BREEDING AND AGRONOMICAL TECHNIQUES TO PRODUCE MEDICAL CANNABIS

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Claire Ashlie Marshall
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1 Universität Innsbruck Institut für Pharmazie/Pharmakognosie BREEDING AND AGRONOMICAL TECHNIQUES TO PRODUCE MEDICAL CANNABIS Gianpaolo Grassi CREA-CI, Rovigo s branch station 15 th of November 2018 (Vienna) gianpaolo.grassi@crea.gov.it

2 Italian pioneers of Cannabis sativa L. breeding Dr. Domenico Allavena (Bologna ) Obtained the varieties: - Fibranova and - Carmagnola Selezionata (C.S.) On the left an original picture made by Allavena of the Pinnatofidofilla

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4 Italian Regions (12/20) that have approved the law that support the cost of the drugs with natural or synthetic cannabinoids. Puglia, Tuscany Liguria, Veneto, Friuli Venezia Giulia Abruzzo Umbria Marche Sicily Emilia Romagna Trento and Bozen Aut. Reg. Piedmont

5 Medicinal Cannabis origin Cannabis sativa L.: Dioecious plant Female flower 5

6 Cannabis accessions collected in Italy (more than 300 strains)

7 Cannabis germplasm collection in Italy Ind. Hemp Landraces Drug type 7

8 Some example of genotypes with interesting characters 8

9 The only one area in open field admitted in Italy to grow Cannabis with high THC concentration ( ) about 2,000 sqm

10 Cannabis genotypes with high THC grown in 2016

11 Breeding method is based on reversion of sex with Ag salt Female side Male side Single female plant sex reverted

12 Natural photoperiod Controlled photoperiod 12

13 Results achieved by the most advanced breeding by the leading pharmaceutical company in the Cannabis sector In: Handbook of Cannabis: Edited by R. Pertwee, (2016),784 pp. 13

Research programs running at the CREA- CI 1. Cannabis without cannabinoids 2. Cannabis with chemotype CBG prevalent 3. Cannabis with chemotype CBD prevalent 4. Cannabis with chemotype THC prevalent 5.

14 Research programs running at the CREA- CI 1. Cannabis without cannabinoids 2. Cannabis with chemotype CBG prevalent 3. Cannabis with chemotype CBD prevalent 4. Cannabis with chemotype THC prevalent 5. Cannabis with chemotype THCV prevalent 6. Cannabis with chemotype CBDV prevalent 7. Cannabis with high content of GLA in seed 8. Cannabis with selected terpenes 9. Cannabis with high yield for indoor and outdoor condition.

15 Gas-chromatogram of Ermo s extract

16 The analysis of Ermo variety could be done using simple test A colorimetric test allows to identify contamination 16

17 Chemotype of Cannabis strain with THC prevalent THC * * = Internal Standard 0.01% conc.

18 Variability of THC content we have available in our accessions

19 Mother plants of CINBOL variety registred in the CPVO and used as medicinal cannabis by the Italian state Cuttings in rooting phase

20 Chemotype of Cannabis strain with CBD prevalent CBD

21 Production of CBD in varieties of Italian cannabis grown in pots *** Uso31 and Santhica, THC = 0 21

22 Chemotype of Cannabis strain with CBG prevalent CBG

23 Chemiotipo di Cannabis con CBDV/CBD prevalente CBDV CBD Ref. CBDV THCV Int. Stand. CBD THC

24 Chemotype of Cannabis strain with THC/THCV prevalent

25 How to grow Cannabis? Vertical farming SCROG Indoor, hydroponic 25

THE EFFECT OF LIGHT SPECTRUM ON THE MORPHOLOGY AND CANNABINOID CONTENT Light Treatments Valoya NS1 had the highest portion of blue light, including some UVA light Valoya AP673L

26 THE EFFECT OF LIGHT SPECTRUM ON THE MORPHOLOGY AND CANNABINOID CONTENT Light Treatments Valoya NS1 had the highest portion of blue light, including some UVA light Valoya AP673L spectrum had the most red light, spectrum designed for high vegetative growth HPS spectrum concentrated in the yellow-orange area of the visible spectrum. Very low blue light content

THE EFFECT OF LIGHT SPECTRUM ON YIELD OF CANNABINOIDS No significant differences found in the total yield of cannabinoids between the light treatments or between the two experiments When comparing

27 THE EFFECT OF LIGHT SPECTRUM ON YIELD OF CANNABINOIDS No significant differences found in the total yield of cannabinoids between the light treatments or between the two experiments When comparing the yield of cannabinoids per Watts of electricity used, LED treatments had the best results in both trial rounds and HPS the lowest values Bar graphs (mean and standard deviation) of yield of cannabinoids per Watts (left) and yield of cannabinoids (right). Solid lines and lower case letters are for trial 1 (blue), and dashed line and upper case letters are for trial 2 (green). Different letters show significant differences (Tukey's HSD, p<0.05). Significant differences between trials are represented by asterisks (* p<0.05, ** p<0.01, *** p<0.001)

29 Color change of

31 Cannabinoid concentration in the flowers during the plant cycle Total Cannabinoids. Top Middle. Basal

32 Flower yield (dry weight) Variety Variety G-170 G-301

33 Accumulation of cannabinoids in Cannabis flowers

$fraction$ obtained

34 Mature plant Principal fraction obtained Leaves could be extracted with CO 2 SCF 34

35 Localization of the three fields where hemp with high CBD content has been grown MEDICINAL CANNABIS IN OPEN FIELD 35

36 Temp. C Environment condition 35,0 Mean temperatures 30,0 25,0 20,0 15,0 Lecce Bari Foggia 10,0 5,0 0,0 Feb Mar Apr May Jun Jul Aug Sep Month 36

37 CBD (% dw) 10,0 CBD concentration in the 3 fields 9,0 8,0 7,0 6,0 5,0 4,0 Lecce Foggia Bari 3,0 2,0 1,0 0, Plant n 37

38 Concen. (% dw) Comparison between CBD and THC content CBD 7,00 Cannabinoid variation THC 6,00 5,00 5,5% CBD 0,30 4,00 0,20 % Limit 3,00 2,00 0,10 1,00 0,00 Lecce Foggia Bari 0,0 38

39 in vitro culture to improve breeding of Cannabis Main tasks 1. Fast multiplication 2. Virus eradication 3. Long term storage Micro-cutting s Pots with cannabis plantlets Root phase Adaptation to normal condition 39 39

40 40 Polyploidy strategy Diploid Three-ploid Tetra-ploid Cell with 2n=2X=20 2n=3X=30 2n=4X=40

41 Polyploid lines of Cannabis are available Three chemotypes have been used CBD CBG THC 41

42 First crop of medicinal Cannabis produced in Italy in the April 2015 Thank you for your attention!

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