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