micropropagation of north american ginseng

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A previously established laboratory-scale micropropagation system (Brown et al. 2001). STAGE. ACTIVITY. EFFICIENCY. 1. Plant and seed selection. 2.
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?

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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 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 welldeveloped 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

and a more uniform formation on the explant surface

Abnormal shoots Solution Tissue recycling Issue 2

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

Low germination and shoot elongation Solution High concentration of GA3 Issue 4

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

2

3

4

5

Conversion (%) of germinated embryos to plantlets in 4 weeks d

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

Concentration (mg l-1) of GA3 b

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

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

• 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 welldeveloped 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 Ginseng garden, Delhi, ON Canada

the overwintering status

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) high quality embryos

Basal medium Optimal GA3 (10-20 mg l-1) Activated charcoal

Plants with welldeveloped root system

Improved efficient protocol

Improved six-stage micropropagation protocol for North American ginseng TIMING STAGE

ACTIVITY

EFFICIENCY 75%

Week 4

1

Embryo induction

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