Abstract
Hemp ( Cannabis sativa L.) is commonly grown for the medicinal secondary metabolites produced by pistillate inflorescences. Micropropagation is a valuable method of propagating hemp plants because of the aseptic process and the production of true-to-type propagules. The hemp cultivar TJ’s CBD was used for a series of experiments to compare media inputs and practices for the clonal micropropagation of hemp. For stage I, shoot tips harvested from stock plants that were grown in a growth chamber produced less endogenous contamination in newly established cultures than shoot tips harvested from the greenhouse. In addition, stage I disinfection treatments with 20%, 40%, and 60% bleach (7.5% sodium hypochlorite) for 10 minutes had no differences in surface contamination rates. All concentrations were able to clean explants equally, and no damage to the explants was observed. For stage II, there were no differences in growth and multiplication rate between shoot tip or nodal explants. In addition, no differences were observed between the gelling agent’s agar, agargellan, and gellan gum at standard rates. When basal nutrient formulations were compared at standard rates and with their respective vitamins, Murashige and Skoog, Linsmaier & Skoog, and Driver & Kuniyuki Walnut media were found to be superior to Lloyd & McCown Woody Plant Medium. Media pH levels of 4.0, 5.0, 5.8, 6.0, and 7.0 were compared, and no differences were observed in final fresh weights, shoot lengths, or quality ratings. The pH levels of 5.8, 6.0, and 7.0 generated a greater number of lateral nodes. Sucrose levels of 0%, 1.5%, 3.0%, 4.5%, and 6.0% (wt/vol) were also compared, with the 1.5% and 3.0% rates showing greater fresh weights, shoot lengths, and quality ratings. Growth room temperatures of 22, 24, 26, and 28 °C were compared, with temperatures of 28 and 26 °C generating greater fresh weights, shoot lengths, numbers of nodes, and quality ratings compared with cooler temperatures. The cytokinins 6-enzylaminopurine (BA), 6-(γ,γ-dimethylallylamino) purine (2iP), and thidiazuron (TDZ) were compared at 1.0, 5.0, and 10.0 μM concentrations. The 5.0-μM TDZ treatment generated greater fresh weights and numbers of lateral nodes; however, it also produced the shortest shoot lengths and lowest quality ratings. The 2iP treatments at 1.0 and 5.0 μM, and the BA treatment at 1.0 μM produced the greatest quality ratings. The 5.0-μM 2iP level was considered the best treatment for stage II multiplication based on high ratings, in addition to the greater final fresh weights, shoot lengths, and numbers of nodes that were produced. For stage III experiments, the auxins indole 3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) were compared at concentrations of 0.25, 0.5, and 2.5 μM. Auxin treatments of 0.25 μM NAA, 0.5 μM NAA, and 2.5 μM IBA generated the greatest final shoot fresh weights, root fresh weights, and numbers of nodes. However, the 2.5-μM IBA treatment resulted in a higher overall rating. For stage IV, ex vitro rooting and acclimation trials compared a dome and an intermittent mist system, as well as treated the unrooted cuttings with an externally applied auxin. Acclimating with a dome produced greater shoot heights, fresh shoot weights, and overall ratings compared with the mist system. The auxin treatment mildly increased fresh root weight, but was not as important to acclimation success as the domed environment. It has been concluded that a micropropagation system that uses lower rates of sucrose, higher growing temperatures, and lower rates of the cytokinins BA and 2iP are optimal for the micropropagation of hemp. In addition, when acclimating hemp plants from tissue culture, an in vitro stage III can be bypassed and plants can be rooted ex vitro during stage IV acclimation with a dome with or without additional auxin treatments.
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Claims 20
Claim · model-written summary
Growth chamber stock plants yielded shoot tips with less endogenous contamination than greenhouse stock plants.
- Taxon
- Hemp
Cannabis sativa subsp. sativat0 - Organ / stage
- shoot tipse0
- Treatment
- grown in a growth chambere0
- Outcome
- endogenous contaminatione0
- Direction
- lesse0
- Comparator
- greenhousee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“shoot tips harvested from stock plants that were grown in a growth chamber produced less endogenous contamination in newly established cultures than shoot tips harvested from the greenhouse”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Bleach concentrations of 20%, 40%, and 60% produced no differences in surface contamination rates.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 20%, 40%, and 60% bleach (7.5% sodium hypochlorite) for 10 minutese0
- Outcome
- surface contamination ratese0
- Direction
- no differencese0
- Comparator
- 20%, 40%, and 60% bleache0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“stage I disinfection treatments with 20%, 40%, and 60% bleach (7.5% sodium hypochlorite) for 10 minutes had no differences in surface contamination rates”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
No explant damage was observed at any tested bleach concentration.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- All concentrationse0
- Outcome
- no damage to the explantse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“All concentrations were able to clean explants equally, and no damage to the explants was observed”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Shoot tip and nodal explants had no differences in growth and multiplication rate.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- shoot tipe0
- Outcome
- growth and multiplication ratee0
- Direction
- no differencese0
- Comparator
- nodal explantse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“there were no differences in growth and multiplication rate between shoot tip or nodal explants”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Agar, agargellan, and gellan gum did not differ at standard rates.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- agare0
- Outcome
- no differencese0
- Direction
- no differencese0
- Comparator
- agargellan, and gellan gume0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“no differences were observed between the gelling agent’s agar, agargellan, and gellan gum at standard rates”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Murashige and Skoog basal medium was superior to Lloyd and McCown medium.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- Murashige and Skooge0
- Outcome
- superiore0
- Direction
- superiore0
- Comparator
- Lloyd & McCown Woody Plant Mediume0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Murashige and Skoog, Linsmaier & Skoog, and Driver & Kuniyuki Walnut media were found to be superior to Lloyd & McCown Woody Plant Medium”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Linsmaier and Skoog basal medium was superior to Lloyd and McCown medium.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- Linsmaier & Skooge0
