|
بهنام خداخواه
|
|
111
Biotechnol. Vol. 18 (1), 2010
In Vitro Propagation of Polystachya sp.
AsPac J. Mol. Biol. Biotechnol. 2010
Vol. 18 (1) : 111-114
Effects of Some Cytokinins, Auxins and Medium Constituents on In Vitro Propagation of Polystachya sp.
Chamchuree Sotthikul*1,3, Parichat Choomporn2, Siriwan Kammuen1, Chuntana Suwanthada1,3
1Department of Plant Science and Natural Resources, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand.
2H.M.the King’s Initiative Centre for Flower and Fruit Propagation, c/o Chiang Mai University, Thailand.
3Huai Hong Khrai Royal Development Study Centre, Doi Saket, Chiang Mai 52200, Thailand.
Proceedings Asia Pacific Conference on Plant Tissue and Agribiotechnology (APaCPA) 17-21 June 2007
* Author for correspondence:
Chamchuree Sotthikul, Department of Plant Science and Natural Resources, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand.
Abstract. A study on in vitro propagation of a wild orchid, Polystachya sp., was carried out by culturing shoots on modified VW (1949) or modified MS (1962) media. It was found that the plants could grow on both modified media supplemented with activated charcoal, 2 mg/L kinetin, or 2 mg/L BAP. MS media with 0.25 mg/L IBA, 0.5-1.0 mg/L NAA promoted root length of 0.40 cm. Combination of 1.0 mg/L NAA and 1.0 mg/L IBA induced 4.10 roots/shoot. The rooting plants could successfully grow in the greenhouse.
Keywords: Polystachya sp.; in vitro propagation; Cytokinin; Auxin.
INTRODUCTION
Polystachya is sympodial and usually epiphytic orchid. Pseudobulbs set close together forming clumps. Many species have fragrant flowers, some have contrasting colors on the lip and anther cap. The name of the genus, Polystachya, is derived from the Greek words, poly (many) and stachy (spike or ear of grain), referring to many spikes of small flowers. There are about 200 species of Polystachya around the world, in Africa, Central and South America, Australia, New Guinea, Madagascar, Philippines, Indonesia. However, many species have limited distributions (Stewart, 1996). In Thailand, the distribution of Polystachya sp. (some have identified this orchid as Polystachya concreata (Jacq.) Garay & Sweet) is across the country from the North, Northeast, to the South. The species has small flowers, 5 mm in size with green sepals, light yellowish green petals, and pale yellow lip (Figure 1). The flowering period is during June to July.
In vitro seed culture of many wild orchids including Polystachya sp. has been continuously performed as part of the conservation project by Huai Hong Khrai Royal Development Study Centre. It has been found that Polystachya sp. has interesting features. The orchid could germinate in basic medium and but rarely produced roots unlike other species in the studies. In addition, it could flower in the general in vitro condition (Figure 2), most plantlets bearing flower spikes would turn brown and die afterwards, and a few could set pods (Figure 3). Therefore, the experiments aimed to find medium formula in combination with some growth regulators to induce better growth, multiplication, and the rooting of plantlets. Further in vitro usage of this orchid may be explored and the plantlets could be successfully transferred into the greenhouse and later in the conservation forest.
Figure 1. Polystachya sp.
112
AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010
In Vitro Propagation of Polystachya sp.
Figure 2. In vitro flowering of Polystachya sp.
Figure 3. In vitro pod forming.
MATERIALS AND METHODS
Plantlets, 1 cm in height, were cultured onto modified MS (Murashige and Skoog, 1962) and VW (Vacin and Went, 1949) media supplemented with 0.1% activated charcoal, 2 mg/L kinetin, 2 mg/L BAP. The second experiment was conducted on modified MS media supplemented with 0, 0.25, 0.5, and 1.0 mg/L IBA, or 0.25, 0.5, and 1.0 mg/L IBA, or in combination of both using 3% sucrose, 0.8% agar. The experiments were carried out in completely randomized designs with each treatment having 10 replicates. The cultures were placed under fluorescent light at an intensity of 30 μmol m-2 s-1, 16 h/day.
