Improvement of Asymbiotic Seed Germination and Seedling Development of Cypripedium Macranthos Sw. With Organic Additives
Improvement of Asymbiotic Seed Germination and Seedling Development of Cypripedium Macranthos Sw. With Organic Additives
Improvement of Asymbiotic Seed Germination and Seedling Development of Cypripedium Macranthos Sw. With Organic Additives
Research Article
Introduction
Members of the Cypripedium genus are commonly called
ladys slippers because of the slipper-like appearance of their
flowers. Cypripedium macranthos Sw. is one of the most
attractive species among the ladys slipper orchids and widely
distributed from Eastern Russia, Northern China, Japan,
Korea and Taiwan (Cribb 1997). Unfortunately they are
becoming extinct due to the destruction of native habitats and
illegal collection (Cribb and Sandison 1998). The most commonly
recommended propagation method for conservation and
commercialization of Cypripedium species is micropropagation,
however, many cultural conditions including physical and
nutritional factors can affect the germination and regeneration
of Cypripedium species critically. Numerous studies on
efficient micropropagation of orchid plants through callus or
protocorm-like bodies (PLBs) have suggested that their
proliferation methods are species-specific and the major
obstacles to successful micropropagation are involved in in
vitro germination of seeds (Arditti 1977; Colli and Kerbauy
1993). So it is very important to find the efficient in vitro seed
germination conditions for each Cypripedium species and
establish their optimum artificial propagation system.
Orchids produce a number of minute seeds but they have
low propagation rate in nature, because ovules are not present
or poorly developed at the time of anthesis, and endosperms
which contain sufficient nutrient reserves for germination are
absent from mature seeds. Therefore, asymbiotic germination
has been a beneficial and common technique for micropropagation
of orchids (Arditti 1967). However, in vitro germination of
terrestrial orchids, such as Cypripedium (Leroux et al. 1995;
Rasmussen 1995), Spiranthes (Zelmer and Currah 1997),
Platanthera (Zettler and MacInnis 1994), Ophrys (Kitsaki et
al. 2004), Calanthe (Lee et al. 2007) and Epipactis (Rasmussen
1992) was found to be more complicated as compared to the
other epiphytic orchids in tropical areas. Because they have
some unfavorable physiological characteristics resulting in
139
140
Sugars
(g)
Total
2.5
2.3
2.8
Sucrose
1.2
0.2
2.3
Glucose
0.8
0.9
0.3
Fructose
0.5
1.2
0.2
24.2
58.6
63.4
2+
Ca
3+
Fe
0.3
0.1
0.6
Mg2+
25.3
11.3
14.5
20.4
6.4
2.7
242.1
120.4
204.2
0.1
1.1
3.3
0.04
0.03
0.7
3-
PO4
+
Inorganic ions K
2+
(mg)
Mn
2+
Cu
Vitamins
(mg)
Na
36.5
5.3
10.4
SO42-
21.4
31.2
38.8
NO3
2.8
3.1
3.5
Thiamin (VB1)
0.03
0.01
Riboflavin (VB2)
0.05
0.01
Niacin (VB3)
0.08
0.03
0.04
0.03
Pyridoxine (VB6)
0.03
0.002
Myo-inositol
0.01
2.4
0.3
0.9
289.3
359.1
141.7
23.7
6.8
15.0
8.4
11.5
12.2
25.6
10.2
8.5
141
Conc.
(B)
0 mlL-1
-1
50 mlL
Coconut
water
-1
100 mlL
-1
200 mlL
-1
Birch sap
14.4 e
60.9 b
62.0 b
70.8 a
74.2 a
64.4 ab
Conc.
