Stevia Breeding

Stevia Breeding at NC State

Stevia rebaudiana (Bertoni) is an herbaceous perennial crop that can be used to produce sweeteners that are non-caloric, natural, and healthful. The leaves contain steviol glycosides, which probably help the plant deter feeding from animals.

The steviol glycosides found in stevia can be classified based on their level of accumulation. Major glycosides include rebaudioside A and stevioside, and minor glycosides include rebaudioside D, and rebaudioside M. Stevioside comprises the majority of the steviol glycosides found in many cultivars of stevia, and is known to have bitter flavor and a negative impact on consumer experience.

However, rebaudioside A has a more desirable taste profile, and currently, is the most studied steviol glycoside and the most used in the commercial market for stevia ingredients.

Project personnel

  • Todd Wehner, project leader
  • Emily Silverman, Research specialist

Funding sources

  • NC Agricultural Research Service
  • USDA-SCRI
  • PepsiCo

Current research

  • Development of cultivars for the southeast U.S.
  • Seed storage and germination
  • Heritability of yield and quality traits
  • Optimization of the breeding program
  • Resistance to disease, lodging, and cold stress
  • Polyploidy and self-incompatibility

Research station locations

  • Raleigh (greenhouses, growth rooms, mist bench)
  • Clinton (fields, greenhouse float beds, dryer)
  • Oxford (fields, greenhouse float beds, dryer)
  • Whiteville (fields, greenhouse float beds, dryer)
  • Our stevia articles

All About Stevia

Introduction

  • Low-calorie sweeteners are important to the food industry. There is extra value if the sweeteners come from natural sources. One of the currently recognized sources of low-calorie, natural sugar replacements is Stevia rebaudiana Bertoni. Others include sorbitol and monk fruit (luo han guo).
  • In the U.S., stevia was approved by the FDA for use as a food in 2008, and is generally recognized as safe (GRAS).
  • In 1899, Swiss botanist M. Bertoni described stevia in 1899 while doing taxonomic studies in Paraguay. Glycosides were identified as the sweetening compounds in 1931.
  • The leaves of stevia are a valuable natural sweetener. The crop is an herbaceous perennial of the Asteraceae family and native to Paraguay. It was originally used by the Guarani people there.
  • Stevia is a potential new crop for North Carolina, especially for those growing tobacco or peanut, since they already have a lot of the production equipment needed.
  • The main compounds in stevia are glycosides: steviosides and rebaudiosides. Rebaudioside A (one of the reb fractions) is commonly extracted and available on the ingredients market. These compounds are 250 to 300 times sweeter than sucrose, but they have inherent challenges when used in drinks (cola and lemon-lime), foods (baked goods), whole leaf (e.g., in tea), or packets (table sweetener) due either to their physical, chemical, or sensory properties.
  • Research is needed to increase the concentration of steviosides in the leaves of stevia, and to change the balance to those steviosides that have high sweetness and away from steviosides that have high sourness or bitterness (directly, or as an aftertaste).

Botany

  • Stevia rebaudiana is an herbaceous perennial in the sunflower family, Asteraceae. It is native to Paraguay, and is grown throughout the world. Production is mainly in east Asia and South America. The shoots, and especially the young leaves of stevia contain natural low-calorie sweeteners. The leaf sweetener content is twice that of the flowers; the stems are lower than the flowers, and the roots have none. The flowers of stevia are hermaphroditic with a 2-lobed stigma and 5 anthers.
  • Stevia belongs to the tribe Eupatorium, and the analysis of chloroplast DNA shows that there has been evolution from a high base chromosome number to a low base chromosome number. Thus, stevia is one of the more adapted species in the tribe.

