Dendrobium Family – Orchidaceae Exhibits a vast diversity in vegetative and floral characteristics 1,600 Dendrobium species are recognized worldwide High value of crop – for flower and medicinal purpose D. husohanense- Anti-tumor and Anti- inflammatry D. longicornu - used to treat fever and coughs Micropropagation Seeds are minute and lack endosperm Micropropagation has been achieved using Shoot tip culture Seed culture (Immature and mature embryo) Auxiliary Bud culture Pseudobulb segment culture Shoot Tip culture Sterlization of Explant Shoot tips(0.5–0.8 cm) harvested from mother plants carefully washed in distilled water surface decontaminated with 0.1% streptomycin (20 s) 70% (v/v) ethanol (50 s) and 0.1% (w/v) HgCl2 (2 min) Thoroughly rinsed with sterilized distilled water Media Subculturing Micropropagation of Dendrobium From Pseudobulb segment Regeneration from Pseudobulb Segment Cultures Pseudobulb segments of about 0.5-1.0 cm excised from the 1 year old in vitro raised seedlings Any leaves or roots, if present, were removed from the segments prior to inoculation Each segment had one or two axillary buds. Single pseudobulb segment was cultured in test tubes (25 mm × 150 mm), each containing 12 mL half-strength MS basal medium supplemented with BAP 1.0 mg L-1 individually or in combination with NAA at 1 mg L-1 The medium was solidified with 4 g L-1 agar Rooting of Regenerated Shoots (Pseudobulbs) Small clumps of shoots having 2-3 pseudobulbs (3-4 cm in length) were cultured in test tubes (25 mm × 150 mm) Each containing 12 mL half-strength MS basal medium supplemented with or without 1.0 mg L-1 IAA or IBA or NAA. The cultures were incubated for 3 months under the conditions as described above. The pH was adjusted to 5.8 before autoclaving at 121°C, 15 lb in-2 for 15 min. Micropropagation of Dendrobium From Auxiliary bud Stem (1–2 cm long), each comprising a node and axillary bud are used as explant- wash in running tape water for 15-20 minute Surface sterilization with- - 10 % (v/v) NaClO solution for 10 minute - 0.1 % (w/v) HgCl2 for 2 min - washing 5–6 times with sterilized distilled water The explants were shortened to 3–4 mm after the removal of leaves, dry sheaths and other external tissues Micropropagation of Dendrobium from immature seeds Capsules collected from hand-pollinated plants after 8–14 wk of pollination Surface-disinfected in 70% ethanol for 30 s, followed by 1.0% sodium hypochlorite with two drops of Tween 20 per 100 ml for 10 min and rinsed five times with sterile distilled water After sterilization, the c

1. Micropropagation of Dendrobium
Manglam Arya
Ad no.-2014-11-102
Centre for Plant Biotechnolgy and Molecular Biology

2. Dendrobium
D. wardianum
• Family – Orchidaceae
• Exhibits a vast diversity in vegetative
and floral characteristics
• 1,600 Dendrobium species are
recognized worldwide
• High value of crop – for flower and
medicinal purpose
 D. husohanense- Anti-tumor and Anti-
inflammatry
 D. longicornu - used to treat fever and
coughs

3. D. crystallinum D. draconis D. formerii
D. latouria
D.polysema
D. spectabile D. pendulum

4. Micropropagation
 Seeds are minute and lack endosperm
 Micropropagation has been achieved
using
a) Shoot tip culture
b) Seed culture (Immature and mature embryo)
c) Auxiliary Bud culture
d) Pseudobulb segment culture

5. Shoot Tip culture
Sterlization of Explant
Shoot tips(0.5–0.8 cm) harvested from mother plants
carefully washed in distilled water
surface decontaminated with 0.1% streptomycin (20 s) 70% (v/v) ethanol
(50 s) and 0.1% (w/v) HgCl2 (2 min)
Thoroughly rinsed with sterilized distilled water
Transverse-thin sections of 1–5 mm thick were taken from shoot tips

6. Media
Composition
A) Full strength MS + 2 mgL-1 BA
B) Basal medium with 3.0% sucrose, 0.7% agar, 0.5 g L-1 mesoinositol, 1.0 g
L-1 casein hydrosylate, 0.5 g L-1 L-glutamine, 250 mg L-1 peptone, 0.2 g L-1
p-aminobenzoic acid and 0.1 g L-1 biotin and 2-7μgL-1TRIA (Triacontanol)
•The pH of the media was adjusted to 5.8 with NaOH or HCl before
agar was added.
Sterilization
•The media were then sterilized by autoclaving at 1210C 15 min
• L-glutamine, biotin, paminobenzoic acid and TRIA were filter sterilized and
added to the media after autoclaving when themedium had cooled to below
50 0C.

7. Subculturing
The cultures were maintained for 6–12 weeks for the
initiation of protocorm-like bodies (PLBs) or proliferating
shoot buds.
 The freshly initiated PLBs transferred to the basal medium
containing 4.0 mg L-1 TRIA.
Healthy shoots with 2–3 leaves developed within 5–10 weeks
The well-developed shoots with 2–3 leaves were further
transferred on fresh basal medium of supplemented with TRIA
2.0 mg L-1 for rooting.

