Performing Department
(N/A)
Non Technical Summary
Saffron (Crocus sativus L.), known for its potential health benefits, is the most expensive spice. However, in the US, the majority of saffron sold is imported from Spain, which originally comes from Iran. This process poses risks to food safety and security, including contamination with toxic chemicals and adulteration with foreign materials. While domestic saffron production is very limited, there is an interests among small farmers and limited-resource farmers because of the relative ease of culture. Despite the need for improving agricultural traits, breeding of saffron remains unattainable due to its triploidy.The exact provenance of saffron remains a mystery. A few species of Crocus such as C. cartwrightianus Herb., and C. pallasii Goldb. were likely involved in the development of the modern saffron. However, a recent findings strongly indicated that saffron may simply be a triploid cultigen of C. cartwrightianus, thus it exhibits a low genetic diversity. Developing hybrids between these saffron relatives could lead to acquisition of breeding lines with agriculturally desirable traits such as higher yield, better climate adaptability, disease and insects resistance, better or unique flavor and aroma profiles.The objectives of the proposed studies are, 1). Creating intraspecific and interspecific hybrids among C. cartwrightianus and its related species; 2) Inducing polyploidy among Crocus species and hybrids; 3) Analyzing phytochemical and characterize aroma profiles of C. cartwrightianus hybrids; and 4) Developing a course module and workbook on saffron chemistry; and 5) Disseminating research findings and visual aids through KSU web page and social network sites.
Animal Health Component
0%
Research Effort Categories
Basic
40%
Applied
10%
Developmental
50%
Goals / Objectives
The objectives of the proposed studies are, 1). Creating intraspecific and interspecific hybrids among C. cartwrightianus and its related species; 2) Inducing polyploidy among Crocus species and hybrids; 3) Analyzing phytochemical and characterize aroma profiles of C. cartwrightianus hybrids; and 4) Developing a course module and workbook on saffron chemistry; and 5) Disseminating research findings and visual aids through KSU web page and social network sites.
Project Methods
Objective 1. Create intraspecific and interspecific hybrids among C. cartwrightianus and its related speciesPlant materials: Corms of C. cartwrightianus and related species will be acquired from the project collaborator and a few other bulb specialists, including Brent and Becky's Bulb. Species that will be used in this part of the project include C. cartwrightianus 'Marcel', C. cartwrightianus, C. mathewii, C. pallasii, and C. thomasii. These corms will be planted in ProMix® and will be kept inside a high tunnel at Kentucky State University. Osmocote® Smart-Release® Plant Food Flower & Vegetable will be placed on the surface of the pot after planting.Cross-pollination: Anthers and tepals will be removed as soon as flower buds emerge from the soil surface, and the remaining flowers will be wrapped with non-woven fabric to prevent accidental pollination while still allowing aeration. Fully mature pollen will be transferred onto the stigma surface via a cotton swab after the flower, which will be wrapped with pieces of new non-woven fabric, is unwrapped. Seeds will be left on plats until fully mature and then will be harvested.Pollen viability: The viability of pollen will be examined by staining with 1% 2,5-diphenyl tetrazolium bromide (MTT) as previously described. We will use a 1% solution of MTT with and without a 5% sucrose solution. Pollen will be incubated for 30 minutes for color development and then the number of viable ones stained red will be counted.Seed culture: Seeds will be surface sterilized with 10% commercial bleach and then washed with double distilled water three times. After surface sterilization, seeds will be placed on ½ Murashige and Skoog (MS) medium containing agar (8g l-1) and sucrose (60 g l-1), and will be placed inside the incubator at 30?C during the day, and 25?C at night in dark. After three months, the temperature will be dropped to 20?C for the day and 15?C for the night to stimulate germination. Once seeds are germinated, seedlings will be cultured with a light intensity of 55 µmol m-2 s-1 with a 16 h/day photoperiod. When corms undergo dormancy, they will be transferred to vials containing new ½ MS medium to accelerate plant growth, and thus corms and plants will be maintained in vitro until they are large enough to flower.Confirmation of interspecific hybridization: The origin of interspecific hybrids will be confirmed with ISSR markers. The genomic DNA of saffron, other Crocus spp. used in hybridization, and the hybrids will be isolated from leaf materials with Promega Wizard® Genomic DNA Purification Kit. The isolated DNA will be quantified with a spectrophotometer and stored at -20°C.ISSR markers-supported analyses: Twenty-seven pairs of previously used ISSR primers for saffron and