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Abstract Tissue culture and radicle excision techniques were employed to evaluate salt tolerance in alfalfa Medicago sativa L. Plant suspension cultures of either seedling root or shoot origin were studied in media with or without supplemental NaCl 3.
To optimize shoot multiplication in vitro, a control medium medium A and four treatment media medium B, C, D, and E , with varying inorganic nutrients and vitamins were tested. The four treatment media differed from the control medium as follows: medium B, 2. Medium C and medium E produced more microcuttings than the control at 6 weeks after the initial subculture with shoot multiplication media and all other treatments at 9 and 12 weeks. Shoots grown on these two media displayed optimal extension and leaf lamina development; however, they exhibited slight chlorosis by 12 weeks after subculture with shoot multiplication media.
Retipping can produce nine-times as many plants in a similar amount of floor space as stem cuttings derived from traditional stock mother plants.
There is increased interest in the production of hemp Cannabis sativa because of its medicinal properties Small, For commercial production purposes, hemp is propagated by seed or stem cuttings to take advantage of superior genotypes Cervantes, Many indoor hemp production facilities propagate cultivars by taking stem cuttings from stock mother plants, which they must maintain Bechtel, Mother plants are large gal container size and require a significant amount of grow space to provide enough cuttings to meet production quotas.
Growers must maintain mother plants in triplicate, with each replicate grown in a separate area of the facility, to reduce the risk of losing valuable cultivars to sudden disease outbreaks. Mother plants lose vigor because of the serial removal of shoots for cuttings, and they must be replaced every 6 months.
Additionally, over time, mother plants accumulate insects and diseases, thus limiting their useful life as donors of cuttings. Overall, this propagation process is labor-intensive and inefficient. Hemp growers are interested in micropropagation as an alternative method of generating clones for commercial production Rosslee, Micropropagation provides unique benefits to growers and has several advantages over traditional plant cloning systems.
These include the production of a large number of genetically clonal plants, uniform plants with enhanced vigor, disease-free plants, and preservation of maternal germ lines Hartmann et al. Micropropagation also requires substantially fewer mother plants to be maintained compared with traditional stem cutting propagation, and in vitro cultures can be stored for longer in a smaller area than mother plants.
There are few published reports of hemp micropropagation. Wang et al. Using nodal stem segments and MS medium, Lata et al. Lata et al. Unfortunately, these published protocols have not translated well to large-scale micropropagation of clones necessary for commercial production. Noted shortcomings of published micropropagation methods include development of hyperhydricity during establishment of shoots in vitro, lack of consistent shoot elongation in culture, and inability of shoot cultures to maintain quality growth for an extended period of time Monthony et al.
Microshoots from in vitro cultures are miniaturized, have altered physiology, and root easily Hartmann et al. Nursery producers use a process called retipping to take advantage of and extend the period of time that micropropagated plants retain this miniaturized physiology so that more cuttings can be rooted Keith and Brand, Retipping is the repeated harvesting of new shoots from recently micropropagated plants. The retipping process stimulates shoot growth from latent buds originating from the region of the stem that was miniaturized in culture.
Retipping is routinely used to substantially increase the yield of propagules for crops such as rhododendron Rhododendron sp. The objective of this work was to enhance hemp micropropagation by reducing hyperhydricity, improving in vitro shoot extension and performance through adjustment of the media nutrient content, and developing a method of ex vitro rooting.
An additional objective was to evaluate retipping of recently micropropagated plants as a method of obtaining large quantities of clones for commercial-scale hemp production. To initiate shoots in vitro, 4-cm-long stem tips were collected from stock plants. After leaves were removed, stems were disinfected by immersion in a solution of 0. Then, stems were aseptically trimmed to remove damaged tissue and cultured in boxes GA-7; Magenta, Lockport, IL with vented lids featuring a vent with a diameter of 10 mm and pore size of 0.