Horticulture

  • Stevia seeds are small and germinate slowly; direct seeding in the greenhouse will result in transplants in 45 to 60 days. Propagation of stevia can also be done with stem cuttings, using 4 internodes per cutting for best results. Propagation is best done in late winter rather than early or late spring. Germination of stevia seeds is commonly less than 50%. Germination speed will be faster on a heating pad set at 40°C for under 24 h, but the total germination percentage will be lower. Thus, breeding progress can be improved (more cycles per year) using methods that promote faster germination (although sacrificing higher germination percentage).
  • Leaf yield in Mississippi was highest from 1 harvest every 180 days, rather than 2 harvests every 90 days or 3 harvests every 60 days. So, research programs needing to take data with fewer labor inputs may want to use just a single harvest at the end of the summer season, rather than 2 or 3 harvests over the summer.
  • Stevia is self sterile, probably due to sporophytic self-incompatibility. Plants produce black and tan seeds. Open pollination results in 0-60% black seeds, controlled cross-pollination results in 86% black seeds, the rest being tan colored. The tan seeds have no germination, but black seeds have 59-86% germination. Thus, stevia breeding programs will need to avoid self-pollination, and use either open-pollination or controlled cross-pollination to produce seeds for breeding and testing. Breeding methods using population improvement or synthetic varieties would be efficient, but not methods using pure lines or inbreds.
  • Black vs. brown seeds (photo)

Production Methods

  • The stevia field can be flat-planted and transplants put in on 15″ row spacing.  The field can be fumigated prior to transplanting.  Immediately after transplanting, 0.75″ of irrigation should be applied to start plants growing, and to settle the soil around the roots to prevent the first herbicide treatments from contacting the roots. This will get the crop to form a canopy quickly.  Weeds can became a problem, even with three hand weedings per year.
  • Stevia can be produced using tobacco facilities, such as greenhouse float beds for transplant production, vegetable transplanters for field establishment, and tobacco barns for stevia leaf drying after harvest. Leaf harvest can be done using a snap bean picker.
  • Greenhouse float beds (photo)
    • Flats with and without float (photo)
  • Vegetable transplanter (photo)
  • Tobacco barn (photo)
  • Snap bean picker (photo)

Production is as follows:

  1. The seedbed (pH 5.5 to 6.5) should be watered to near the limit for the soil before seeding, and kept at a temperature of 15 to 25 C.
  2. Seed the soil bed with: 10 to 15 gr/m2.
    1. Thin plants to: 1 plant/16.7 cm2 (600 plants/m2).
    2. Seedbed dimensions: 10 m x 1 m with a separation of 50 cm among seedbeds.
    3. Approximately 166-170 m2 of seedbed will plant 1 ha of production.
    4. The texture of the seedbed should be sandy, with organic material and phosphorus added.
  3. Distribute the seeds on the surface. Do not cover them with soil. Apply pressure slightly on the surface using the palm of your hands to attach the seeds to the surface. Stevia seeds need sunlight for germination. It is a photoblastic species, so seeds will not germinate if they are covered by soil.
  4. Cover the seedbed with a shade cloth (50%) until the plants are 1 to 2 cm high. The fabric will protect the seeds from wind and desiccation.
  5. Day 1 to 4: when watering seed displacement should be avoided. The seedbed should stay moist near the limit of the soil until germination. Water 2 to 5 times/day, watering through the shade cloth to prevent seed displacement.
  6. Day 5 to 7: the seeds will begin to germinate.
  7. Day 8 to 60:
    1. The plants should be 1 to 2 cm high. Raise the shade cloth to 40 cm and water the plants through the cloth 3 times/day.
    2. Eliminate any poor-growing stevia seedlings as well as weeds.
  8. Day 60 to 90: Plants that are 10 to 15 cm high, with a minimum of 20 leaves and strong branches are ready to be transplanted.

Weed control

  • Research on weed control in North Carolina is progressing, in the IR-4 program. Several herbicides have been identified that work in stevia for both annual and perennial crops.
  • Recommendations will be added to the NC Agricultural Chemicals Manual.
  • Follow the stevia research in the IR-4 program at NC State.

