A HIGHLY EFFICIENT PROTOCOL FOR MICROPROPAGATION OF NORTH

AMERICAN GINSENG

Sijun Zhou and Daniel C.W. BrownSouthern 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

A previously established laboratory-scale micropropagation system (Brown et al. 2001)

STAGE ACTIVITY EFFICIENCY

1 Plant and seed selection

2 Introduction into culture 95%

3 Embryo induction 90%

4 Embryo development 20 / explant

5 Shoot production 19 / explant

6b Tissue recycling 47%

6a Shoot elongation 25%

7 Root production 50%

8 Acclimatization 65%

9 Field performance 11%

Overwintering 3%

Improvement of the previous system• Issues

1) Low quality and fused embryos 2) Multiple and abnormal shoots3) Development of embryos staid on cotyledonary stage4) Low germination of embryos and elongation of shoots5) Plants whithout a taproot or a well-developed root system

Issue 1 FusedembryosEmbryos 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 pretreatmentThe pretreatment of cotyledons with 1.0 M sucrose solution at 4oCfor 48-72 h resulted in more individual somatic embryos

and a more uniform formation on the explant surface

Issue 2 Abnormal shootsSolution Tissue recycling

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 explantMost of the somatic embryos were single

Issue 3Development of embryos staid on cotyledonay stageSolution 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

Issue 4 Low germination and shoot elongationSolution High concentration of GA3

Concentration (mg l-1) of GA3

b

Cumulative germination (%) at various times (weeks)c

Conversion (%) of germinated embryos

to plantlets in 4 weeks d2 3 4 5

5 0 12.00 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

Table 1. Effect of GA3 concentration on germination of somatic embryos and conversion of germinated embryos a

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 5Difficulty to obtain plants with well-developed root systemSolutionBasal medium and activated charcoal

• 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

Effect of basal media on development of seed-derived plants

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 statusGinseng garden, Delhi, ON Canada

Field performance of 3-year-old plants

Plants transplanted in May to July, 2004Pictures taken on July 28, 2006 to show the mature plants

Roots from 5-year-old plants

Summary of improvement of the system

Improvedefficientprotocol

high quality embryos

Plants with well-developed root system

Plasmolysis at 4oC

Maturation culture(activated charcoal)

Basal medium

Optimal GA3(10-20 mg l-1)

Activated charcoal

Improved six-stage micropropagation protocol for North American ginseng

TIMING STAGE ACTIVITY EFFICIENCY

Week 4 1 Embryo induction 75%

Week 8 2 Embryo maturation

Week 12 3 Embryo germination 54%

Week 16 4a Plant development 85%

Week 16 4b Tissue recycling 80%

Week 18 5 Acclimatization 90%

Week 24 6 Transfer to field 94%

Overwintering 80%

300 plants in 31 weeks vs 30 plants in 4 years