061575572

0.41151436450.4115143645

0.25600112040.2560011204

0.41404911320.4140491132

FW-DW

0.16254152760.1625415276

0.15623737380.1562373738

0.12810166220.1281016622

0.24937622370.2493762237

0.09291034020.0929103402

0.20496264380.2049626438

FW:DW

0.07299712890.0729971289

0.1499435740.149943574

0.11853607120.1185360712

0.07240340430.0724034043

0.0919443590.091944359

0.11245446920.1124544692

Dry weight

0.39889557640.3988955764

1.30713472321.3071347232

0.8805079640.880507964

0.61323441930.6132344193

0.57694608360.5769460836

0.87295970560.8729597056

FW-DW

0.27114408390.2711440839

0.42684978930.4268497893

0.34958001760.3495800176

0.34653541760.3465354176

0.17261738440.1726173844

0.27958176250.2795817625

FW:DW

0.07299712890.0729971289

0.1499435740.149943574

0.11853607120.1185360712

0.07240340430.0724034043

0.0919443590.091944359

0.11245446920.1124544692

Dry weight

0.39889557640.3988955764

1.30713472321.3071347232

0.8805079640.880507964

0.61323441930.6132344193

0.57694608360.5769460836

0.87295970560.8729597056

FW-DW

0.27114408390.2711440839

0.42684978930.4268497893

0.34958001760.3495800176

0.34653541760.3465354176

0.17261738440.1726173844

0.27958176250.2795817625

FW:DW

TIMEDWWaterTubT1 DT2 DT3 DT1-T2T2-T3T1-T3T1 FT2 FT3 FT1-T2T2-T3T1-T3

10.2751110.1520.14220.2322-0.00163333330.01285714290.00616923080.18110.07990.2112-0.01686666670.01875714290.0023153846

10.256110.16540.22740.31190.01033333330.01207142860.01126923080.18560.24420.27880.00976666670.00494285710.0071692308DroughtFull water

10.1679110.16790.36840.01542307690.19970.26020.70260.01008333330.06320.0386846154

10.1926110.17680.22960.38850.00880.02270.01628461540.19970.31740.70460.01961666670.05531428570.0388384615

10.2482110.18180.25280.39790.01183333330.02072857140.01662307690.2050.3270.75160.02033333330.06065714290.0420461538

10.1867110.18670.39790.01624615380.22120.33680.77160.01926666670.06211428570.0423384615

10.1868110.18680.25820.41130.01190.02187142860.01726923080.24080.35870.8020.019650.06332857140.0431692308

10.2066110.19260.25830.41990.010950.02308571430.01748461540.24950.36160.81410.01868333330.06464285710.0434307692

10.1818110.19280.42550.01790.26350.38060.82380.01951666670.06331428570.0431

10.2122110.19350.2860.43690.01541666670.02155714290.01872307690.26980.38370.82750.01898333330.06340.0429

10.238110.19520.29160.44020.01606666670.02122857140.01884615380.3890.82810.0627285714

10.1928110.20010.44760.01903846150.27690.39760.86490.02011666670.06675714290.0452307692

10.304220.20660.30740.45780.01680.02148571430.01932307690.27870.40580.86870.02118333330.06612857140.0453846154

10.3639220.20840.31320.46080.01746666670.02108571430.01941538460.28790.41780.87850.021650.06581428570.0454307692

10.2995220.20980.46470.01960769230.290.42070.90860.02178333330.06970.0475846154

10.3169220.21110.31410.46570.01716666670.02165714290.01958461540.2910.4210.91980.02166666670.07125714290.0483692308

10.2769220.21140.31550.49260.017350.02530.02163076920.29220.42190.93120.02161666670.07275714290.0491538462

1220.21220.5070.02267692310.29370.43630.93910.02376666670.07182857140.0496461538

10.3742220.2150.32910.51040.01901666670.02590.02272307690.29690.44730.94780.02506666670.07150.0500692308

10.2635220.21840.32930.51610.01848333330.02668571430.02290.29740.47220.95130.02913333330.06844285710.0503

10.3822220.21850.51620.02290.29950.48080.95380.03021666670.06757142860.0503307692

10.3175220.22140.33910.52330.01961666670.02631428570.02322307690.3040.97930.0519461538

10.2974220.22270.34610.5260.02056666670.02570.02333076920.31110.48510.98950.0290.07205714290.0521846154