Chemistry

    • The sweetness obtained from stevia leaves is due to stevioside and rebaudioside. Stevioside has a bitter aftertaste, making it less suitable for use as a sweetener. Rebaudioside-A is sweet without the bitter aftertaste, so it is the preferred compound for isolation from dried leaves. Other rebaudiosides (such as reb-D) have less off flavors than reb-A, and may enhance the sweetness of reb-A. Stevia glycosides are diterpenoids whose biosynthetic pathways share four steps in common with gibberellic acid formation. It appears that stevia has an altered biochemical pathway that favors the production of the intermediate compounds rather than the final product, gibberellic acid.
    • None of the sweet compounds metabolize in the human body, so are non-caloric, cause no diabetes, and cause no dental cavities. Steviosides are natural, low-calorie sweeteners. This makes stevia a valuable crop for world agriculture, looking for replacements for sucrose and fructose. Stevia became useful as a sweetener crop when it was commercialized by the Japanese in the 1970s. It is now available on the market as leaves, or powdered extracts for use in the preparation of food and drink.
    • The stevia glycosides are present in different forms in stevia leaves, and have different tastes on the human tongue. Stevioside is 250 to 300 times the sweetness of sucrose, reb-A is 350 to 450 times the sweetness of sucrose, reb-B (may be an artifact of measurement) is 300 to 350 times the sweetness of sucrose, reb-C is 50 to 120 times the sweetness of sucrose, reb-D is 200 to 300 times the sweetness of sucrose, reb-E is 520 to 300 times the sweetness of sucrose, dulcoside-A is 50 to 120 times the sweetness of sucrose, and steviolbioside is 100 to 125 times the sweetness of sucrose.
  • Genetics
    • Both genotype and environment affect the content of steviol glycosides in the stevia crop. Broad-sense heritability was measured for yield and quality of 10 varieties of stevia tested in 1 location with 3 replications. Heritability was high for leaf yield (99.0%), stevioside content (92.8%), and plant height (97.0%).
    • Stevia has various leaf types, including:

Breeding

Cultivars and Germplasm

A stevia germplasm collection was made from Paraguay for the Institute of Himalayan Bioresource Technology in India. Paraguay is the center of diversity for the crop. India has a large breeding program for stevia, and breeders there have introduced several elite varieties.

Breeding Methods

Stevia is induced to flower by short days, which occurs in September to December in the northern hemisphere, and usually occurs 54 to 104 days after transplanting. Photoperiod sensitivity ranges from 8 to 14 hours requirement for flowering depending on the variety. Flowering can be induced at the 4-leaf-pair stage using 2 short-day cycles. Therefore, seedlings of the stevia accessions of interest can be treated with 2 short-day cycles before transplanting to the field, and they should flower when needed in the intercross blocks for the plant breeding program.

  • Greenhouse with supplemental light (photo)
  • Pollination with greenhouse cages (photo)
  • Propagation in tissue culture (photo)
  • Seed increase using field cages (photo)

Stevia is a relatively new crop for agriculture, but has found an important place due to the recent emphasis on natural low-calorie sweeteners. There is already a significant body of research covering the biology, genetics and breeding of stevia from institutes around the world, including Brazil, Canada, China, India, and the U.S. Progress has been made in areas including seed germination, stand establishment, crop production, shoot harvest, and leaf processing.

New varieties have been developed that improve on the performance of previous varieties for yield and steviosides. More importantly, it is possible to reduce the bitter compounds, and increase the sweet compounds through selection.

  • Yield data from field plots (photo)

Resistance

  • Disease resistance has been studied for Septoria. A screening method was developed to test for resistance, and stevia clone 598-1 was identified as resistant in both field and greenhouse tests.
  • Tomato spotted wilt virus has been identified as a problem.
  • Most diseases of tobacco do not affect stevia, so stevia makes a good rotational crop for tobacco.
  • Cold tolerance and overwintering ability are important in most areas of the U.S.

Seed Companies

  • Baker Creek
  • Botanical Interest
  • Everstevia
  • Germany Seeds
  • Gurney’s
  • Harris Seed
  • Hirt’s Botanical
  • Johnny’s
  • Jung Seed
  • Park Seed
  • RH Shumway
  • Richter’s
  • Seed Buddy
  • Seed Savers
  • Seedway
  • Stevia Store (Eirete I)
  • Stevia Store (Eirete II)
  • Stevia Store (Katupyry)
  • Stevia Store (Morita II)
  • Stevia Store (Morita III)
  • Stevia Store (Native)
  • Stokes
  • Super Sweet
  • Swallowtail
  • Territorial
  • Tradewinds

Stevia Organizations