Methods of regenerating and transforming cannabis

Abstract

Methods of in vitro clonal propagation, regeneration and transformation in Cannabis are provided. Also provided is the use of such methods in improvements of cannabis cultivars such as via breeding.

1 . A method of in-vitro propagating  cannabis , the method comprising:
 (a) culturing a  cannabis  plant part comprising a meristem on a solid culture medium so as to obtain an explant; and subsequently   (b) subjecting said explant to a cell wall disrupting agent;   (c) culturing said explant in a liquid medium; and optionally subsequently wherein steps (a)-(c) are effected for 1 to n times until emergence of leaves suitable for regeneration.   
     
     
         2 . The method of  claim 1 , further comprising sterilizing said explant prior to (a). 
     
     
         3 . The method of  claim 1 , wherein said liquid medium comprises said cell wall disrupting agent. 
     
     
         4 . The method of  claim 1 , wherein said step (c) is performed while shaking. 
     
     
         5 . The method of  claim 1 , wherein step (a) is performed for 7-30 days. 
     
     
         6 . The method of  claim 1 , wherein step (c) is performed for 5-30 days. 
     
     
         7 . The method of  claim 1 , wherein said meristem is an apical meristem or an axillary meristem. 
     
     
         8 . The method of  claim 1 , wherein said explant comprising said meristem is of a stem. 
     
     
         9 . The method of  claim 1 , wherein said explant is from a seedling. 
     
     
         10 . The method of  claim 1 , wherein said explant is from a mature plant. 
     
     
         11 . The method of  claim 1 , further comprising removing leaves and necrotic regions from said explant between steps (a) to (c). 
     
     
         12 . The method of  claim 1 , wherein said cell wall disrupting agent is selected from the group consisting of a chemical, an enzyme and a physical treatment. 
     
     
         13 . The method of  claim 1 , wherein said enzyme comprises a plurality of enzymes. 
     
     
         14 . The method of  claim 12 , wherein said enzyme is provided at a sub-lethal concentration. 
     
     
         15 . The method of  claim 1 , wherein said enzyme is selected from the group consisting of pectinase, cutinase and a combination thereof. 
     
     
         16 . The method of  claim 1 , wherein in said steps (a) said solid medium is devoid of said cell wall disrupting agent. 
     
     
         17 . The method of  claim 1 , wherein said emergence of leaves suitable for regeneration is manifested by rooting and acclimatization. 
     
     
         18 . (canceled) 
     
     
         19 . A method of  cannabis  regeneration in a tissue culture, the method comprising culturing regenerable  cannabis  explant in-vitro in a solid medium comprising at least one regeneration agent and a cell wall disrupting agent so as to regenerate  cannabis.    
     
     
         20 . The method of  claim 19 , wherein said regenerable  cannabis  explant is obtained by a method of in-vitro propagating  cannabis , the method comprising:
 (a) culturing a  cannabis  plant part comprising a meristem on a solid culture medium so as to obtain an explant; and subsequently   (b) subjecting said explant to a cell wall disrupting agent;   (c) culturing said explant in a liquid medium; and optionally subsequently wherein steps (a)-(c) are effected for 1 to n times until emergence of leaves suitable for regeneration.   
     
     
         21 - 63 . (canceled)