8. Initiation of PLBs from thin
sections of shoot tip on the
Formation of healthy shoots
from PLBs
Healthy shoots with well-developed
leaves ready for rooting

9. Micropropagation of Dendrobium From Pseudobulb
segment
Regeneration from Pseudobulb Segment Cultures
 Pseudobulb segments of about 0.5-1.0 cm excised from the 1 year
old in vitro raised seedlings
 Any leaves or roots, if present, were removed from the segments
prior to inoculation
 Each segment had one or two axillary buds. Single pseudobulb
segment was cultured in test tubes (25 mm × 150 mm), each
containing 12 mL half-strength MS basal medium supplemented
with BAP 1.0 mg L-1 individually or in combination with NAA at 1
mg L-1
The medium was solidified with 4 g L-1 agar

10. Rooting of Regenerated Shoots (Pseudobulbs)
 Small clumps of shoots having 2-3 pseudobulbs (3-4 cm in length) were
cultured in test tubes (25 mm × 150 mm)
 Each containing 12 mL half-strength MS basal medium supplemented with
or without 1.0 mg L-1 IAA or IBA or NAA.
 The cultures were incubated for 3 months under the conditions as described
above.
 The pH was adjusted to 5.8 before autoclaving at 121°C, 15 lb in-2 for 15
min.
 The culture is incubated for 60 days-
- Temperature- 25_+20C
- Photoperiod- 16 hrs(white fluorescent light)

11. Flower of D. transparens Axillary bud from a pseudobulb
segment developing into callus
Multiple shoot induction
In vitro rooting of the regenerated
microshoots (pseudobulbs)
Flowering of the plantlets under
shade net house (50%) environment

12. Micropropagation of Dendrobium From
Auxiliary bud
• Stem (1–2 cm long), each comprising a node and axillary bud
are used as explant- wash in running tape water for 15-20
minute
• Surface sterilization with-
- 10 % (v/v) NaClO solution for 10 minute
- 0.1 % (w/v) HgCl2 for 2 min
- washing 5–6 times with sterilized distilled water
• The explants were shortened to 3–4 mm after the removal of
leaves, dry sheaths and other external tissues

13.  Then cultured on media -
- MS medium + 3% sucrose + 0.8% agar
- the growth regulators - NAA, 2,4-D and BAP 0 and 50 mM,and in various
combinations
 Culture condition
- cultures were incubated at 25+2 8C under a 12-h photoperiod of 50 μmol m-2 s2
photon flux density
 After 20–25 weeks of culture these plantlets were transferred to perforated
‘Thermocol’ or propylene plastic pots (10 × 7 cm size) containing a range of composts:
(i) Crushed brick and charcoal (1 : 1)
(ii) Crushed brick and charcoal (1 : 1) + a top layer of moss
(iii) Crushed brick and charcoal, and decaying litter (1 : 1 : 1)
(iv) Crushed brick and charcoal, and decaying litter (1 : 1 :1) + a top layer of moss
(v) Crushed brick and charcoal,and shredded bark (1 : 1 : 1)
(vi) Crushed brick and charcoal chunks, and shredded bark (1 : 1 : 1) + a top layer
of moss

14. (A)Mature plant in the natural
habitat.
(B) Initiation of PLBs on explants
cultured on MS medium containing
15 mM BAP and 15 mM 2,4-D for
15 days
(C) Initiation of shoots from an
axillary bud on medium containing
5 mM BAP and 15 mM NAA in 15
days
(D) As in (C) but after 30 days
(E) Rooted plantlets after 70 days in
culture
(F) Hardened plantlets after 90 days

15. Micropropagation of Dendrobium from immature
seeds
 Capsules collected from hand-pollinated plants after 8–14 wk of pollination
 Surface-disinfected in 70% ethanol for 30 s, followed by 1.0% sodium
hypochlorite with two drops of Tween 20 per 100 ml for 10 min and rinsed five
times with sterile distilled water
 After sterilization, the capsules were dried in a laminar airflow cabinet before
dissecting
 Capsules were dissected longitudinally in the laminar airflow cabinet, Seeds
were scooped out and sown by spreading as thinly over the surface of the
culture medium in a glass test tubes each containing 10 ml of medium

16. The medium consisted of full-strength MS-medium or half-
strength Knudson’s (KC) medium or Vacin and Went’s (VW)
medium supplemented with 3% sucrose and 0.9% agar
pH of all media was adjusted to 5.7 with 1 N NaOH or HCl
before autoclaving at 1210C, 15psi for 15 min
culture at 250C under cool white fluorescent light at 40mmolm-2
s-2 with a 16 hrs photoperiod per day
The green and white PLB were subcultured onto fresh medium
of similar composition. The seedlings derived from the 12-wk-old
capsules cultured on half-strength MS +1mgL-1 were used for
rooting

17. Advantage
 Produces numerous propagules in relatively short
period of time
 Propagation can be done all year round independent
of seasonal changes
 Produces large number of disease-free planting
materials, free from viruses, fungus, bacteria
 Produces clones of plants that are slow and difficult
or impossible to propagate vegetatively

18. References
1) Shu-fung, L., Nalawade, S.M., Chao-Lin, K., Chung-Li, C.,and Hsing-
Sheng, T. 2004. Asymbiotic germination of immature seeds, plant
development and ex vitro establishment of plants of Dendrobium tosaense
makino – a medicinally important orchids. Society for In Vitro Biology.
40:528–535.
2) Sunitibala, H., and Kishor, R. 2009. Micropropagation of Dendrobium
transparens L. from axenic pseudobulb segments. Indian J. of
Biotechnol.vol.8: 448-452.
3) Dohling, S., Kumaria, S., and Tandon, P. 2012. Multiple shoot induction
from axillary bud cultures of the medicinal orchid, Dendrobium longicornu.
AoBPLANTS[ejournal].Available:http//www.aobplants.oxfordjournals.org/p
ls032;doi:10.1093/aobpla/pls032. [6 December 2014].
4) Ravindra, B. M., Gangadhar, S. M., and Nataraja, K. 2004.
Micropropagation of Dendrobium nobile from shoot tip sections. J. of
Plant Physiol. 162 (2005) 473—478.

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