- Outcome
- superiore0
- Direction
- superiore0
- Comparator
- Lloyd & McCown Woody Plant Mediume0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Murashige and Skoog, Linsmaier & Skoog, and Driver & Kuniyuki Walnut media were found to be superior to Lloyd & McCown Woody Plant Medium”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Driver and Kuniyuki Walnut medium was superior to Lloyd and McCown medium.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- Driver & Kuniyuki Walnut mediae0
- Outcome
- superiore0
- Direction
- superiore0
- Comparator
- Lloyd & McCown Woody Plant Mediume0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Murashige and Skoog, Linsmaier & Skoog, and Driver & Kuniyuki Walnut media were found to be superior to Lloyd & McCown Woody Plant Medium”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Tested medium pH levels did not differ in final fresh weight, shoot length, or quality ratings.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- Media pH levels of 4.0, 5.0, 5.8, 6.0, and 7.0e0
- Outcome
- final fresh weights, shoot lengths, or quality ratingse0
- Direction
- no differencese0
- Comparator
- 4.0, 5.0, 5.8, 6.0, and 7.0e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Media pH levels of 4.0, 5.0, 5.8, 6.0, and 7.0 were compared, and no differences were observed in final fresh weights, shoot lengths, or quality ratings”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Medium pH 5.8, 6.0, and 7.0 produced more lateral nodes.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- pH levels of 5.8, 6.0, and 7.0e0
- Outcome
- number of lateral nodese0
- Direction
- greatere0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The pH levels of 5.8, 6.0, and 7.0 generated a greater number of lateral nodes”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Sucrose at 1.5% and 3.0% produced greater fresh weights, shoot lengths, and quality ratings.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 1.5% and 3.0% ratese0
- Outcome
- fresh weights, shoot lengths, and quality ratingse0
- Direction
- greatere0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“1.5% and 3.0% rates showing greater fresh weights, shoot lengths, and quality ratings”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Temperatures of 28 and 26 °C produced greater fresh weights, shoot lengths, node numbers, and quality ratings than cooler temperatures.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- temperatures of 28 and 26 °Ce0
- Outcome
- fresh weights, shoot lengths, numbers of nodes, and quality ratingse0
- Direction
- greatere0
- Comparator
- cooler temperaturese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“temperatures of 28 and 26 °C generating greater fresh weights, shoot lengths, numbers of nodes, and quality ratings compared with cooler temperatures”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
The 5.0-μM TDZ treatment produced greater fresh weight and lateral node number.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 5.0-μM TDZ treatmente0
- Outcome
- fresh weights and numbers of lateral nodese0
- Direction
- greatere0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The 5.0-μM TDZ treatment generated greater fresh weights and numbers of lateral nodes”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
The 5.0-μM TDZ treatment produced the shortest shoot lengths and lowest quality ratings.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 5.0-μM TDZ treatmente0
- Outcome
- shoot lengths and lowest quality ratingse0
- Direction
- shorteste0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The 5.0-μM TDZ treatment generated greater fresh weights and numbers of lateral nodes; however, it also produced the shortest shoot lengths and lowest quality ratings”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
2iP at 1.0 and 5.0 μM gave the greatest quality ratings.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 2iP treatments at 1.0 and 5.0 μMe0
- Outcome
- quality ratingse0
- Direction
- greateste0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The 2iP treatments at 1.0 and 5.0 μM, and the BA treatment at 1.0 μM produced the greatest quality ratings”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
BA at 1.0 μM gave the greatest quality ratings.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- BA treatment at 1.0 μMe0
- Outcome
- quality ratingse0
- Direction
- greateste0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The 2iP treatments at 1.0 and 5.0 μM, and the BA treatment at 1.0 μM produced the greatest quality ratings”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
The 5.0-μM 2iP level was judged the best stage II multiplication treatment because of high ratings, fresh weight, shoot length, and node number.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 5.0-μM 2iP levele0
- Outcome
- best treatment for stage II multiplicatione0
- Direction
- beste0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The 5.0-μM 2iP level was considered the best treatment for stage II multiplication based on high ratings, in addition to the greater final fresh weights, shoot lengths, and numbers of nodes that were produced”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
The three named auxin treatments gave the greatest shoot and root fresh weights and node numbers.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 0.25 μM NAA, 0.5 μM NAA, and 2.5 μM IBAe0
- Outcome
- final shoot fresh weights, root fresh weights, and numbers of nodese0
- Direction
- greateste0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Auxin treatments of 0.25 μM NAA, 0.5 μM NAA, and 2.5 μM IBA generated the greatest final shoot fresh weights, root fresh weights, and numbers of nodes”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
The 2.5-μM IBA treatment had a higher overall rating.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- 2.5-μM IBA treatmente0
- Outcome
- overall ratinge0
- Direction
- highere0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the 2.5-μM IBA treatment resulted in a higher overall rating”
t0“Hemp (Cannabis sativa L.)”
Claim · model-written summary
Dome acclimation produced greater shoot height, shoot weight, and overall ratings than mist acclimation.
- Taxon
- HempCannabis sativa subsp. sativat0
- Treatment
- Acclimating with a domee0
- Outcome
- shoot heights, fresh shoot weights, and overall ratingse0
- Direction
- greatere0
- Comparator
- mist systeme0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Acclimating with a dome produced greater shoot heights, fresh shoot weights, and overall ratings compared with the mist system”
t0“Hemp (Cannabis sativa L.)”
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