Plant Cell Reports (1998) 17: 810–813 © Springer-Verlag 1998
S. K. Roy · M. S. Islam · S. Hadiuzzaman
Micropropagation of Elaeocarpus robustus Roxb.
Received: 17 March 1995 / Revision received: 30 December 1997 / Accepted: 9 January 1998
Abstract Multiple shoots were obtained from shoot tips and seed production orchards (Dunstan et al. 1992). Mi-
and nodal explants of 20-year-old trees of Elaeocarpus ro- cropropagation methods are specifically applicable to spe-
bustus on Murashige and Skoog’s medium supplemented cies for which clonal propagation is required. Thus, it is
with 0.5 mg l–1 each of BA and Kn. Explants taken from necessary to develop the essential micropropagation tech-
in vitro-proliferated shoots subsequently produced multi- nology and then proceed to scale up to meet the needs of
ple shoots when cultured on the same basal medium con- a particular operation (Gamborg and Phillips 1995).
taining 0.5 mg l–1 each of BA and Kn. Repeated subcul- Elaeocarpus robustus Roxb. (Family, Elaeocarpaceae)
ture resulted in rapid shoot multiplication at an average is an evergreen species commonly found in tropical coun-
rate of 10 new shoots per subculture. The addition of CM tries and native to Bangladesh (Das 1987). The fleshy sour
(10%) and CH (100 mg l–1) to the medium enhanced the fruits provide vitamin C and are eaten raw or cooked and
number of shoots up to 20 per subculture and increased the pickled. The tree has a fine-textured, moderately hard and
length of shoots. In vitro-raised shoots were rooted on half- strong wood which is suitable for making furniture and mu-
strength MS medium containing 1.0 mg l–1 IBA and 0.5 sical instruments. Due to cross pollination, propagation
mg l–1 IAA. Following transplantation in the field 85% of through seeds dose not conserve true-to-type fruit in this
the plantlets survived and grew uniformly. plant, and clonal propagation through conventional meth-
ods like cuttings or grafting have not been successful. The
Key words Elaeocarpus robustus · Fruit plant · objective of the study reported here was to establish an ef-
Micropropagation ficient and reproducible method for the micropropagation
of Elaeocarpus robustus from mature trees.
Abbreviations NAA α-Naphthalenacetic acid · IBA in-
dole-3-butyric acid · IPA indole-3-propionic acid · IAA
indole-3-acetic acid · Kn kinetin · BA 6-benzylaminopu-
rine · CN coconut milk · CH casein hydrolysate Materials and methods
Plant materials and culture media
Small stem twigs were collected in the spring from coppiced branch-
Introduction es of 20-year-old trees of E. robustus growing in a field of the Ja-
hangirnagar University Campus Dhaka. Expanded leaves were first
Vegetative or clonal propagation provides several advan- removed, and stems were washed in an agitated solution of liquid
tages for the improvement of perennial fruit crops. Clonal detergent for 15 min and later washed in running tap water for 1 h.
Surface sterilization was carried out with 0.1% mercuric chloride
multiplication of superior phenotypes and valuable breed- (BDH) for 5 min. Stems were thoroughly rinsed with sterile distilled
ing stocks can be used for establishing tree improvement water and cut into pieces (1.0–1.5 cm long) before being implanted
into the culture medium.