Louis, MO at pH 5. After 3 weeks, sterile shoots were subcultured with the same medium and maintained for another 3 weeks, at which time they were subcultured with shoot multiplication medium.
Various Murashige and Skoog MS -based media formulations for initiation and shoot multiplication of hemp. Two studies were conducted to optimize the nutrient composition of MS medium for in vitro shoot growth and performance.
For these studies, in vitro shoots were initiated as described. The first shoot multiplication study evaluated the mesos components of MS, which included the calcium chloride anhydrous , magnesium sulfate anhydrous , and potassium phosphate monobasic nutrients. In addition to control medium A, there were four other treatment media formulations provided in Table 1 : medium B, 2. For both studies, the experimental unit was a box.
At each subculture, four microcuttings were placed per box. There were 10 and 8 boxes per treatment for the mesos and NH 4 NO 3 studies, respectively.
For each study, experimental units were arranged in a completely random design. Cultures were subcultured every 3 weeks and maintained as described for in vitro shoot initiation. At each subculture, shoot extension and leaf lamina development were visually assessed by the lead author.
Data were collected at 6, 9, and 12 weeks after shoots were initially subcultured with shoot multiplication media. For prerooting, 8 to 10 microshoots were placed per box and cultures were maintained as described for in vitro shoot initiation. Microcuttings rooted at 21 d and initiated new shoot growth. Rooted microcuttings were acclimated to greenhouse conditions by gradually decreasing atmospheric relative humidity around the microcuttings and gradually increasing light levels.
This was accomplished by melting 30 holes diameter, 0. After 4 d with 60 holes in the domes, rooted microcuttings were potted in mL containers filled with a peatmoss-based potting mix Promix BK25; Premier Tech Horticulture, Quakertown, PA.
Plants were top-dressed with 15N—3. Plants received a 20N—8. For each of the three experimental time replications of retipping, 10 recently potted micropropagated plants were used. Vacuum infiltration. The vac- uum pump was turned on to decrease the pressure, and the agroinfiltration time was calculated after the vacuum reached 80 mbar.
Sonication was carried out using a Analysis of transient GUS expression. The relative GUS expression was calculated with 30 pollen sacs per treatment to obtain the total expression value stained area x color intensity with ImageJ Rasband — as previously reported Photos of the strongest GUS-stained regions in the pollen sacs were taken using bright field illumination.
The intensity of the GUS staining was measured in the saturation chan- nel. The mean gray values were measured and compared among different treatments.
All analyzed tissues were thoroughly cleared to correlate the color information with the degree of blue staining. In addition, the number of stained pollen sacs was counted from 10 pollen sacs to calculate the percentage of GUS staining. This calculation included three repetitions. Image collection and analysis of GFP fluorescence. The F-box gene was used as an internal reference for hemp, and three independent biological replications were conducted.
All primer sequences are listed in Supplemental Table S1. Andre, C. Cannabis sativa: The plant of the thousand and one molecules. Frontiers in Plant Science. Chandra, S. Cannabis Sativa L. Switzerland: Springer. Izzo, A. Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends Pharmacological Science. Gagne, S. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.
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Journal of Visualized Experiments. King, J. Development and optimization of agroinfiltration for soybean. Plant Cell Reports. Wahby, I. Agrobacterium infection of hemp Cannabis sativa L. Journal of Plant Interactions. Farag, S. Cannabinoids production by hairy root cultures of Cannabis sativa L. American Journal of Plant Sciences. Jefferson, R. On the other hand, many species are rare and endangered, and the need for conserving them from extinction is urgent.
However, details of their propagation in general is hampered by the slow rate of vegetative multiplication. Hay, as a beginning to more detailed studies on the propagation of Aroid in general. Protoplasts were successfully isolated. A successful protocol for the rapid micropropagation of both Aroid species was developed, which combined callus induction and a high rate of shoot multiplication, followed by root development.
Plantlets could then be directly transferred to green house acclimatization conditions, with high survival rates.
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