Explants were evaluated in terms of height, number of leaves, number of roots and root length after 6 weeks of culturing.
RESULTS AND DISCUSSION
It was found that plantlets showed no significant differences in all treatments in terms of height, number of leaves, number of roots and root length. Shoot numbers were also not different (data not shown). However, plantlets on MS-based media tended to produce better root numbers and root length than those on VW-based media (Table 1). The quality of plantlets i.e. leaf color and general appearance on MS media was also better. MS medium with 0.25 mg/L IBA induced plantlets with 0.40 cm of root length, which did not differ significantly from plantlets growing on media with 1.0 and 0.5 mg/L NAA (0.31 and 0.28 cm, respectively) while 2.0 mg/L NAA induced only 0.08 cm of root length. The medium with 1.0 mg/L NAA +1.0 mg/L IBA promoted 4.10 roots having 0.12 cm in length (Table 2, Figure 4). Though it was not significantly different from other treatments and the length of roots is considerably short in the duration of 6 weeks, but the medium could increase the number of roots on this orchid which is normally difficult to induce. Shoot and root forming of plants is influenced by the balance of both endogenous and exogenous and auxin (Miller and Skoog, 1957). There are many studies on cytokinin, auxin, and media on various kinds of orchids such as Dendrobium (Devi and Laishram, 1998; Pathania et al, 1998; Prasad et al, 2001), Geodorum (Roy and Banerjee, 2002), Phaius (Nagaraju and Parthsarathy, 1995). It has been shown that successful propagation of orchids depends on many factors i.e. species or cultivar, age, explant, and media. This study showed that rooting of Polystachya sp. was induced but further adjustment of auxin types, concentrations, or auxin combination are still needed to improve the number of roots and root length. The interesting features such as in vitro flowering and pod setting may be very useful for future breeding projects or physiology studies and more.
Non-flowering Polystachya sp. plantlets could then be successfully grown in the greenhouse with survival rate of 96.89 %.
AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010 In Vitro Propagation of Polystachya sp. 113
Figure 4. Polystachya sp. cultured on MS media supplemented with NAA and IBA for 6 weeks.
Medium
Height (cm)
Number of Leaves
Root Length (cm)
Number of Roots
VW
2.00
1.50
0.20
0.40
VW+AC
2.32
1.74
0.00
0.00
VW + AC + kinetin
1.68
1.24
0.40
0.20
VW + AC + BAP
1.76
1.28
0.38
0.80
MS
1.74
1.32
0.50
1.80
MS+AC
2.26
1.56
0.38
1.40
MS + AC + kinetin
2.48
1.82
0.28
1.40
MS + AC + BAP
2.54
1.80
0.56
1.60
ns
ns
ns
ns
Table 1. Height, number of leaves, number of roots and root length of Polystachya sp. after cultured onto different treatments for 6 weeks.
ns = non significant differences (P = 0.05).
114 AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010 In Vitro Propagation of Polystachya sp.
PGR (mg/L)
Height (cm)
No. of Leaves
Root Length (cm)
No. of Roots
NAA
0
1.56
2.20
0.16
bcd
1.00
0.25
1.46
1.66
0.12
cd
1.50
0.5
1.41
2.08
0.28
abc
1.90
1.0
1.33
1.56
0.31
ab
2.10
2.0
1.24
1.58
0.08
d
1.80
IBA
0
1.18
2.78
0.21
bcd
1.70
0.25
1.28
2.42
0.40
a
2.70
0.5
1.12
2.16
0.19 bcd
2.10
1.0
1.19
1.73
0.13
bcd
1.80
NAA 0.25+ IBA 0.25
1.29
2.07
0.15
bcd
2.30
NAA 0.5+ IBA 0.5
1.25
1.83
0.15
bcd
2.90
NAA 1.0+ IBA 1.0
1.25
2.52
0.21
bcd
4.10
ns
ns
*
ns
Table 2. Height, number of leaves, number of roots and root length of Polystachya sp. after cultured onto different treatments for 6 weeks.
ns = non significant differences (P = 0.05).
* = Means followed by the same letter within the same column were not significantly different by LSD test (P = 0.05).