(B)
0 mlL-1
-1
50 mlL
Coconut
water
-1
100 mlL
-1
200 mlL
60.8 b
-1
Fresh
weight
(mg)
Dry
weight
(mg)
76.8 ez
7.8 e
228.1 b
24.2 bc
296.4 a
34.1 a
221.9 b
23.7 bc
13.1 e
0 mlL
65.3 e
6.4 e
55.8 c
62.1 b
50 mlL-1
210.3 c
21.9 c
65.2 ab
68.2 ab
260.5 ab
28.7 ab
Birch sap
-1
100 mlL
-1
200 mlL
56.9 c
53.4 bc
200 mlL
205.6 c
22.0 c
0 mlL-1
10.9 g
12.7 e
0 mlL-1
71.4 e
6.9 e
207.6 c
22.4 c
100 mlL
273.2 ab
30.9 ab
200 mlL-1
231.7 b
25.5 b
72.9 e
7.2 e
-1
50 mlL
-1
59.1 b
100 mlL
66.4 ab
66.9 ab
61.1 b
61.4 b
0 gL
-1
15 gL
11.1 g
25.5 f
21.3 de
30.6 ef
35.4 d
60 gL-1
36.8 e
41.9 c
-1
Maple sap
50 mlL
-1
-1
0 gL
12.2 e
30 gL
-1
-1
60.5 b
200 mlL-1
-1
-1
15 gL
Banana
powder
126.7 d
13.3 d
30 gL
178.4 cd
18.6 cd
60 gL-1
168.6 cd
16.9 cd
-1
-1
0 gL
9.6 g
10.3 e
0 gL
68.4 e
6.8 e
1 gL-1
35.2 e
38.2 cd
1 gL-1
135.1 d
14.3 d
43.3 d
43.7 c
169.9 cd
18.0 cd
173.3 cd
18.2 cd
**
**
-1
2 gL
-1
4 gL
40.9 d
Peptone
-1
2 gL
-1
4 gL
35.0 d
Significance
A
Type
(A)
12.8 g
-1
Peptone
12.6 gz
Organic additives
50 mlL-1
-1
Banana
powder
Protocorm
formation
(%)
0 mlL
100 mlL
Maple sap
Germination
(%)
Significance
**
**
**
**
**
**
AB
**
**
AB
**
**
142
Table 4 Effects of organic additives on the growth of seedlings developed from protocorm of C. macranthos Sw. after 5 months
of culture
Organic additives
Type
(A)
Root
Conc.
(B)
No.
Length
(cm)
Diameter
(mm)
Formation
(%)
3.1 d
1.0 d
1.0 a
6.7 ab
2.7 b
0.9 a
100 mlL
7.1 a
2.9 ab
200 mlL-1
7.0 a
2.8 ab
3.2 d
0 mlL-1
Coconut
water
-1
50 mlL
-1
-1
0 mlL
Birch
sap
-1
50 mlL
20.2 g
3.7 e
1.4 b
62.5 c
6.8 b
2.0 a
0.9 a
74.6 a
8.0 a
2.1 a
0.9 a
69.5 abc
8.1 a
2.1 a
0.9 d
0.8 a
23.9 g
3.9 e
1.4 b
2.6 b
0.9 a
57.1 cd
6.4 bc
1.7 ab
3.0 ab
1.0 a
70.4 ab
7.8 a
2.2 a
6.1 b
2.7 b
0.9 a
67.3 b
7.4 ab
2.0 a
0 mlL
3.2 d
1.1 d
1.0 a
23.0 g
3.5 e
1.4 b
50 mlL-1
5.4 c
2.5 b
0.8 a
61.1 c
7.0 b
1.6 ab
6.7 ab
3.3 a
0.9 a
73.5 a
7.9 a
2.1 a
-1
-1
100 mlL
-1
200 mlL
6.6 ab
2.9 ab
0.9 a
68.0 b
7.8 a
2.1 a
0 gL-1
3.0 d
1.4 cd
0.8 a
21.3 g
3.4 e
1.3 b
15 gL-1
4.0 cd
1.8 c
1.0 a
31.5 f
4.2 de
1.5 ab
-1
30 gL
5.2 c
2.1 bc
1.0 a
36.9 ef
4.4 de
1.6 ab
60 gL-1
4.6 cd
1.7 c
0.9 a
29.0 f
5.3 d
1.5 ab
0 gL-1
2.8 d
1.2 d
1.0 a
21.0 g
3.7 e
1.3 b
4.4 cd
1.9 c
0.9 a
44.2 e
5.2 d
1.6 ab
5.1 c
2.3 bc
0.8 a
56.8 cd
6.0 c
2.0 a
4.5 cd
1.9 c
0.8 a
52.6 d
5.3 d
1.9 a
-1
Peptone
Diameter
(mm)
5.9 b
200 mlL
Banana
powder
Length
(cm)
6.6 ab
-1
100 mlL
-1
Maple
sap
Bud
1 gL
-1
2 gL
-1
4 gL
Significancey
A
**
**
NS
**
**
**
NS
**
**
AB
NS
**
**
NS
143
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