Recipient Organization
CHI BOTANIC INC.
1962 MAIN ST STE 200 # 234
SARASOTA,FL 342369519
Performing Department
(N/A)
Non Technical Summary
Aloe vera is a widely used plant found in hundreds of products. Most commercial aloe cultivation has been offshored. U.S. manufacturers of aloe containing products must import this crop but this material often has issues with seasonal variation, dilution, contamination with pesticides, and even adulteration. Additionally, aloe produces carcinogens, such as aloin, that must be removed before use through costly processing steps.We aim to solve the current issues with aloe by the development of a commercial Aloe vera plant cell culture. The liquid culture of plants is an established technology in which plants are grown as a fine suspension of small clumps or single cells. Unlike Aloe vera plants, an aloe cell culture can be domestically-produced in a safe and high quality way without pesticides or adulterants. Aloe cell culture also allows the application of techniques to isolate aloe cell lines that do not produce the carcinogen aloin. Chi Botanic has recently established an aloe cell culture.Our objectives for this Phase I proposal is to (1) optimize growth of our aloe plant cell cultures, (2) determine the quality of this plant material (polysaccharide and aloin content), and (3) develop aloe plant cell lines that do not produce the carcinogen aloin.To accomplish these objectives, we will subject our aloe cell cultures to culture optimization methods commonly used to optimize growth of microorganisms. To generate aloin free plant cell cultures, we will apply a gene editing approach to disrupt known genes involved in aloin biosynthesis. This research will establish aloe cell culture technology as an innovative and domestic way to produce products from aloe.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Goals / Objectives
Technical Objectives.The overall objective of the proposal is to generate a toxin-free aloe cell culture for the use in foods, drinks, or other products that contain aloe. While the generation of plant cell cultures is guided by standard tissue culture practices, the specific tissues, genotypes, hormone concentrations, and optimal medium are plant species specific. Our Technical Objectives are to further expand the application of plant cell culture to aloe and the production of valuable polysaccharides. The specific objectives of the program can be enumerated as follows:Optimize Aloe vera plant cell suspension culturesDetermination of aloin and polysaccharide content in Aloe vera callus and cell cultures.Demonstrate aloin-free Aloe vera cell cultures by disruption of anthraquinone biosynthesis.
Project Methods
Task 1.1: Secure appropriate Aloe vera germplasm.Commercially cultivated Aloe vera (Aloe barbadensis miller-stockton) germplasm has been secured by Chi Botanic from Stockton Aloe (Pompano Beach, FL). This elite line of aloe is cultivated commercially in Florida and the Dominican Republic. Seeds/embryos and leaf tissues are being used to establish in vitro culture of callus.Task 1.2: Establish in vitro aloe callus and shoot culture production methods.Callus is sterile, undifferentiated plant tissue cultured on solidified growth medium. The generation of callus can be induced by the addition of plant growth regulators, specifically auxins and cytokinins to plant growth medium. Loosely-organized and rapidly growing callus is commonly used to establish plant liquid cell suspensions. A variety of plant growth regulator compositions have been used to establish aloe callus for clonal propagation of aloe. Over the past year, we have evaluated 6 published media for generating callus20-23 from aloe stem cross-sections and apical meristems of juvenile plants. Task 1.3: Optimization of liquid cell suspensions from aloe callus.Growth regulator concentration is of primary importance to the maintenance of liquid plant cell cultures. Informed by the results from Task 1.2, a subset of concentrations and ratios of cytokinins and auxin will be used to empirically determine the optimum plant growth regulators for cell proliferation in liquid plant growth medium. A preliminary 4x4 experiment testing NA (0.2-5mg/L) and BA (0.2-2) concentrations showed optimal growth of suspended aloe cells in MS Medium (pH 5.7) with Gamborg vitamins, 3% sucrose, 2mg/NA and 2mg/L BA. However, with the minimal liquid culture optimization to date, aloe cell suspensions currently grow slowly and are darkly pigmented, common indicators of stress remedied through additional culture method optimization.Further optimization of aloe cell suspension culture will investigate the impact of growth regulator composition, culture conditions, and media adjuvants. First, in vitro aloe shoot explants will be subjected to a full factorial design testing growth medium containing varying concentrations of two auxins (indole-3-acetic acid, IAA; NA); 2,4D; and two cytokinins (benzyladenine BA; and