10.2212220.22890.52880.02306923080.31160.49160.99330.030.07167142860.0524384615

10.2377130.23190.37550.53090.02393333330.02220.0230.49180.99680.0721428571

10.215130.23560.37890.53790.02388333330.02271428570.02325384620.31690.49191.00410.02916666670.07317142860.0528615385

10.2485130.23770.53840.02313076920.31750.52711.01050.03493333330.06905714290.0533076923

10.1952130.2380.38820.53860.02503333330.02148571430.02312307690.32030.53071.01780.03506666670.06958571430.0536538462

10.2114130.24420.39370.55380.02491666670.02287142860.02381538460.32180.53091.0190.034850.06972857140.0536307692

10.2356130.24820.55960.02395384620.32250.58451.0960.04366666670.07307142860.0595

10.2084130.24850.42160.56340.028850.02025714290.02422307690.34950.58911.16550.03993333330.08234285710.0627692308

10.2214130.2560.44240.57340.03106666670.01871428570.02441538460.36390.60161.21430.03961666670.08752857140.0654153846

10.2227130.27280.57570.02330.37310.60261.29130.038250.09838571430.0706307692

10.1654130.27510.4760.580.03348333330.01485714290.02345384620.37420.64151.37770.044550.10517142860.0771923077

10.1935130.27870.60140.02482307690.38220.71011.54240.054650.11890.0892461538

10.152130.30510.48190.60730.02946666670.01791428570.02324615380.43810.79211.64070.0590.12122857140.0925076923

10.269824Median0.21360.32230.50870.0179750.02160714290.02270.29530.43630.943450.02376666670.06958571430.0498576923

10.287924STDEV0.03409593980.08194430940.08311050970.00804789240.00384062560.00404484720.05918265450.13614082780.27170019220.01388297560.02107303260.0172360575

10.20524VAR0.00116253310.00671486980.00690735680.00006476860.00001475040.00001636080.00350258660.0185343250.07382099440.0001927370.00044407270.0002970817

10.199724STERR0.00097416970.00356279610.0023745860.00034990840.00016698370.0001155671

10.181124

10.2924

10.291240.3062088659

10.240824

10.321824

124

10.185624

10.199724

10.244215

10.211115

10.176815T1 BiomassT2 BiomassT3 BiomassT1-T2 RGRT2-T3 RGRT1-T3 RGR

10.200115Median0.21360.32230.50870.0179750.02160714290.0227

10.305115ST DEV0.03409593980.08194430940.08311050970.00804789240.00384062560.0040448472

10.278715% ST DEV of median15.962518634825.424855526816.337823809144.772697906717.774796066817.8187100752

10.218415

10.228915Variance0.00116253310.00671486980.00690735680.00006476860.00001475040.0000163608

