Date of Award
12-2024
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Plant and Environmental Science
Committee Chair/Advisor
Jeffrey Adelberg
Committee Member
Jim Faust
Committee Member
Raghupathy Karthikeyan
Abstract
Micropropagation is a technique used to propagate clean, high-quality clonal stock plants using plant growth regulators (PGR) in a single harvest batch system. This two-part study uses an in vitro multiple harvest system “hedging” combined with the fed- batch media process, to observe (1) the effects of modified physical state and (2) LED light quality to improve shoot production and quality. In Experiment 1, four genotypes of Cannabis sativa (‘Cherry 1’,‘BaOx’,‘T1’,‘Peach’) were observed in three different physical states: stationary agar (A), stationary Oasis® infused with liquid (OILs) and agitated Oasis® infused with liquid (OILa). Fifteen explants (nodes and shoot tips) were planted in treatment physical states consisting of 120 mL DKW medium (liquid or agar) without cytokinin (liquid or agar) and later harvested on repeated 3-week cycles where 15 mL media was supplemented as a treatment factor. The harvested number of shoots, shoot length, and dry shoot mass was recorded from repeated cutting cycles. Some genotypes (‘T1’ and ‘Peach’) did not produce shoots on a multiple harvest cutting schedule, although during cycle one harvest showed that OIL treatments produced more shoots with greater length. ‘T1’ and ‘Peach’ were eliminated from the experiment after one cycle. Over multiple cycles, the remaining genotypes (‘BaOx’ and ‘Cherry 1’) showed that both types of OIL treatments with media supplements grew better than A treatments, regardless of if A received any supplement or not. Shoots harvested increased from initial 15 explants per vessel during cycle 1 to a maximum of 30 shoots in the third cycle, in both OIL media but in A there were approximately 20 shoots per vessel. Shoot length was also highest in both OIL treatments for both genotypes, but OILa were larger during cycle 1, especially for the genotype ‘Cherry 1’. By the third cycle, shoots harvested, shoot length, and dry mass decreased. By cycle 5, the only vessels that produced above 7 shoots per vessel were those in OILa treatments, but internodes were too short (less than 10 mm) to plant ex vitro. In Experiment 2, light quality was observed to affect in vitro shoot growth with Cannabis sativa (‘BaOx and ‘Cherry 1’) by comparing growth in polychromatic white and dichromatic red:blue with and without supplemental far-red light (white, high red:blue and medium red:blue, white with 5% far-red, high red:blue 5% far-red, medium red:blue with 5% far-red, and low red:blue with 5% far-red). Fifteen explants (nodal and apical) were cultured in RV750 vessels containing OILs with 120 mL DKW medium with similar PPFD (190-240 µmol m-2 s-1) for a 16 hour photoperiod. Vessels also received 15 mL liquid media supplements biweekly during the time of harvest. Shoot number, shoot length, leaf number, shoot fresh and dry mass were recorded during each two-week harvest period. Five randomly selected shoot tips per vessel were rooted ex vitro on the greenhouse mist bench for 16 days. Over multiple repeated cycle harvests, 5% far-red increased shoot number and length with any polychromatic or dichromatic light source, while higher blue light percentages increased dry shoot mass. Shoot numbers increased in both genotypes from initial 15 at cycle 1 to a maximum 28 in cycle 3 with far-red, but only 18 without far-red. By the end of cycle five, both far-red and no far-red treatments had decreased to approximately 18 shoots per vessel. The accumulated number of shoots over 5 cycles (10-week period) was 108 shoots with far-red, and 84 without. Shoot length in far-red treated plants increased from 19 mm during cycle 1 to 25 mm in cycle 3. Plants without far-red light were only 15 mm length in cycles 1-3. By cycle 5, shoot length in both far-red and non-far-red treated plants were reduced to 10 mm. Shoot dry mass during cycle 1, was greatest at 7 mg for ‘BaOx’ and 6mg for ‘Cherry 1’ for plants with the highest percentage of blue light before decreasing 50% in cycle 3 where it remained around 4 mg for the duration of 5 cycles. Sixty eight percent of shoots rooted on the mist bench, regardless of prior in vitro treatment. OIL with media supplementation allowed shoots to be successfully harvested on a repeated schedule for five cycles, and the signaling response of far-red light enhanced in vitro productivity and length.
Recommended Citation
McKay, Molly, "Multiple Cut Systems of Cannabis Sativa L. For Micropropagation Without the Use of Cytokinins" (2024). All Theses. 4422.
https://open.clemson.edu/all_theses/4422