kinetin) to determine the optimal combination to obtain rapidly growing cell suspensions. Other factors, which may also be adjusted to improve growth include carbon source (eg. sucrose, mannose, maltose), nitrogen source (nitrate vs. ammonium, peptone/yeast extract), pH, temperature, and light.23 Lastly, media adjuncts have been shown to promote the growth of plant cell suspension cultures and minimize browning. Media additions that have been used for this purpose include antioxidants (e.g. pycnogynol, citric acid, vitamin E) and the use of 2-aminoindane-2-phosphonic acid (AIP), a phenylalanine ammonia lyase enzyme (PAL) inhibitor.Figure 4. (A) In vitro germinated aloe seed (B) Aloe shoot culture (C) Early callus formation (D) Callus on low hormone media (E) Callus on high hormone medium (F) Early-stage aloe suspension culture (G) Late-stage aloe suspension culture.Determination of aloin and polysaccharide content in Aloe vera callus and cell cultures.Task 2.1: Develop analytical methods to measure aloin.Aloin is a bitter, yellow-brown colored anthraquinone found in aloe that is no longer generally recognized as safe.24 Aloin can be detected by three primary ways: 1) spectroscopically by measuring absorption of UV radiation at 293 nm,25 2) measuring natural fluorescence,26 and 3) by mass spectrometry.27 Aloin can be separated from other chemical constituents found in an aloe plant by reverse phase HPLC. The spectroscopic method established by Park 1998 will be the primary method followed to determine aloin content. Briefly, phenolics are extracted from freeze dried plant material using ethanol and sonication. This extract is then subjected to reverse phase HPLC on a C18 column and eluted with methanol. Aloin is detected by monitoring UV at 293 nm. Standard phenolic compounds, including aloin and aloe-emodin, will be dissolved in ethanol and run as standard solutions. When aloin content is extremely low, such as after generation of low- or no-aloin aloe cultures (Task 3) mass spectrometry will be used in addition to UV absorption to verify aloin content. Mass spectrometry will be conducted with electrospray ionization in the negative ion mode. Aloin ions (monoisotopic mass of 418.126) will be detected with the most abundant product ion observed for aloin at m/z 297, which results from the loss of [C4H8O4]−.27Task 2.2: Evaluate aloin content in Aloe vera callus.Aloe vera callus tissue will be removed from growth media and freeze dried. Freeze dried plant cell material will be disrupted via sonication and then extracted with ethanol. If callus tissue is not completely disrupted by sonication, tissue will be manually ground in a mortar and pestle. Extracts will be subjected to the analytical methods found in Task 2.1.Task 2.3: Evaluate aloin content in Aloe vera cell cultures.Aloe vera cell cultures will be dewatered via centrifugation and freeze dried. Freeze dried plant cell material will be evaluated the same as callus tissue (Task 2.2).Task 2.4: Testing of callus and cell culture for polysaccharide gel (polyacetylated mannan) and aloin content with a third party analytical lab.Freeze dried aloe samples will be sent for analysis.Demonstrate aloin-free Aloe vera cells by disruption of anthraquinone pathwayTask 3: Directed disruption of anthraquinone pathwayTask 3.1: Identify key genes in anthraquinone pathway that lead to aloin biosynthesis.Using literature references and genome data,steps in the biosynthesis of aloin and closely related anthraquinones will be investigated.Task 3.2: Validate CRISPR-Cas9 approach to disrupt target genes in anthraquinone pathway.CRISPR-Cas9 will be used to disrupt key genes in aloe plant cell cultures. Aloe vera has a diploid (2n =14) karyotype, making targeted gene editing much simpler than plants with more complex genomes. sgRNAs will be designed to disrupt the coding sequence of each gene. Synthetic gene fragments will be synthesized to validate sgRNAs. Over 20 PAM sites have been identified for each. The efficiency of Cas9 ribonuclease activity targeting synthetic PAM sites will be tested in vitro.Task 3.3: Create gene edited plant cell culture lines using Cas9 ribonucleotide proteins.The most efficient sgRNA will be produced in vitro and combined with in vitro produced Cas9 protein to form a ribonucleotide protein complex (RNP). This protein complex will be delivered into aloe plant cells via 1) particle bombardment or 2) electroporation following methods developed by Woo 2015 31. Single gene disruptions as well as multigene disruptions from a single transgenic event will be attempted. Transformed cells will be evaluated for gene editing events Mutant cell cultures will be evaluated in a fluorescent plate reader..Task 3.4: Evaluate gene edited cell line for aloin productionMutant aloe cell lines with reduced aloin fluorescence will have aloin content measured using analytical methods established in Task 2. Mutants with low- or no-aloin content will be genotyped,