The basal medium consisted of the mineral salts and organic nu-
Communicated by G. Phillips trients of the MS medium (Murashige and Skoog 1962), 3% sucrose
and 0.8% Difco agar. Depending on the experiment, the basal medi-
S. K. Roy (½) · M. S. Islam · S. Hadiuzzaman 1
um was variously supplemented with factorial combinations of dif-
Department of Botany, Jahangirnagar University, Savar, Dhaka,
ferent growth regulators such as BA, Kn, IPA, IBA, NAA, IAA at
Bangladesh
different concentrations (0.2–2.5 mg l–1). The effects of CM and CH
Fax: 880-2-83 30 54
on shoot growth and development were also determined. All of the
e-mail: ju@[Link]
supplements were added to the molten agar, and the pH of the me-
Present address: dium was adjusted to 5.8, before autoclaving at 1.06 kg cm–1 and
1
Department of Botany, Dhaka University, Dhaka, Bangladesh 121 °C for 15 min in culture tubes (150 × 25 mm) or 100-ml conical
811
flasks (Corning brand). The cultures were maintained at 24° ±2 °C Table 2 Effects of coconut milk (CM) and casein hydrolysate (CH)
under a 16-h photoperiod with a light intensity of 60–70 µE m–2 s–1 on the number of shoots regenerated when shoots were cultured in
from Phillips cool-white fluorescent tubes. There were 20 explants MS medium with constant concentrations (0.5 mg l–1 each) of BA
per treatment and all experiments were repeated thrice. and Kn
CH CM Number of shoots a
Acclimatization and establishment of plants in soil (mg l–1) (% v/v) ± SE
Four-to six-week-old regenerants with well-developed roots were 0 0 10.2 ± 1.6
removed from the culture tubes and washed free of agar. They were 5 11.6 ± 1.3
then dipped in 0.2% cupravit (Cu-oxychloride) (Bayer) fungicide 10 12.2 ± 1.7
for 2 min and subsequently transplanted into plastic trays contain- 15 12.8 ± 1.9
ing sterilized alluvial soil and river sand (1 : 1). The tray contain- 20 9.3 ± 1.5
ing plantlets was covered with a transparent polyethylene lid to main- 50 0 12.6 ± 1.7
tain high humidity, and the microcuttings were misted thrice each 5 12.8 ± 1.3
day. Following 3 weeks at 24° ±2 °C under a 16-h photoperiod the 10 15.6 ± 1.9
lid was removed and the plantlets were transplanted singly to earth- 15 13.5 ± 1.8
en pots containing sterile sand, soild and humus (1 : 2 : 1) at ambient 20 10.6 ± 2.1
room temperature (28 ° ±2 °C) with indirect sunlight. After 2 months
they were planted out. 100 0 12.9 ± 1.6
5 18.5 ± 2.2
10 20.2 ± 1.7
15 15.7 ± 2.3
20 11.8 ± 1.5
Results and discussion 150 0 7.5 ± 0.9
5 10.3 ± 1.2
Initiation of shoot cultures 10 15.2 ± 1.8
15 12.1 ± 1.6
The surfcae-sterilization procedure for explants described 20 8.3 ± 1.4
in the Materials and methods yielded 90% aseptic cultures. a
The values represent the mean (± SE) of three independent expe-
Media containing Kn alone did not form multiple shoots riments. Ten explants were used for each experiment
(data not presented). BA alone induced multiple shoots
but at a low frequency (Table 1). On the combinations
of 0.5–1.0 mg l–1 BA + 0.5 mg l–1 Kn, 76–85% of the cul- NAA combinations (Table 1); other auxins tested failed to
tures showed an induction of shoots within 3 weeks with promote shoot formation (data not shown).
4–5 shoots per culture (Table 1). The only auxin-cytokinin
combinations that promoted shoot induction were BA-
Shoot multiplication
Table 1 Effect of growth regulators in MS basal medium on shoot At the time of subculturing, newly formed shoots were sep-
induction and number of shoots per culture established from apical
and nodal bud explants of E. robustus. Data taken after 28 days of arated and transplanted to fresh medium of the same com-
culture position (0.5 mg l–1 each of BA and Kn). The shoots con-
tinued to proliferate through several subcultures with an
Growth Percentage Number average of 10 new shoots per transfer (Figs. 1, 2). In an at-
regulators of cultures of shoots
(mg l–1) with induced per explant a
tempt to enhance shoot proliferation, we added CH
shoots (%) a (50–200 mg l–1) and CM (5–20% v/v) to the medium.
The addition of 100 mg l–1 CH to the medium in-
BA creased the number of new shoots per transfer to 20
0.5 15.7 ± 2.6 1.8 ± 0.6 (Fig. 3, Table 2). Thus, the optimal medium routinely used
1.0 17.9 ± 1.6 1.7 ± 0.7
1.5 32.1 ± 4.3 2.0 ± 0.8 for multiplication of a large number (20) of shoots with the
2.0 26.2 ± 2.1 2.1 ± 0.6 proper length was MS basal medium with 0.5 mg l–1 BA
2.5 20.6 ± 7.2 1.8 ± 0.4 + 0.5 mg l–1 Kn + 10% CM + 100 mg l–1 CH. Minocha
BA + NAA (1987) reported that the addition of casein hydrolysate at
1.0+0.2 41.3 ± 4.6 1.7 ± 0.6 500 mg l–1 to the medium for the culture of shoot tips of
1.5+0.5 49.6 ± 3.4 1.9 ± 0.7 paper bark birch resulted in significant increases in growth.