REFERENCES
Devi, Y.S. and Laishram, J.M. 1998. In vitro Propagation of Dendrobium hybrids through Shoot-tip and Axillary Bud Culture. Journal of Orchid Society of India. 12(1-2): 79-81.
Nagaraju, Y. and Parthsarathy, V.A. 1995. In vitro Propagation of Phaius by Shoot Tip Culture. Annals of Plant Physiology. India. 9(2): 102-104.
Pathania, N.S., Sehgal, O.P.; Debojit, P.; and Dilta, B.S. 1998. Studies on Micropropagation in Dendrobium cv. Sonia. Journal of Orchid Society of India. 12(1-2): 35-38.
Prasad, G.V.S.S, Rao, I.V.S. and Reddy, P.V. 2001. In vitro Propagation of Orchid-Dendrobium ‘Sonia’. Indian Journal of Plant Physiology. 6(3): 284-288.
Roy, J. and Banerjee, N. 2002. Rhizome and Shoot Development During in vitro Propagation of Geodorum densiflorum (Lam.) Schlt. Scientia Horticulturae. 94(1-2) 181-192.
Stewart, J. 1996. Orchids of Kenya. Timber Press Inc. Portland. Oregon. USA. 176p.
پاسخ
مشاهده
|
|
0
|
|
|
|
0
|
|
بهنام خداخواه
|
111
Biotechnol. Vol. 18 (1), 2010
In Vitro Propagation of Polystachya sp.
AsPac J. Mol. Biol. Biotechnol. 2010
Vol. 18 (1) : 111-114
Effects of Some Cytokinins, Auxins and Medium Constituents on
In Vitro
Propagation of
Polystachya
sp.
Chamchuree Sotthikul*
1,3
, Parichat Choomporn
2
, Siriwan Kammuen
1
, Chuntana Suwanthada
1,3
1
Department of Plant Science and Natural Resources, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand.
2
H.M.the King’s Initiative Centre for Flower and Fruit Propagation, c/o Chiang Mai University, Thailand.
3
Huai Hong Khrai Royal Development Study Centre, Doi Saket, Chiang Mai 52200, Thailand.
Proceedings Asia Pacific Conference on Plant Tissue and Agribiotechnology (APaCPA) 17-21 June 2007
Abstract.
A study on
in vitro
propagation of a wild orchid,
Polystachya
sp., was carried out by culturing shoots on modified VW (1949) or modified MS (1962) media. It was found that the plants could grow on both modified media supplemented with activated charcoal, 2 mg/L kinetin, or 2 mg/L BAP. MS media with 0.25 mg/L IBA, 0.5-1.0 mg/L NAA promoted root length of 0.40 cm. Combination of 1.0 mg/L NAA and 1.0 mg/L IBA induced 4.10 roots/shoot. The rooting plants could successfully grow in the greenhouse.
Keywords:
Polystachya
sp.;
in vitro
propagation; Cytokinin; Auxin.
INTRODUCTION
Polystachya
is sympodial and usually epiphytic orchid. Pseudobulbs set close together forming clumps. Many species have fragrant flowers, some have contrasting colors on the lip and anther cap. The name of the genus,
Polystachya
, is derived from the Greek words, poly (many) and stachy (spike or ear of grain), referring to many spikes of small flowers. There are about 200 species of Polystachya around the world, in Africa, Central and South America, Australia, New Guinea, Madagascar, Philippines, Indonesia. However, many species have limited distributions (Stewart, 1996). In Thailand, the distribution of
Polystachya
sp. (some have identified this orchid as
Polystachya
concreata (Jacq.) Garay & Sweet) is across the country from the North, Northeast, to the South. The species has small flowers, 5 mm in size with green sepals, light yellowish green petals, and pale yellow lip (Figure 1). The flowering period is during June to July.
