A HIGHLY EFFICIENT PROTOCOL FOR MICROPROPAGATION OF NORTH AMERICAN GINSENG Sijun Zhou and Daniel C.W. Brown Southern Crop Protection and Food Research Centre Agriculture and Agri-Food Canada

Introduction Why micropropagation? http://www.thefoodclub.org.uk

Introduction In Ontario, ginseng is the fifth most important cash crop, but it hasn’t been genetically improved.

Introduction Ginseng’s improvement is difficult because it has a long production cycle A micro-propagation method could contribute to its genetic improvement by reducing the generation cycle time The seeds are usually produced after a 3-year cultivation and must be stratified for an additional 12-18 months before germination

A previously established laboratory-scale micropropagation system (Brown et al. 2001) Although the embryogenesis frequency is relatively high, the efficiency of the system is low, because of the low efficiency of the late stages. Especially the field performance and the overwintering stages have a very low efficiency.

Improvement of the previous system Issues 1) Low quality and fused embryos 2) Multiple and abnormal shoots 3) Development of embryos staid on cotyledonary stage 4) Low germination of embryos and elongation of shoots 5) Plants whithout a taproot or a well-developed root system

Issue 1 Fused embryos Embryos could be induced on MS medium without growth regulators, but most of them were abnormal and fused together. The fused embryos gave only multiple shoots, from which quality roots couldn’t be obtained.

Solution Plasmolysis pretreatment The pretreatment of cotyledons with 1.0 M sucrose solution at 4oC for 48-72 h resulted in more individual somatic embryos This problem in Korea ginseng was solved by pre-treating cotyledon explants with 1.0 M sucrose solution for 72 h. However, When cotyledons of North American ginseng were pre-treated with 1.0 M sucrose solution at room temperature following the same protocol, they died even with a 12 h treatment. After the failure of pre-treatment at room temperature, we pre-treated cotyledons at 4 0C. The cotyledons survived even after a 1 week treatment. and a more uniform formation on the explant surface

Issue 2 Abnormal shoots Solution Tissue recycling The abnormal, fused and small-sized somatic embryos only produce enlarged cotyledons, abnormal and multiple shoots after culture on germination medium. These tissues can be used as explants to produce secondary somatic embryos. Abnormal shoots from germination of embryos were used as material to produce new embryos

Efficiency of the system was enhanced tremendously by tissue recycling Formation of somatic embryos with a frequency up to 90% on MS medium with 2,4-D and NAA Up to hundreds of embryos per explant Most of the somatic embryos were single

Issue 3 Development of embryos staid on cotyledonay stage Solution Maturation culture on medium containing charcoal Embryos developed well on SH or ½ MS containing 1% activated charcoal and 3% sucrose Embryos didn’t develop on other tested media without activated charcoal On induction medium, somatic embryos, when reaching the cotyledonary stage, stopped growing, grew very slowly or began undergoing secondary embryogenesis, while small-size of immature embryos did not germinate into plants. For maturation of the somatic embryos, different basal media in different strengths and with different concentrations of sucrose were tested but none of the combinations resulted in development of somatic embryos without activated charcol.

Issue 4 Low germination and shoot elongation Solution High concentration of GA3 Table 1. Effect of GA3 concentration on germination of somatic embryos and conversion of germinated embryos a Concentration (mg l-1) of GA3 b Cumulative germination (%) at various times (weeks)c Conversion (%) of germinated embryos to plantlets in 4 weeks d 2 3 4 5 12.00 16.67±5.46 -- 10 12.63 26.32 44.21 52.70±0.63 83.81±2.35 20 12.86 32.86 47.14 56.04±1.35 85.41±2.24 30 10.96 30.97 41.29 52.36±4.41 74.40±2.91 40 9.68 29.68 47.10 53.45±1.72 63.84±5.84 After 1-2 months on maturation medium, the well-developed embryos were transferred onto germination medium containing GA3. The optimal concentration of GA3 was 10-20 mg l-1. A lower concentration of GA3 gave a much lower germination frequency. In the old protocol, the concentration of GA3 used was 1-2 mg l-1. That is why the germination and shoot elongation were so low. A higher concentration of GA3 resulted in a weak and abnormal shoot and a subsequent low conversion rate of plants. a Somatic embryos produced from tissue recycling; b Basal medium was SH; c Germination was based on the presence of shoots more than 0.5 cm in length; d Plantlets developed on half-strength SH medium containing 0.5% activated charcoal

Germination of embryos on MS medium containing 10-20 mg l-1 GA3 54% of the matured embryos germinated with a normal shoot in 2 to 4 weeks on this medium

Issue 5 Difficulty to obtain plants with well-developed root system Solution Basal medium and activated charcoal Effect of basal media on development of seed-derived plants After germination, the root did not develop well in elongation media using MS as basal medium. To solve this problem, the effect of three basal media, MS, B5 and SH on development of plants was tested using stratified seeds….. According to this result, we changed the basal medium from MS to SH for plant development and activated charcoal was added. Poor embryo germination and death on MS; slower plant development and brown roots on ½ and 1/3 MS Fast development of plants on 1/3 SH; thickened roots with normal color

Development of plants on ½ SH with 0.5% activated charcoal About 85% of the germinated embryos developed into plants with well-developed taproots The taproots did not develop well in any of the tested elongation media without activated charcoal.

Acclimation of plants After elongation, the plants with well-developed root system were established in soil mix (Promix BX) at a 90% efficiency

Transplant to the field About 400 plants were transplanted into the field during the summer season of 2004, 2005 and 2006. Field establishment rate was 94%

Field performance Plants transplanted in May to July, 2004 Pictures taken on June 3, 2005 to show the overwintering status Ginseng garden, Delhi, ON Canada

Field performance of 3-year-old plants Plants transplanted in May to July, 2004 Pictures taken on July 28, 2006 to show the mature plants

Roots from 5-year-old plants

Summary of improvement of the system Plasmolysis at 4oC Maturation culture (activated charcoal) Improved efficient protocol high quality embryos Basal medium Optimal GA3 (10-20 mg l-1) Activated charcoal Plants with well-developed root system

Improved six-stage micropropagation protocol for North American ginseng Overwintering 80% 300 plants in 31 weeks vs 30 plants in 4 years