related Crocus spp. will be ordered from IDT Inc. and labeled with fluorescence. The parameters for polymerase chain reaction (PCR) will follow as previously described. A negative control will be added in each run to test contamination in all analyses. Amplification products for all samples will be separated by capillary electrophoresis with an ABI 3130 Genetic Analyzer. Results will be analyzed with Gene-Mapper software. The hybrids will be confirmed by the presence of alleles from both parents. Replicate amplifications will be performed when necessary.Objective 2. Induce polyploidy among Crocus species and hybridsPolyploidization and plant regeneration: Crocus calli will be initiated and transferred to fresh MS medium containing MS major salts, agar (8g l-1), sucrose (60 g l-1), and oryzalin at 5, 10, and 20 µg l-1 for 5 days. After treatment, calli will be transferred to MS medium containing full concentrations of major salts, agar (8 g l-1), sucrose, (30g l-1), and GA3 (1 mg l-1). Subsequent development and growth occurs at 20°C with a 16 h/day photoperiod.Flow cytometric analyses of regenerants: Young leaves will be macerated and stained with propidium iodide, and sample preparation and measurement of the ploidy level will be performed as previously described for ploidy analyses of Crocus series Verna.Objective 3. Analyze phytochemicals and characterize aroma profiles of C. cartwrightianus hybridsPhytochemical analyses of stigmata: Stigma threads will be harvested from saffron, all species used in this project, and hybrids developed in this project. After harvesting, stigma threads will be dried in a food processor at 60?C for 30 min.; then they will be placed in air-tight containers before analyses. After grinding the samples, they will be sieved through a 0.5 mm mesh. Approximately 100 mg of dry macerated stigmata will be extracted with 5 ml of cold 50% ethanol in mortar. The macerate will be transferred and the volume will be adjusted to 20 ml with 50% ethanol, followed by sonication for 20 min. The content will be centrifuged at 4000 rpm for 10 min and washed with 50% ethanol (5 ml ea.) twice to collect the supernatant. Analysis of the main compounds of saffron extract (picrocrocin, crocins, crocetin, and safranal) will be performed on the Shimadzu LC-MS-IT-TOF (Tokyo, Japan) with a SPDM20A PDA detector using a CORTECS C18 column (150 mm × 4.6 mm × 2.7 μm, Waters, Milford, MA, USA) as our group previously described. The quantification of individual compounds will be calculated based on peak area measurements in comparison to curves constructed with reference standards.Phytochemical analyses of saffron essential oils: Three types of samples will be used for extraction of essential oils from saffron, species of saffron relatives, and hybrids: whole flowers, flowers without stigma threads, and stigma threads. Analyses of these will be carried out on gas chromatography-mass spectrometry equipment with a triple axis detector. Gas chromatographic conditions, including carrier gas linear velocity, will be adjusted and optimized for each type of plant species. Mass spectrometer parameters will be as follows: interface and source temperatures, 250°C and 200°C, respectively; ionization mode, EI with a voltage of 0.94 kV applied; and acquisition mass range, 40-400 m/z with a scan speed of 1666 amu/s and a scan interval of 0.25 s.Objective 4: Develop a course module and workbook on saffron chemistryAn undergraduate course on medicinal herbalism was offered as a "Special Topics" course in the spring of 2020. After approval by the faculty senate at KSU, this course will be formally offered in 2024 and 2025. At least one lab activity in this course will involve essential oils and another will involve saffron and medicinal plants. Additionally, a lab exercise will be designed for a chemistry course, and a workbook on essential oil extraction and use will be developed for STEM education at KSU. The developed lab exercises and workbook will be uploaded to Multimedia Education Resource for Learning and Online Teaching, a free website development tool, which currently indexes tens of thousands of discipline-specific learning materials, and learning exercises.Objective 5: Disseminate research findings and visual aids through KSU web page and social network sites A webpage on research findings and associated project activities will be created via the KSU website. Concurrently, research findings, updates, and photographs will be disseminated through social network sites such as Facebook and YouTube. Videos on growing, harvesting, extracting, and using essential oils and saffron products will be filmed and placed on the YouTube website and KSU's CACS homepage. Undergraduate students will also be recruited for installation and maintenance of the demonstration garden with medicinal plants and herbs, featuring saffron and Crocus species. Finally, trained graduate and undergraduate students will participate in CACS and KSU activities, including AFE Day, KSU Field Day, and Third Thursday Things, and also go to local schools by request.