In vitro
seed culture of many wild orchids including
Polystachya
sp. has been continuously performed as part of the conservation project by Huai Hong Khrai Royal Development Study Centre. It has been found that
Polystachya
sp. has interesting features. The orchid could germinate in basic medium and but rarely produced roots unlike other species in the studies. In addition, it could flower in the general in vitro condition (Figure 2), most plantlets bearing flower spikes would turn brown and die afterwards, and a few could set pods (Figure 3). Therefore, the experiments aimed to find medium formula in combination with some growth regulators to induce better growth, multiplication, and the rooting of plantlets. Further in vitro usage of this orchid may be explored and the plantlets could be successfully transferred into the greenhouse and later in the conservation forest.
Figure 1.
Polystachya
sp.
* Author for correspondence:
Chamchuree Sotthikul, Department of Plant Science and Natural Resources, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand.
112
AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010
In Vitro Propagation of Polystachya sp.
Figure 2.
In vitro
flowering of
Polystachya
sp.
Figure 3.
In vitro
pod forming.
MATERIALS AND METHODS
Plantlets, 1 cm in height, were cultured onto modified MS (Murashige and Skoog, 1962) and VW (Vacin and Went, 1949) media supplemented with 0.1% activated charcoal, 2 mg/L kinetin, 2 mg/L BAP. The second experiment was conducted on modified MS media supplemented with 0, 0.25, 0.5, and 1.0 mg/L IBA, or 0.25, 0.5, and 1.0 mg/L IBA, or in combination of both using 3% sucrose, 0.8% agar. The experiments were carried out in completely randomized designs with each treatment having 10 replicates. The cultures were placed under fluorescent light at an intensity of 30 μmol m-2 s-1, 16 h/day.
Explants were evaluated in terms of height, number of leaves, number of roots and root length after 6 weeks of culturing.
RESULTS AND DISCUSSION
It was found that plantlets showed no significant differences in all treatments in terms of height, number of leaves, number of roots and root length. Shoot numbers were also not different (data not shown). However, plantlets on MS-based media tended to produce better root numbers and root length than those on VW-based media (Table 1). The quality of plantlets i.e. leaf color and general appearance on MS media was also better. MS medium with 0.25 mg/L IBA induced plantlets with 0.40 cm of root length, which did not differ significantly from plantlets growing on media with 1.0 and 0.5 mg/L NAA (0.31 and 0.28 cm, respectively) while 2.0 mg/L NAA induced only 0.08 cm of root length. The medium with 1.0 mg/L NAA +1.0 mg/L IBA promoted 4.10 roots having 0.12 cm in length (Table 2, Figure 4). Though it was not significantly different from other treatments and the length of roots is considerably short in the duration of 6 weeks, but the medium could increase the number of roots on this orchid which is normally difficult to induce. Shoot and root forming of plants is influenced by the balance of both endogenous and exogenous and auxin (Miller and Skoog, 1957). There are many studies on cytokinin, auxin, and media on various kinds of orchids such as
Dendrobium
(Devi and Laishram, 1998; Pathania et al, 1998; Prasad et al, 2001), Geodorum (Roy and Banerjee, 2002), Phaius (Nagaraju and Parthsarathy, 1995). It has been shown that successful propagation of orchids depends on many factors i.e. species or cultivar, age, explant, and media. This study showed that rooting of
Polystachya
sp. was induced but further adjustment of auxin types, concentrations, or auxin combination are still needed to improve the number of roots and root length. The interesting features such as
in vitro
flowering and pod setting may be very useful for future breeding projects or physiology studies and more.Non-flowering
Polystachya
sp. plantlets could then be successfully grown in the greenhouse with survival rate of 96.89 %.
113 AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010 In Vitro Propagation of Polystachya sp.
Figure 4.
Polystachya
sp. cultured on MS media supplemented with NAA and IBA for 6 weeks.
|
Medium
|
Height (cm)
|
Number of Leaves
|
Root Length (cm)
|
Number of Roots
|
|
VW
|
2.00
|
1.50
|
0.20
|
0.40
|
|
VW+AC
|
2.32
|
1.74
|
0.00
|
0.00
|
|
VW + AC + kinetin
|
1.68
|
1.24
|
0.40
|
0.20
|
|
VW + AC + BAP
|
1.76
|
1.28
|
0.38
|
0.80
|
|
MS
|
1.74
|
1.32
|
0.50
|
1.80
|
|
|
|
|
|