Commercial Micropropagation of Alocasia species*

By Jorge Sahagun

(*Presented during the Philippine Association for Plant Tissue Culture 5th Scientific Convention, Grand Caprice
Convention Center, Lapasan, Cagayan de Oro City, 2-6 May 2005)

ABSTRACT

Philippine Alocasias were tissue cultured for conservation and commercial purposes.
Explants were obtained from both tip and axillary buds. Commercial bleach (Zonrox) was used
to sanitize tissues. Two (2) mm. terminal buds and 3-5mm. axillary buds were excised and
cultured in modified MS medium supplemented with 2 ppm 6-Benzylanimopurine (6-BA) and
2g/l peptone for 2 to 3 weeks followed by subcultures every 3 weeks.
Alocasia micholitziana, A. sanderana, A. boyseana, A. heterophylla, A. scalprum, A.
wenzelli, A. zebrina and 3 other Philippine species (no taxonomic nomenclature yet pending
botanic description) were isolated and grown.
The same technique was used on some other commercial varieties of Alocasia
successfully. Three months after isolation, regenerated plantlets were ready for planting in plugs
or in community pots.

Key words: Alocasia, Conservation, Commercial tissue culture

Introduction

Among the cultivated Araceae plants, the genus Alocasia is considered among the
favorites of both collectors and landscapers around the world. Over the past 150 years,
Alocasia has been the height of fashion in cycles of roughly 30 years (Burnett, 1984). A
lot of species of this enduring ornamental foliage has not been described. Continuous
collection may cause extinction in the wild if not properly monitored. Habitat destruction
is one of the main causes of the different wild Alocasias to be threatened. Artificial
propagation of these species will relieve the pressure of wild collections. Propagation of
horticulturally desirable individuals thru tissue culture will then serve as a powerful tool
for conservation.
Shoot tip culture has been widely used to produce a lot of disease-free clones of
aroids since 1974 (Hartman). Various groups of people and companies adapt the same
technique for mass-producing ornamental aroids. In Malaysia for instance, Chan et al
(2003) designed a Micropropagation protocol for multiplying the population of their local
Araceae ornamental crops. In the Philippines, where the level of endemism of Alocasia is
very high, there is no published reports on how to conserve and commercialized these
plants. Among the Philippine Alocasia species that needs to be conserved are: Alocasia
sanderana, A micholitziana, A. heterophylla, A. scalprum, A. zebrina, A. wenzelii, and
A. boyseana.

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Materials and methods

Establishment of Explants

Alocasia mother plants were conditioned in a cool dry place for two weeks prior
to isolation. Tip cuttings (containing both axillary and terminal buds) of Alocasia
micholitziana, A. heterophylla, A. sanderana, A. wenzelii, A. zebrina, A. scalprum and
two commercial Alocasia varieties were used in determining the appropriate sterilization
techniques for Alocasia tissue culture. Explants were washed in running tap water
followed by thorough cleansing by soap and water. Tissues were then subjected to three-
stage bleach surface sterilization. To establish an appropriate bleach sterilization
technique, two modifications were done and tested for efficiency. The first modification
was done by immersing the explants into 15% (v/v) commercial bleach (Zonrox)
solution, with the addition of powder detergent (Tide) and agitating the tissues for 15
minutes. This was followed by a second stage sterilization using 10% bleach for 10
minutes and final stage using 5% bleach for 5 minutes. The second modification was
done by replacing the 15% bleach with only 1% for 40 to 50 minutes as the first stage
sterilization. The rest is just like the second and third stage of the first modification.
Tissues were rinsed after each stage with sterile water. After rinsing, dead tissues were
removed until the 2mm terminal buds and 3-5 mm axillary buds were left (a tissue
allowance of about 1-2mm). The explants were then inoculated on a modified Murashige
and Skoog (1962) medium supplemented with 2ppm 6-Benzylamino purine + 2 g/L
peptone for 3 weeks.

Effect of Modified MS supplemented with 2ppm 6-BA + 2g/L peptone to Alocasia tissue
cultures

Alocasia explants were inoculated on modified MS supplemented with 2ppm 6-

BA (Chan et al,2003) + 20g/L sugar + 2g/L peptone. The following Alocasia species
were used for this experiment: A. sanderana, A. micholitziana, A. heterophylla, A.
boyseana, A. wenzelii, A. zebrina, A. scalprum, and 3 other Philippine species. The
medium was also tested for two commercial varieties of Alocasias: Alocasia xCorazon
Aquino and Alocasia xMark Campbell. After 2-3 weeks, tissues were observed for their
cultural response.

Rooting and Acclimatization

Multiple shoots and plantlets formed from the calli were transferred to MS
medium with the addition of 2g/L peptone every three weeks. After 3-5 months
depending on the species, healthy plantlets were planted in community pots and
acclimatized in a net house that provides relative humidity.