10.218515ST ERR0.00097416970.00356279610.0023745860.00034990840.00016698370.0001155671

10.272815

10.23191513.656387665217.180616740124.6696035242

10.209815

10.2937218Detectable effect size based on T1-T3 RGR

10.3111218n=3613.66%

10.3731218n=2417.18%

10.2787218n=1224.67%

10.3225218

10.3203218

10.2969218

10.2495218

10.4381218

10.3495218

10.2922218

10.3116218

20.307417

20.481917

20.252817

20.291617

20.378917

20.388217

20.329117

20.339117

20.375517

20.227417

20.421617

20.28617

20.260228

20.383728

20.079928

20.32728

20.491628

20.436328

20.42128

20.317428

20.710128

20.485128

20.38928

20.380628

20.300411

20.409711

20.395911

20.19611

20.499311

20.199811

20.389711

20.445411

20.54511

20.474511

20.501711

211

20.4219210

20.5307210

20.4722210

20.4808210

20.4473210

20.4178210

20.3587210

20.3616210

20.4058210

20.2442210

20.3976210

20.4207210

20.3141113

20.2582113

20.3293113

20.3461113

20.1422113

20.2583113

20.476113

20.2296113

20.4424113

20.3132113

20.3937113

20.3155113

20.7921216

20.5309216

2216

20.4918216

20.6016216

20.5271216

20.5891216

20.6415216

20.6026216

20.5845216

20.3368216

20.4919216

30.573419

30.538419

30.388519

30.368419

30.50719

30.419919

30.516219

30.464719

30.553819

30.5819

30.440219

30.465719

30.996826

30.931226

30.939126

31.017826

30.953826

30.211226

30.771626

30.828126

31.010526

30.908626

31.165526

30.823826

30.5757115

30.5233115

30.4476115

30.3979115

30.5309115

30.5104115

30.5596115

30.5161115

30.5379115

30.5288115

30.4926115

30.2322115

30.8687212

30.8785212

30.9478212

31.096212

30.9933212

30.8141212

30.7026212

30.8649212

31.2143212

30.8275212

30.9895212

30.9198212

30.6014117

30.4578117

30.4369117

30.3979117

30.4255117

30.4113117

30.6073117

30.5634117

30.5386117

30.3119117

30.4608117

30.526117

30.9793214

31.2913214

31.0041214

30.7046214

31.5424214

30.9513214

31.019214

30.7516214

31.3777214

31.6407214

30.2788214

30.802214

0.00804789240.00804789240.00384062560.00384062560.00404484720.0040448472

0.01388297560.01388297560.02107303260.02107303260.01723605750.0172360575

T1-T2

T2-T3

T1-T3

RGR (gDW/day)

Relative Growth Rate (RGR)

0.03409593980.03409593980.08194430940.08194430940.08311050970.0831105097

0.05918265450.05918265450.13614082780.13614082780.27170019220.2717001922

T1 Biomass

T2 Biomass

T3 Biomass

Biomass (g DW)

Biomass

0.00804789240.0080478924

0.00384062560.0038406256

0.00404484720.0040448472

Drought RGR

© PharmOut 2019

Summary

Introduction

History

Applications

Benefits and constraints

Tissue culture facility requirements

Micropropagation workflows and media

Summary

© PharmOut 2019

Introduction to plant tissue culture

• In vitro propagation of plant parts, such as single cells, tissues and organs under sterile conditions.

• Used routinely in ornamental and horticultural crop nurseries:

• Industry-wide application of tissue culture: potato seed certification schemes mandate the use of tissue

culture to produce healthy primary stock. tissue culture of banana provides Panama disease-free stock.

• Begonia• Rosa• Chrysanthemum

• banana• potato• grapevine

© PharmOut 2019

History of plant tissue culture

© PharmOut 2019

Applications of plant tissue culture

Disease elimination from unhealthy plants• Meristem culture to re-establish healthy stock after viral,

fungal or bacterial infection.Transport and quarantine inspection• Low volume & ease of observation, preferred form of

cultivation by DAWR.Germplasm Storage• Ease of manipulation & miniaturised plant organs

convenient for preservation of live genetic resources. Genetic improvement• Biotech approaches to genetic modification and the

creation of novel variation. Micropropagation• Rapid multiplication of clean, clonal stock.

© PharmOut 2019

Advantages of micropropagation?

• Rapid and efficient plant multiplication at scale.• Genetic and phenotypic uniformity (clonal multiplication).• Production of high health plants, TC process is sterile & disease free. • Pathogen testing for certification is readily applied in vitro.

• Note: Recent phytoplasma infection in Cannabis report in USA.• Reduced space requirements to store hundreds of varieties.• Reduced-risk maintenance of high value stock genotypes.

© PharmOut 2019

Disadvantages of micropropagation?

• Set-up costs for specialised laboratory and equipment.• Skilled labour (aseptic technique).• Cannabis may be difficult to establish in vitro and may require considerable

time and effort to optimise growth conditions.• Losses due to infection; potential for off-types/spontaneous mutations.

© PharmOut 2019

Micropropagation: critical factors

1. Facilities: laboratory, tissue culture growth room, nursery. 2. Mother plants: a source of healthy tissue to bring in vitro.3. Workflow and sterile media formulation: optimisation, per variety.4. Skilled staff: majority operational costs.

© PharmOut 2019

1. Micropropagation: facility requirements

• Media prep room: autoclave, accurate balance, pH meter, dispenser, 4 °C storage.

• Transfer room (clean room): laminar flow hoods, instruments and sterilisers.• Growth room: temperature & humidity control, horticultural lighting. • Nursery: acclimatisation and transplantation of rooted plantlets.

© PharmOut 2019

2. Micropropagation: mother stock

• A source of optimal health, elite phenotype explants (cuttings) is required. • Not all genotypes of a species are amenable to in vitro propagation. • Explants are nodal cuttings with axillary buds (meristem culture).• After initial establishment, mother plants can be maintained in vitro. • Establish & maintain a genotype library of high-health stock plants efficiently.