1.5+1.0 38.7 ± 4.2 2.1 ± 0.6
2.0+1.0 37.8 ± 3.2 2.1 ± 0.8 There are other reports on the effects of the addition of
complex organic substances on the growth in culture of
BA + Kn
0.2+0.2 51.6 ± 5.9 2.5 ± 0.6 woody species (Gamborg et al. 1976; Thorpe 1992).
0.5+0.5 85.3 ± 5.3 4.6 ± 0.9
1.0+0.5 76.4 ± 6.2 4.2 ± 0.8
1.0+1.0 71.4 ± 3.5 3.8 ± 0.8 Rooting
1.5+1.0 59.5 ± 4.1 3.1 ± 0.5
a
The values represent the mean (± SE) of three independent expe- Without auxin treatment, shoots obtained from the multi-
riments. Twenty explants were used for each experiment plication culture failed to root. Well-developed shoots were
812
Table 3 Effects of auxins in half-strength MS medium with 3%
sucrose on root formation from regenerated shoots of E. robustus.
Data were recorded after 28 days for culture
Growth regulators Shoots rooted (%) a
(mg l–1)
IBA 0.5 40.2 ± 5.3
IBA 1.0 51.3 ± 3.2
IBA 1.5 41.8 ± 7.2
NAA 0.5 0
NAA 1.0 0
NAA 1.5 0
IAA 0.5 29.6 ± 7.2
IAA 1.0 22.2 ± 3.6
IAA 1.5 26.4 ± 8.2
IBA 0.5+IAA 0.5 86.2 ± 8.6
IBA 1.0+IAA 0.5 100.0 ± 0
IBA 1.0+NAA 1.0 76.6 ± 7.3
IBA 0.5+NAA 0.5 46.8 ± 8.3
IBA 1.0+NAA 0.5 58.7 ± 5.6
IBA 1.0+NAA 1.0 56.5 ± 7.4
IBA 1.5+NAA 1.0 48.7 ± 5.8
a
The values represent the mean (± SE) of three independent expe-
riments. Twenty explants were used for each experiment
viding 100% rooting in 23 days (Table 3, Fig. 4). The first
roots began to emerge after 12 days. In contrast, only NAA
produced rooted shoots from Eucalyptus citriodora (Gupta
et al. 1981), while IBA+NAA was effective for Artocar-
pus heterophyllus (Roy et al. 1993). Three auxins in com-
bination, IBA, IAA and IPA, were essential for inducing
roots in Tectona grandis (Gupta et al. 1980).
Acclimatization and establishment of plantlets in soil
After 4–6 weeks in rooting medium, the rooted shoots
were transferred to plastic trays. About 92% of the trans-
planted plants survived. At the time of acclimation the
shoots elongated, and the leaves expanded and turned deep
green. The plant also grew more vigorously when trans-
planted to the potting mix. After 2 months the plants were
planted out into the field, where 85% of the plants sur-
vived. To date they are still growing with full vigor and
uniformity.
The results of this investigation clearly show that api-
Figs. 1, 2 Multiple shoot formation from the shoot-tip explant cal and nodal buds of coppiced shoots from selected ma-
(Fig. 1) and nodal explant (Fig. 2) of E. robustus on MS medium ture trees of Elaeocarpus robustus are capable of produc-
with 0.5 mg l–1 BA+0.5 mg l–1 Kn ing multiple shoots in vitro, which can then be rooted to
form complete plantlets. Moreover, the multiplication po-
Fig. 3 Elongation and multiplication of shoots cultured in MS me-
dium supplemented with 0.5 mg l–1 each of BA and Kn+10% tential continued for an extended time, and the survival of
CM+100 mg l–1 CH regenerants in the fild was satisfactory. The technique de-
scribed here provides a promising method for propagation
Fig. 4 Root formation on a regenerated shoot using half-strength on a commercial scale as well as for the conservation of
MS with 1.0 mg l–1 IBA+0.5 mg l–1 IAA
superior genetic strains.
excised from the culture flask and implanted individually Acknowledgements Financial support for the purchase of labora-
on root induction medium containing half-strength MS ba- tory equipment through a research grant to S. K. Roy from the Inter-
national Foundation for Science, Sweden, is gratefully acknowl-
sal medium with different concentrations and combina- edged.
tions of IBA, NAA and IAA. IBA at 1.0 mg l–1+0.5 mg l–1
IAA was found to be the best combination of auxins, pro-
813
Gupta PK, Mascarenhas AF, Jagannathan V (1981) Tissue culture of
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