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Results

Figure 1. Contamination Rate after the Two Modifications of the Three-

Stage Bleach Sterilization
(I= 15%-10%-5%; II=1%-10%-5%)

100 A. micholitziana
A.heterophylla
A. sanderana
50 A. scalprum
A. zebrina
A. wenzellii
0 A. Mark Campbell
I II A. Cory Aquino

Table 1. Performance of the Second Modification (1%-10-5%) of Three Stage Bleach

Sterilization to Alocasia Tissue Cultures

CONTAMINATION % OF ASEPTIC SURVIVAL

Alocasia species RATE EXPLANTS RATE
A. micholitziana 8.33 91.67 100
A.heterophylla 46.15 53.85 75
A. sanderana 62 38 80
A. scalprum 58 42 80
A. zebrina 33 67 67
A. wenzelli 36.36 63.64 67
A. Mark Campbell 44 56 100
A. Cory Aquino 9.1 90.9 100
Average 37.12 62.88 83.62

Table 2. Effect of Modified MS+2ppm 6BA+2g/L peptone in Alocasia Tissue

Cultures

Time of
Alocasia species Response appearance
A. micholitziana Callus; Multiple Shoots 2 wks
A.heterophylla Callus 3wks
A. sanderana Multiple Shoots 2wks
A. scalprum Callus 3wks
A. zebrina Callus 3wks
A. wenzelli Callus 3wks
A.boyseana Callus 3wks
A.species A Callus 3wks
A. species B Callus 3wks
A.species C Callus; Multiple Shoots 2wks
A. Mark Campbell Callus; Multiple Shoots 3wks
A. Cory Aquino Callus; Multiple Shoots 3wks

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Discussion

Establishment of Explants
Figure 1 shows the contamination rate of Alocasia tissue cultures after the two
modifications of three-stage bleach sterilization technique. Contamination rate is
significantly lower after the second modification (1%-10%-5%) with an average of
37.12% compared to the first modification (15%-10%-5%). In the works of Chan et al.
(2003), they used 50mg/L mercury chloride and a two-stage bleach sterilization to
establish aseptic cultures of Araceae. In the present study the modified three-stage bleach
surface sterilization was used. The works of Chan et al. with A. sanderana (37.5%) is
almost identical to the results using the second modification of bleach sterilization (1%-
10%-5%) in terms of aseptic plants produced (Table 1). This sequence of bleach
sterilization gave a high percentage of survival (83.63%).

Effect of modified MS supplemented with 2ppm BA+2g/L peptone in Alocasia tissue

cultures
Alocasia explants responded differently on the modified MS + 2ppm 6-BA (Chan
et al, 2003). To enhance further growth, 2g/L peptone was added to the medium. Most of
the Philippine Alocasia species tested formed calli after inoculation on MS supplemented
with 2ppm 6-BA + 20g sugar + 2g/L peptone. For very responsive species like A.
micholitziana and A. sanderana, evidence of growth is observed as early as two weeks. In
Table 2, only A. sanderana and Alocasia species C formed only multiple shoots after 2-3
weeks in culture. There were cultures that formed both calli and multiple shoots. Most
calli produced in these cultures were manifested in the cultures of terminal buds while
multiple shoots were observed in the axillary buds. Plantlets grew from the calli after
about 3 to 4 weeks after the observance of the calli. This result indicates that various
genetic make up respond differently to certain kind of culture medium.

Conclusion and Recommendation

Alocasia tissues are difficult to clean up because growing points are found near or
below the ground. As stated by Dodds and Roberts (1982), aseptic tissues that were most
difficult to establish were those found in the soil from tropical regions. To establish an
aseptic culture, a three-stage modification (1%-10%-5%) was found to be effective. This
series of bleach concentration gave a high percentage of survival (83.63%).
Most of the commercially cultivated Alocasia species (A. sanderana, A.
micholitziana, and Alocasia xCory Aquino) tested may be propagated using the modified
MS+2ppm 6-BA+2g/L peptone. Plantlets were still produced on some of the calli but not
all turn into plantlets immediately. This indicates cultural responses varied from species
to species, although a lot of the native Philippine species tested responded almost the
same. It might be interesting to try a series of experiments using the different
concentrations of 6BAand its combinations with other growth hormone (i.e. auxins) to
determine the optimum medium for the possible production of multiple shoots for
Philippine Alocasia species.

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References:

Burnett, David 1984. The Cultivated Alocasia. AROIDEANA. Vol. 7 (no. 3 & 4)

Chan, Lai-keng, C.M. Tan and G.S. Chew 2003. Micropropagation of the Araceae
Ornamental Plants. Proc. 1st IS on Accl. & Estab. Microprop. Plants. Eds. A.S.
Economou and P.E. Read Acta Hort. 616, ISHS

Hartman, R. D. 1974. Dasheen Mosaic virus and other phythopathogens eliminated from
caladium, taro and coco yam by culture of shoot tips. Phytopath 64: 237-240

Murashige, T. and Skoog, F. 1962. A Revised medium for rapid growth and bioassay
with tobacco tissue cultures. Physiol. Plantar. 15: 473-497.

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Figure 2. Multiple Shoot formation from the axillary bud of some Alocasia species A:
after 3 weeks B. after 6 weeks

A B

Figure 3. Callus formation in some Alocasia cultures. A. Callus after 3weeks B.

formation of plantlets from the callus after another 3 to 4 weeks

A B