© PharmOut 2019

3. Micropropagation: workflows & media

1. Initiation (day 0)• Nodal cutting surface sterilisation• node isolated & embedded

2. Shoot multiplication (day 25)• aseptic sub-culture of shoots• shooting is plant hormone enhanced: cytokinin

3. Rooting of shoots (day 50)• rooting is plant hormone enhanced: auxin

4. Acclimatisation/de-flask (day 75)• plant nursery, humidity gradually reduced• transplant into soil, rockwool, coco coir…

© PharmOut 2019


1. Initiation

2. Shoot multiplication

3. Rooting of shoots

4. Acclimatisation

© PharmOut 2019


Gelling agent Macro & micro nutrients Carbon sourceHormones: cytokinins

auxinsProvides physical support

Synthetic nutrients N, P, K…

Sucrose replaces light

Steer organogenesis

Media formulation, key ingredients

Additional considerations & ingredients: pH, osmotic potential, charcoal, vitamins, other organics…

© PharmOut 2019


• Shoot & root proliferation is induced by specific media formulations.

• Hormones (plant growth regulators; PGRs) at the appropriate concentration promote organogenesis:• Cytokinins: shoot induction/elongation • Auxins: root induction

• Transfer between shooting & rooting media is required.

• Too much PGR can have unexpected effects.Auxins

Cyto

kini

ns

Shoot promotion

Root promotion

callus

© PharmOut 2019


(A) Mother (stock) plant. (B) Shoot formation. (C-E) Rooting. (F-G) Established plants in nursery. (H) Field production.

Reference: Lata et al. (2016) J Applied Research on Medicinal and Aromatic Plants 3: 18-26

© PharmOut 2019


Vegetative bud

• Micropropagation output is dependant on the:• sub-culture interval, approximately 25 days.• multiplication rate (MR).

• MR dependant on nodes per shoot and vegetative buds per node.

© PharmOut 2019

3. Micropropagation: workflows & media• MR dependant on nodes per shoot and vegetative buds per node.

1 shoot

© PharmOut 2019


Day Sub MR=4 MR=5 MR=8 MR=10Day 0 1 1 1 1 1Day 25 2 4 5 8 10Day 50 3 16 25 64 100Day 75 4 64 125 512 1,000Day 100 5 256 625 4,096 10,000Day 125 6 1,024 3,125 32,768 100,000Day 150 7 4,096 15,625 262,144 1,000,000Day 175 8 16,384 78,125 2,097,152 10,000,000Day 200 9 65,536 390,625 16,777,216 100,000,000Day 225 10 262,144 1,953,125 134,217,728 1,000,000,000Day 250 11 1,048,576 9,765,625 1,073,741,824 10,000,000,000Day 275 12 4,194,304 48,828,125 8,589,934,592 100,000,000,000Day 300 13 16,777,216 244,140,625 68,719,476,736 1,000,000,000,000Day 325 14 67,108,864 1,220,703,125 549,755,813,888 10,000,000,000,000Day 350 15 268,435,456 6,103,515,625 4,398,046,511,104 100,000,000,000,000

• Very rapid multiplication possible but not realistic or necessary! • Competent technician could manage 1500-2000 cuttings per day.

• How productive can micropropagation be?

© PharmOut 2019

3. Micropropagation: workflows & media• How much space is required to store in vitro stock plants?• Assume we can hold 10 shoots in a 700 mL tub…

Metric MR=4 MR=5 MR=8 MR=10# shoots stored 250 200 125 100# shoots derived 1,000 1,000 1,000 1,000SQM required 0.360 0.288 0.180 0.144

For comparison…• 1,000 cuttings per month from vegetative

propagation requires minimum of 2.5-5 SQM and a lot more inputs!

© PharmOut 2019

Summary

Micropropagation is:• well established in horticulture.• requires optimisation for Cannabis.• a specialised activity with specialised.

facility requirements & applications.• requires investment.• suits large scale facilities.

Slide Number 1My back groundSummaryIntroduction to plant tissue cultureHistory of plant tissue cultureApplications of plant tissue cultureAdvantages of micropropagation? Disadvantages of micropropagation? Micropropagation: critical factors1. Micropropagation: facility requirements2. Micropropagation: mother stock3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & media3. Micropropagation: workflows & mediaSummary

2019 Medicinal Cannabis Conference · 2019-04-02 · • In vitro propagation of plant parts, such...

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